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Theses & Dissertations Graduate Studies

Fall 12-14-2018

Developing of Micellar Drug Carriers for the Treatment of Breast Developing of Micellar Drug Carriers for the Treatment of Breast

Cancer Bone Metastasis Cancer Bone Metastasis

Tong Liu University of Nebraska Medical Center

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DEVELOPING OF MICELLAR DRUG CARRIERS FOR THE TREATMENT OF

BREAST CANCER BONE METASTASIS

by

Tong Liu

A DISSERTATION

Presented to the Faculty of

The Graduate College in the University of Nebraska

In Partial Fulfillment of the Requirements

For the Degree of Doctor of Philosophy

Pharmaceutical Sciences Graduate Program

Under the Supervision of Professor Tatiana K. Bronich

University of Nebraska Medical

Center Omaha, Nebraska

November 2018

II

ACKNOWLEDGMENT

The journey in the past five years was an unforgettable experience for me to

explore the scientific researches. Many ups and downs, joys and sorrows challenged

every aspect of my personal and professional to extreme. I'm glad that I went through

finally and now look forward to moving to the next step for a new career. It could never

be possible without the contribution of people who lend their hands to the shapes the

research projects and my graduation. Please allow me at this special moment to express

the sincere appreciation and the gratitude for the invaluable support.

My heartfelt appreciation goes to my mentor, Dr. Bronich, who is a brilliant and

dedicated scientist and has been guiding me constantly throughout the Ph.D. training. It

could never be imagined for me to finish the researches without her countless support

and patience. She's been critical and inspiring during each discussion, through which I

grew my profession steadily. Many researching opportunities would be missed if she did

not provide innovative and sharp comments and suggestion. From her, I learned to

conduct researches with the attention to details and to present data concisely. She also

granted me plenty of opportunities and flexibilities to explore and evaluate my ideas. It

has been a great pleasure to be her student. Dr. Rakesh Singh has been enormously

helpful in the biological study in the bone metastases, and I was able to gain the skill of

intracardiac injection under his instruction. He was so kind that no matter how busy the

schedule was, he always found the time to sit down with me and shared his insight. I feel

lucky to have him in the committee. I would extend my thankfulness to other committee

members, Dr. Band, Dr. Li, and Dr. Vetro. They have been effective co-mentors and

III

shaped the achievement with valuable advice and constructive criticism. Special thanks

to Dr. Li for his kindness and suggestion on my life as a student. I would also like to

thank my current and previous lab members. Dr. Svetlana Romanova made the efforts in

the synthesis of the polymers and helped to prepare the material in the animal studies.

Dr. Hangting Hu brought me aboard of the cell-based experiments and transferred the

essential techniques, which was the foundation of many of the researches.

I thank Dr. Swapnil Desale, Dr. Jinjin Zhang, Dr. Krtui Soni, Dr. Fan Lei and Dr.

Shaheen Ahmed, Xinyuan Xi, Nan Zhao, Fei Wang and Zi Wang for their kind help,

friendship, and the opportunity to work with them. The Ph.D. study is focused on a

pinpoint and dives deeply. When reaching an interdisciplinary field like pharmaceutical

sciences, one’s knowledge is always limited. Thanks to the colleagues in UNMC, who

are willing to help at every moment. They are Dr. Ying Xie, Michelle Varney, Linyun Wu,

Dr. Xiaobei Wang, Dr. Xin Wei, Zhifeng Zhao, Dr. Kumar Virender, Yang Peng, Hongjun

Wang, Dr. Di Wen, Lin Feng, Zhiyi Lin, Hang Su, Dr. Dongwei Guo, Zhihao Mao,

Nicholas Wojtynek, Bowen Qi, Dr. Beth Clymer, Yu Hang, Weiming Tang. Also, I would

like to acknowledge the assistance from UNMC core facilities, IACUC, comparative

medicine and financial support from NIH, UNMC Program of Excellence Graduate

Assistantship, and the China Scholar Council. The acknowledgment would be

incomplete without mentioning my friends on and off the campus, Dr. Chaojun Wang, Dr.

Xiaoyan Yang, Dr. Miaorong Yu, Chenjun Shi, Dr. Shengqi Hou, Dr. Jiaqiang Wang, Dr.

Nimin Wu, Yike Wang, Yuxiang Zhu, Chunyi Zhou, Qingfeng He, Jian Zhang, Yuning

Zhang and Junming Zhou.

In the end, I'm so grateful for my parents and their unconditioned love over the

past three decades. They are the backbones and ready to be supportive whenever it is

IV

needed. Their encouragement and willingness to share any burden make me brave on

the road chasing my dream. I feel extremely blessed to be their child.

Tong Liu

November 2018

V

DEVELOPMENT OF MICELLAR DRUG CARRIERS FOR THE

TREATMENT OF BREAST CANCER BONE METASTASIS

Tong Liu, Ph.D.

University of Nebraska Medical Center, 2018

Advisor: Tatiana K. Bronich, Ph.D.

Breast cancer (BC) remains one of the most frequently diagnosed cancer

worldwide. Bone is one of the most common sites and often the first clinical indication of

metastatic progression of BC. Current treatments including radiation, surgery, and

chemotherapy are rarely curative with limited effect in overall survival. The challenge is

true especially for patients with triple-negative BC that are not responsive to receptor-

targeted therapy, or patients who had exposure to chemotherapy before and might

already gain the resistance to some of the drugs.

Here we took advantage of the aminobisphosphonate, alendronate (ALN), that

can bind to the bone mineral efficiently and engineered ALN-decorated polymeric

micelles to target Docetaxel (DTX) to bone metastasis. DTX/ALN-m showed high affinity

to hydroxyapatite in vitro, exhibited similar cytotoxic activity as free drug and

demonstrated the potential to intervene the tumor microenvironment by inhibiting bone

resorption and macrophage recruitment. Systemic treatment with the DTX/ALN-m led to

VI

significant attenuation of bone metastatic tumor burden and improved survival time in the

immunocompetent mouse model of BC dissemination to the bone.

To further improve the efficacy of the metastasis treatment, we developed a

micellar formulation of DTX and Dasatinib (Das), an inhibitor of multiple tyrosine kinases.

Dual drug-loaded micelles inhibited the osteoclast differentiation, migration of cancer

cells, exhibited strong synergy in metastatic BC cell lines and retained the comparable

efficacy even in DTX-tolerant cells. Mechanistic studies revealed the connection of drug

tolerance to the activation of AMP-activated protein kinase and the role of both drugs in

mitigating adaptive resistance. We also demonstrated that this micellar drug

combination exerted enhanced antitumor activity delaying the progression of metastatic

disease. Overall, the micellar drug carriers provide an effective platform for the

treatment of breast cancer bone metastasis by targeting tumor and their

microenvironment.

VII

TABLE OF CONTENT

ACKNOWLEDGMENT ..................................................................................................... II

LIST OF FIGURES ........................................................................................................ IX

LIST OF TABLES ....................................................................................................... XIII

LIST OF ABBREVIATION ........................................................................................... XIV

LIST OF CONTRIBUTORS ....................................................................................... XXIII

CHAPTER I INTRODUCTION ........................................................................................ 1

1.1 CANCER AND METASTASES......................................................................................... 2

1.2 BONE METASTASES ...................................................................................................... 4

1.3 BONE-TUMOR MICROENVIRONMENT ......................................................................... 8

1.4 CURRENT TREATMENT OPTIONS IN THE CLINIC FOR BONE METASTASES ........ 14

1.5 CHEMO DRUG RESISTANCE....................................................................................... 20

1.6 NANOCARRIERS-BASED DRUG DELIVERY SYSTEM FOR CANCER THERAPY .... 29

1.7 CONCLUSION ............................................................................................................... 40

1.8 REFERENCES ............................................................................................................... 41

CHAPTER II TARGETED POLYMERIC MICELLES FOR THE TREATMENT OF

BREAST CANCER BONE METASTASIS ....................................................................55

2.1 INTRODUCTION ............................................................................................................ 56

2.2 MATERIALS AND METHODS........................................................................................ 59

VIII

2.3 RESULTS AND DISCUSSION ....................................................................................... 74

2.4 CONCLUSION ............................................................................................................. 111

2.5 REFERENCE ............................................................................................................... 112

CHAPTER III COMBINATION THERAPEUTIC PLATFORM FOR BREAST CANCER

BONE METASTASIS TREATMENT ........................................................................... 116

3.1 INTRODUCTION .......................................................................................................... 117

3.2 MATERIALS AND METHODS...................................................................................... 120

3.3 RESULT AND DISCUSSION ....................................................................................... 135

3.4 CONCLUSION ............................................................................................................. 197

CHAPTER IV SUMMARY AND FUTURE STUDY....................................................... 204

4.1 SUMMARY ................................................................................................................... 205

4.2 FUTURE STUDY .......................................................................................................... 208

4.3 REFERENCE ............................................................................................................... 210

IX

LIST OF FIGURES

Scheme 2.1: Synthesis of the bone-targeted copolymer. ...............................................76

Figure S2.1: 1H-NMR spectra (400 MHz, DMSO-d6) of synthesized block-copolymers.77

Figure S2.2: GPC traces................................................................................................79

Figure S2.3: The plot of fluorescence intensities ratio of I3/I1.. ......................................80

Figure 2.1: In vitro characterization of ALN-m and ALN-m/DTX micelles. ......................86

Figure S2.4: Binding of the ALN(37%)-m to the bone mineral HA in DI water and in the

presence of 3.4 mM CaCl2 in the solution. ....................................................................89

Figure S2.5: In vitro toxicity of free ALN in 4T1 cancer breast cells as determined by

MTT assay. ...................................................................................................................93

Figure 2.2: Effects of ALN-m on osteoclastogenesis. .....................................................96

Figure 2.3: ALN-m inhibits the resorption activity of osteoclasts. ...................................98

Figure 2.4: Inhibition of tumor-induced macrophage migration by ALN-m. ................... 100

Figure 2.5:. Anti-metastatic efficacy of ALN-m/DTX in syngeneic 4T1 breast cancer bone

metastasis model. ....................................................................................................... 105

Figure S2.6: BLI captures and individual mouse respose. ........................................... 106

Figure 2.6: Treatment efficiencies of sequential chemotherapy with ALN-m/DTX and

radiotherapy (RT). ....................................................................................................... 109

X

Figure S2.7: Quantification of bone metastasis burden in individual mice treated ........ 110

Figure 3.1: Representative image of the morphology change in DTX and Das treated

4T1 cells. ..................................................................................................................... 136

Figure 3.2: Dose-responsive profile of DTX to 20 nM 36 h pre-treated 4T1. ................ 138

Figure:3.3: Adapted response to DTX treatment. ......................................................... 140

Figure 3.4: Adaptive response to Das. ......................................................................... 141

Figure 3.5: Recovery of DTX adapted 4T1 cells. ......................................................... 142

Figure 3.6: The regimen of different drug treatments for the analysis of the corresponded

adaptive responses. .................................................................................................... 145

Figure 3.7: Adaptive response of p-AMPK level to the drug treatments. ...................... 146

Figure 3.8: Parent 4T1 and drug adapted 4T1 in 96-well scanned by Licor Odyssey. .. 148

Figure 3.9: Representative fluorescent images parent 4T1 and drug adapted 4T1. ..... 149

Figure 3.10: Quantification of p-AMPK level/total AMPK change in DTX and Das adapted

cells by In-Cell ELISA. ................................................................................................. 150

Figure 3.11: 1H-NMR (400 MHz, DMSO-d6) of synthesized block-copolymers. ............ 155

Figure 3.12: 1H-NMR (400 MHz, TFA-d1) of synthesized block-copolymers. ................ 156

Figure 3.13: In vitro release profile of DTX/m, Das/m and (DTX+Das)/m. .................... 163

Figure 3.14: Relative sensitivity to Das. ....................................................................... 167

XI

Figure 3.15: Growth inhibition of AICAR and Compound C. ......................................... 171

Figure 3.16: Cell viability after 24 h treatment of Das/m. .............................................. 176

Figure 3.17: 4T1 migration assay. ............................................................................... 177

Figure 3.18: 4T1 migrated through the transwell membrane. ....................................... 178

Figure 3.19: Migrated MDA-MB-231 using preOB-CM. ................................................ 179

Figure 3.20: Das inhibited osteoclast at nanomolar concentration. .............................. 182

Figure 3.21: Representative image of the differentiated osteoclast under different

treatment groups. ........................................................................................................ 183

Figure 3.22: Tumor growth in each mouse. ................................................................. 185

Figure 3.23: Average tumor growth of mice with different treatment. ........................... 186

Figure 3.24: Body weight loss along with tumor growth. .............................................. 188

Figure 3.25: Acute toxicity study based on H&E staining of the major organs collected 24

h after the final treatment. ............................................................................................ 189

Figure 3.26: Kaplan-Meier survival curves of tumor-bearing mice following the

treatments. .................................................................................................................. 191

Figure 3.27: Correlation between initial tumor burden (bioluminescence) and the survival

time within each treatment groups. .............................................................................. 193

Figure 3.28: Blood count 24 h and five days after the final treatment. .......................... 195

XII

Figure 3.29: Representative image of the blood smear from the tumor-bearing mice. . 196

XIII

LIST OF TABLES

Table S2.1: Polymer characteristics determined by 1H NMR and GPC ..........................78

Table 2.1: Physicochemical characteristics of DTX-loaded micelles ..............................84

Table 2.2: IC50 values of free DTX and DTX-loaded micelles in 4T1 breast cancer cell

line. ...............................................................................................................................92

Table S2.2: PK parameters of DTX in plasma in tumor-free nude mice. ...................... 104

Table 3.1: Polydispersity of synthesized polymers by gel permeation chromatography.

.................................................................................................................................... 157

Table 3.2: Yield of preparing drug-loaded micelles with DTX, Das and the combinations.

.................................................................................................................................... 159

Table 3.3: Size, PDI and distribution modal of the drug-loaded micelles. ..................... 161

Table 3.4: In vitro tumor growth inhibition of drug-loaded micelles. .............................. 166

Table 3.5: Growth inhibition in MDA-MB-231 and PC-3 IC50 (µM). ............................... 169

Table 3.6: Growth inhibition of the combination with AICAR and Compound C, IC50 µM.

.................................................................................................................................... 173

XIV

LIST OF ABBREVIATION

%ID Percentage of injected dose

1H-NMR Proton nuclear magnetic resonance

4T1/Luc Luciferase-expressing 4T1

ABC ATP-binding cassette

Abs Absorbance

ACC Acetyl-CoA carboxylase

ACN Acetonitrile

AICAR 5-Aminoimidazole-4-carboxamide

ribonucleotide

AKT v-Akt Murine thymoma viral oncogene

ALK Alkynyl

ALK-m Non-conjugated micelles

ALN Alendronate

ALN-m Alendronate-conjugated micelles

XV

ALK-PEG-NH2 α-Amino-ω-ALK polyethylene glycol

AMPK 5’ AMP-activated protein kinase

ANOVA Analysis of variance

ATCC American type culture collection

AUC Area under the curve

BCA Bicinchoninic acid assay

BCRP Breast cancer resistance protein

CI Combination index

CMC Critical micelle concentration

Das Dasatinib

Das/m Das-loaded micelles

Deff Hydrodynamic diameters

DLS Dynamic light scattering

DMEM Dulbecco's modified Eagle medium

DMF N, N-dimethyl formamide

DMSO-d6 Deuterated Methyl sulfoxide

XVI

DTX Docetaxel

ALK-m/DTX DTX-loaded non-conjugated micelles

ALN-m/DTX DTX-loaded alendronate-conjugated micelles

DTX/m DTX-loaded micelles

DTX/m+Das/m Drug loaded micellar cocktail

DTX+Das/m Combinational drug-loaded micelles

EC50 Half-maximal effective concentration

EDC 1-3-dimethylaminopropyl-3-ethylcarbodiimide

hydrochloride

EDTA Ethylenediaminetetraacetic acid

EGFR Epidermal growth factor receptor

EGTA Ethylene glycol-bisβ-aminoethyl ether-

N,N,N’,N’-tetraacetic acid

ELISA Enzyme-linked immunosorbent assay

Em Emission

EPR Enhanced permeability and retention

Ex Excitation

XVII

F-12K Ham's F-12K Kaighn's Medium

FBS Fetal bovine serum

GAS Growth arrest-specific

VCAM Cell adhesion molecule

Glu L-glutamic acid γ-benzyl ester

GPC Gel permeation chromatography

H&E Hematoxylin and eosin

HAP Hydroxyapatite

HCl Hydrochloride

HPLC High-performance liquid chromatography

HPMA N-2-hydroxypropylmethacrylamide

HSC Hemopoietic stem cells

i.p. Intraperitoneal

i.v. Intravenous

IACUC Animal care and use committee

IC50 Half-maximal inhibitory concentration

XVIII

IL Interleukin

IR Irradiation

IVIS In vivo imaging system

L.C. Loading capacity

M-CSF Macrophage colony-stimulating factor

MDR Multidrug resistance protein

MEM Minimum essential medium eagle

mPEG Methoxy polyethylene glycol5k

mPEG-PLE Methoxy polyethylene glycol5k-block-polyL-

glutamic acid

mPEG-PLE-PLF Methoxy polyethylene glycol5k-block-polyL-

glutamic acid-block-polyL-phenylalanine

MRM Multiple reaction monitoring

MRP Multidrug resistance-associated protein

MRT Main drug residual time

MTD maximum tolerant doses

MTT 3-4,5-Dimethylthiazol-2-yl-2,5-

XIX

diphenyltetrazolium bromide

MW Molecular weight

MWCO Molecular weight cut off

MWw Weight-averaged molecular weight

Na2SO4 Sodium sulfate anhydrous

Na3VO4 Sodium orthovanadate

NaF Sodium fluoride

NaOH Sodium hydroxide

NBF Neutral buffered formaldehyde

NCA N-carboxy anhydride

NCA Non-compartmental analysis

NHS N-hydroxysuccinimide

OPN Osteopontin

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate buffered saline

PDI Polydispersity index

XX

PE Plating efficient

PEG Polyethyleneglycol

PhA L-phenylalanine

PI Propidium iodide

PI3K Phosphoinositide 3-kinase

PLE PolyL-glutamic acid

PLF PolyL-phenylalanine

PMSF Phenylmethane sulfonyl fluoride

preOB-CM MC3T3-conditioned medium

QTRAP Triple quadrupole linear ion traps

RANK Receptor activator of NF Kappa B

RANKL Receptor activator of NF Kappa B ligand

Raptor Regulatory associated protein of mTOR

TFA-d1 Deuterated trifluoroacetic acid

RBC Red blood cells

RIPA Radioimmunoprecipitation assay

XXI

RPMI Roswell park memorial institute medium

r.t. Room temperature

Rt/R0 Post-treatment radiance over initial radiance

RTK Receptor tyrosine kinase

SD Standard deviation

SDS Sodium dodecyl sulfate

SF Surviving fraction

STAT Signal transducers and activators of

transcription

T1/2 Half-life time

TAM Tumor-associated macrophages

THPTA Tris3-hydroxypropyltriazolylmethylamine

TRAP Tartrate-resistant acid phosphatase

Tris Trishydroxymethylaminomethane

UV Ultraviolet–visible spectroscopy

v/v% Volume percentage

XXII

VD Volume distribution

WBC White blood cells

XXIII

LIST OF CONTRIBUTORS

1. Chapter II – Dr. Svetlana Romanova, Dr. YuXiang Dong, Dr. Xinming Liu assisted in the

polymer synthesis and conjugation. Dr. Rakesh guided the osteoclast differentiation

study and the establishment of the mouse model. Ms. Michelle Varney helped in TRAP

assay and cell maintenance. Mr. Hongjun Wang provided suggestions in the left-

ventricle injection. Dr. Larisa Poluektova supported the bone decalcification. Mr. Hang

Su helped in blood collection in the pharmacokinetic study. Dr. Samuel M Cohen

examined the tissue samples. Michelle Varney provides technique support for osteoclast

staining. Dr. Chi Zhang, Dr. Wang Shuo and Dr. Megan Hyun conduct IR treatment.

2. Chapter III – Dr. Svetlana Romanova performed the polymerization reaction and GPC

characterization. Dr. Beth K Clymer ran the western blot for AMPK/pAMPK analysis. Dr.

Michael A. Hollingsworth and Ms. Jenea Sweeter helped in-cell ELISA quantification.

Nicholas Wojtynek performed the fluorescent microscopic imaging. Dr. Xin Wei provided

the suggestion on bone tissue fixing and decalcification. Dr. Samuel M Cohen examined

the tissue samples and assisted in animal study design.

3. Tong Liu made major contributions in all chapters. The overall project was designed

under the guidance of Dr. Tatiana K. Bronich. Dr. Rakesh Singh provided valuable

direction to the projects.

XXIV

4. The work was financially supported by National Institutes of Health (U01 CA198910),

Nanomaterials Core Facility of the Center for Biomedical Research Excellence (CoBRE).

Award from the National Institute of General Medical Sciences of the National Institutes

of Health under grant number P20GM103480. The stipend support for Tong Liu was

provided by research assistantship from Nebraska Center for Nanomedicine (2013-

2014), Nanomaterials Core Facility of the Center of Biomedical Research Excellence

(CoBRE) (2014-2018), and China Scholar Council (2013-2017).

1

CHAPTER I

INTRODUCTION

2

1.1 CANCER AND METASTASES

1.1.1 Improved clinical care in cancer

Since its first documentation, cancer has a long history for more than three

thousand years[1]. Projected by the American cancer society, over 1.7M people are

newly diagnosed for cancer in the US, and 600 thousand patients will die in 2018.

Among all cases, breast cancer, lung cancer, and prostate cancer were forecasted to be

the most common ones[2]. Over the last few decades, the fundamental of the disease

was explored extensively with advanced molecular biological technologies. Researches

identified numerous pathways, genetic regulation, and mutations that associate with the

tumor cells and novel drugs were designed to target the discovered mechanism.

Diagnostic also evolved drastically, allowing the detection of the tumor cells at very early

stages[3] as well as personally profiles phenotypes that could be sensitive to specific

drug molecules[4]. Deep learning and artificial intelligence facilitated tools that emerge

since 2014 made the way into the clinics successfully, helping to build the most effective

treatment regimen for individual patients based on their bassline characterization[5].

With all the efforts above, people have achieved substantial improvement of the overall

therapeutic outcome, longer survival time, and better qualities of life.

3

1.1.2 The deadly challenge by cancer metastases

Even with so many achievements, cancer is still considered incurable. The

disease is found refractive even after complete remission and tumor-free for several

years. Once comes back, tumors are frequently found in distant organs other than the

primary location (metastases), for example in lung, liver, and bone[6]. Also, quite a few

populations were found to have the metastases at initial presentation due to the

relatively late diagnose. The forming of the bone metastases generally takes the steps of

disseminating from the primary site, gaining access to the systemic circulation, homing

to a distant organ and developing into metastatic lesions[7]. The metastases are usually

not dissectible by surgery as they disperse widely to multiple sites and diffuse deep into

the healthy tissue. If from recurrence, metastases are generally less sensitive to the

same drug, because the tumor that survives from the previous treatment has been

positively selected for the "right" gene or drug-resistant phenotypes. Tumor

microenvironment also supports the colonization of the metastases, making it extremely

challenging to combat[8].

4

1.2 BONE METASTASES

1.2.1 Preferential metastasis to bone

The selection of organs for circulating tumor cells to reside does not appear to be

random. Tumors with different origins have shown their preference. Among the cases,

bone tends to be one of the most popular places to settle, especially in patients with

breast and prostate cancer[9,10]. Both tumors will likely metastasize to the bone at the

late stage, and the bone undergoes pathological destruction and results in severe pain

and spinal paralysis if in the vertebrae. The reason why bone is attractive to the

disseminated tumor cells were not fully clarified. The widely acknowledged hypothesis is

the “seed and soil” theory[11]. Tumor cells were believed to seed itself to the bone due

to the favorable environment that has the stem cell niches to support the initial

colonization and host cells that can remodel the bone further by interacting with the

tumor cell. When looking into the details of how metastases were distributed in the

skeletons, it is interesting that a large portion of the lesion forms in the axial skeletons

which are consist of head bones and trunk of a vertebrate[12]. Both hematopoietic

condition and the way blood is drained into the skeletons are accredited for the frequent

incidence of metastases in the axial skeletons and the girdles as suggested by

Batson[13].

5

1.2.2 Prognosis of the bone metastases

The prognosis of the bone metastases varies, depending, on the origin of the

diseases as well as the existence of other metastases. For breast cancer bone

metastases at the first relapse, the median survival time from the diagnose is close to

two years[14]. It could be more than four years, in the bone metastases from prostate

cancer with only axial skeleton affected. However, lung cancer metastasized to bone has

a much shorter survival period that is counted by months[10]. Other metastases taking

places at the same time (e.g., liver, lung) can drastically deteriorate the prognosis and

shorten the survival time from years to only a few months. Besides, patients with

exclusive metastases in bones at the initial relapse might also be followed with the

secondary lesions in visceral organs, which could also substantially differentiate the

prognosis from those who do not. In a study of 17,251 patients with bone metastases

from multiple types of cancers, Elisabeth found the prognosis depends on both the

synchronous metastases as well as the types of cancer. However, it is interesting that

the report was suspected to have selection bias due to misclassification of the patients

with bone metastases of mild symptoms or lesser extent than other metastases to be

recorded. Hence, the modality risk could be potentially higher than estimated[15].

6

1.2.3 Major Clinical symptoms

Bone fracture is the symptom commonly occurs in bone metastases, especially

for breast cancer bone metastases. More than half of the patients with pathological

fracture were the one with breast cancer[16]. The symptom commonly happens in the

long bone, femur, rib and vertebral and lead to disability (broken long bone) or lung

diseases (vertebral collapse)[17,18]. Tumor-induced osteolytic lesion by the excessive

osteoclast resorptive activity contributes most to fractures. The process is associated

with unlocalized pain that does not relieve during sleep or by laying down and

significantly affect the quality of life[19]. Part of the pain could also be attributed to the

movement of the fractured bones. Pain in return is considered as an indication of

developing bone metastases[18].

Hypercalcemia is another frequently observed symptom. The increase calcium

level in serum is contributed mostly by the enhanced calcium reabsorption. The PTHrP

secreted from tumor cells can promote the process in Henle ascending limb and distal

convoluted tubule[20]. Pathological bone resorption by osteoclast also releases

additional soluble calcium to the serum. The factors like PTHrP, IL-6, IL-11, and VEGF

produced by tumor cells are known to affect the osteoblast cell and lead to elevated

expression of the RANKL, which is the differentiating stimuli for osteoclast maturation.

Some cell lines were also reported to produce the RANKL directly or secrete proteases

that cleave the anchored RNAKL into the soluble form[21]. Hypercalcemia leads to pains,

cognition difficulties, vomiting, etc., and requires immediate control. Hypercalcemia can

7

be severe and a sign of the disease progression. It generally comes with the poorer

prognosis, and about half of the patients with hypercalcemia died within one month[18].

The spinal cord compression is also frequently diagnosed in the clinic that

reduces the survival time substantially[22]. The affection of the spinal cord can result in

severe pain, vertebral collapse motility issues, bladder, bowels dysfunction. About one

out of five patients with bone metastases will end up with the spinal cord compression,

and it is common in breast, prostate and lung cancer[23]. Multiple evolvements at the

different location are frequently reported: thoracic spine accounts for 60-80% of all the

cases whereas the cervical and lumbar share the rest[24].

8

1.3 BONE-TUMOR MICROENVIRONMENT

The microenvironment of bone is a complicated and well-balanced biological

condition maintained by the extracellular matrix, hematopoietic cells, mesenchymal stem

cells bone marrow stromal cells and infiltrated immune cells. In 1889, Stephen Paget

highlighted the importance of the bone microenvironment and believed it to be the

proper "soil" that is used by cancer "seed." The follow-up studies have revealed more

details about how cancer cell turn the bone to its favor.

9

1.3.1 Hemopoietic stem cells (HSC) niche

The hemopoietic stem cells (HSC) niche was suggested to be the first

component in the bone that attracts circulating tumor cells to home, establish the initial

colony and protect it from chemotherapy. In the physiological condition, the CXCR4 is

produced by osteoblast and bone marrow stromal cells to direct hemopoietic cells to the

niches. The receptor of CXCR4, CXCR12 was also found on tumor cells. Studies in cells

and animals have validated that the CXCR12 is overexpressed in the highly metastatic

lesion and CXCR4/CXCR12 is one of the critical factors that regulate the tumor

migration and help the tumor cells to penetrate the basement membrane[25]. Once

arrived at the bone, the adhesive receptor Anxa2-R expressed on tumor cells can be

used for their retention and survival in the bone marrow, just as HSCs. Other ligands,

growth arrest-specific 6 (GAS-6), vascular cell adhesion molecule 1 (VCAM-1) and

matrix protein osteopontin (OPN) were also reported to be associated with the first stage

invasion. Just as mentioned above, tumor cells share a lot in similar to the HSCs, who

are regulated to go to bone niches via the physiological mechanisms[26]. Another way of

how tumor cells “hijack” the biological system is to compete with HSCs to occupy the

niches. It is reported that HSCs alter their expression of adhesive ligand Notch-1 and

Tie-2 and exit the niches, vacating the space for tumor cells to reside[27]. As a result of

such invasion, more unmatured hemopoietic cells were found in the circulation in a

metastatic prostate cancer model in bone.

10

1.3.2 Bone remodeling and the "vicious cycle."

Bone is a living organ and constantly under remodeling. In healthy adults,

osteoclast and osteoblast derived from monocyte linage work in conjunction to remove

the “outdated” mineral composition and replace it with the fresh one immediately. The

balance of both resorption and formation is critical for maintaining the bone mass and its

normal function[28,29]. One of the widely acknowledged mechanisms that bone

remodeling process relies on is the receptor activator of NF Kappa B (RANK) ligand

(RANKL) system, which is further regulated by osteoprotegerin (OPG). In vitro and in

vivo studies confirmed that RANKL is essential for differentiating macrophage into

matured osteoclast and maintain its resorptive function. While OPG serves as a

neutralizer and prevents the binding between RANK and RANKL, the relative

OPG/RANKL is suggested to control the bone remodeling process.

Tumor cells are found to secrete various cytokines that directly or indirectly affect

the RANKL/OPG level. These include parathyroid hormone, interleukins, and tumor

necrosis factors and can promote the production of RANKL in osteoblast while

suppressing the OPG, moving the balance toward bone resorption[30]. Research

observed higher expression level of PTHrP in metastases in bone than other visceral

organs. The finding fits into the tumor-induced bone resorption theory and hints the

association was not solely intrinsic but evolved along the time[31]. Other factors from

tumor cells, like colony stimulating factor (M-CSF), can also facilitate the osteoclast

formation[30].

11

The increased level of osteoclast and bone degradation will create the physical

space for the expansion of metastatic colony and more importantly release the

"nutrients" that was embedded inside the matrix when the bone was modeled. Bone is

known to be rich in growth factors like TGF-beta, IGFs, FGFs, PDGF and BMPs[32].

They are believed to influence the tumor growth from the following aspects: directly

promoting tumor cells survival and proliferation, stimulating the secretion of osteolytic

mediates by tumor cells, and even changing the tumor cells’ phenotype. In animal

studies, it has been uncovered that the Smad mediated pathway was involved in the

progression of the bone metastases. Mice inoculated with tumor cells that have Smad4

knockdown or express inhibitory Smad7 showed decreased bone metastases[33]. TGF-

beta also induces the production of osteolytic factors, e.g., PTHrP. Previous research

uncovered that high-level of expression in breast cancer bone metastases is not intrinsic

but rather the result of the stimulation by TGF-beta released from the matrix, or in other

words, a phenotypical change happened due to the interaction between tumor and

bone[34]. The PTHrP subsequently promote the expression of the RANKL in bone host

cells and consequently lead to enhanced osteoclast resorption. More interestingly, it is

not the only factor that represents the alteration of the tumor phenotype to utilize the

bone microenvironment. The interleukin-8 (IL-8) releasing level was found to be greater

in cells derived from metastatic lesion than in the primary tumor and suggested the

correlation between the metastatic potential and IL-8[35].

Tumor-associated macrophages (TAM) has been intensely investigated for

shaping the tumor microenvironment. The macrophage is generally classified into two

12

main phenotypes, M1 and M2: M1 is the phenotype driven by IFNγ or bacterial products

and is considered as "cytotoxic" phenotype that suppresses tumor growth; M2 is the one

induced by IL-4 or IL-13 and is believed to serve as a support role for tumor

progression[36]. The tumor-associated macrophages are thought to originate from

chemotactic recruitment from both peripheral blood circulation (monocyte or monocyte-

related cells) and residential macrophages[37,38]. It has been discovered that various

factors could act as the chemoattractant for the recruitment, including CSF‑ 1, VEGF,

IL‑ 34, CCL2, CCL5, C5a. Interestingly, these molecules were found not only produced

by tumor cells, but also by the TAM that already infiltrated inside the tumor tissue[39],

suggesting that tumor-TAM works as a whole entity in maintaining the stable population

of TAM in the tumor lesion as the tumor grows[40]. M2-like subtype has attracted

substantial attention and been studied as it promoted some of the key processes in

tumor development, including angiogenesis, immunosuppression, metastasis and

genetic instability[41]. When it comes to the effect on chemo drug treatment, mixed

results were reported with both improved efficacy and the rise of resistance[36], which

correlated with the researches that suggested the relationship of M1/M2 ratio to the

disease prognosis[42]. Evidence also indicated that the TAM is not the same as

standard format as seen in non-neoplastic tissue but has rather board heterogenicity (a

subpopulation of skewed M2), shaped by the metabolic re-programming in different

tumors[43,44]. Most of the studies in TAM focused on the primary tumors and soft organ

metastases while few have been explored in cancer bone metastases. One important

role that TAM involves in the metastatic cancer development is to follow the “scene” set

by the tumor cells and provide the trail that leads the circulating tumor cell to settle down

in the bone[45].

13

14

1.4 CURRENT TREATMENT OPTIONS IN THE CLINIC FOR BONE METASTASES

The overall paradigm in skeleton metastases treatment aims to target three

aspects of the disease: kill tumor cells (or suppress their proliferation), prevent bone loss

and symptom control. The tumor development is the cause and key of the complication,

thus controlling the tumor growth and even eliminate them is the essential and most

demanding need. The purpose could be achieved to some extent by the treatment of

antiproliferative chemo drug, targeted kinase inhibitors and androgen deprivation agents.

As discussed in the previous section, the tumor does progress alone, but also "hijack"

host cells into helpers that remodel the microenvironment in favor of tumor growth. Anti-

resorptive drugs are used in conjunction with the chemotherapy to interfere one of the

most crucial pathological process, the bone destruction, and showed beneficial in

prolonging patient survival time. Pain is one of the symptoms that are frequently seen in

cancer, especially in bone metastases where pain could be severe. Palliative therapies

are adopted to relief the symptom and improve patients’ quality of life.

15

1.4.1 Treatment directly against tumor growth:

Taxane (e.g., Docetaxel) and anthracyclines (e.g., Doxorubicin) remains the first-

line therapy in the chemotherapy system for patients with bone metastases. These small

cytotoxic molecules were used as a single agent or in combination or sequentially. For

breast cancer metastases, the median time to progression after chemotherapy ranges

from 3 months to a year and the median survival time ranges from 8 months to two

years[46]. Patients who had prior exposure to anthracycline tended to respond in a lower

rate to taxane, suggesting the emergence a possible multidrug resistance. Both classes

of drugs were limited by their toxicities: bone marrow suppression in taxane-based

treatment[47] and cardiotoxicity in anthracycline[48]. In metastatic breast cancer,

another commonly used therapy is androgen deprivation (or suppression), as more than

seventy percent of the patients are hormone receptor-positive[49]. This type of drugs

showed the generally good response the first time however the disease relapsed after

several cycles of the treatments. Hormonotherapy is also used in combination of with

chemotherapy. In a study of prostate bone metastases, 17-month improvement in

median survival time was observed when adding the docetaxel to the androgen

deprivation therapy[50]. For the subpopulation with HER2 positive tumors, a monoclonal

antibody that targets HER2 can be used in combination with chemotherapy. In a study of

186 patients with metastatic breast cancer, the median survival time increased from 6

months (Docetaxel alone) to 31months (Docetaxel and Trastuzumab)[51]. By 2018, over

30 receptor tyrosine kinase (RTK) inhibitors have been approved in the clinic for various

cancers[52]. This class of drugs is considered as the targeted therapy to intervene the

many aberrantly regulated signal transduction[53] and has proved the efficacy not only

16

as a single agent but also as the adjuvant to overcome the drug resistance[54]. Several

RTKs were also tested in patients with bone metastases[55]. For instance, sunitinib was

shown to prolong the survival in patients with bone metastases from cancer of unknown

primary[56].

17

1.4.2 Treatments for bone resorption:

Bisphosphonates were mainly used to treat osteoporosis and to prevent the bone

loss[57]. The chemical structure of bisphosphonate is compromised by two phosphorus

groups that covalently bound to the one carbon atom. This conformation grants it high

binding affinity to the hydroxyapatite which is the mineral compartment of the bone[58].

Systemically administered bisphosphonate will "shoot" to the bone, and upon bone

resorption, the drug will be uptake by osteoclast cells. The accumulation of

bisphosphonate in osteoclast cell will inhibit farnesyl pyrophosphate synthase and finally

cause the osteoclast cell detaching from the resorption surface. Depending on the

concentration and the efficacy of specific bisphosphonate, the osteoclast could also

become apoptotic after drug exposure[59]. The ability of bisphosphonates to target bone

and to limit the resorptive activity of osteoclast makes them good candidates to intervene

the bone remodeling in metastatic lesion. Indeed, zoledronate was found to delay the

occurrence of skeleton-related symptom in patients with prostate cancer bone

metastases. However the drug did not prolong the overall survival time[60], nor did they

decrease the risk of developing metastases when used as a prevention medicine[61].

Apart from directly acting on osteoclast cellular function, another approach is to

control the differentiation and activation of the osteoclast. As discussed in the previous

section RANKL is the key to regulate osteoclast-mediated bone resorption. Denosumab

is a human monoclonal IgG2 antibody that binds to RANKL with high specificity and

further prevents the contact between RANKL and RANK, cutting off the activation

18

signal[62]. A clinical study showed Denosumab had an even better response and could

delay the skeleton related events even more than what zoledronate did[63].

19

1.4.3 Treatment for pain control:

Non-invasive beam radiotherapy and radiopharmaceuticals are two major

approaches used in the clinic for palliating skeletal metastases. External beam

radiotherapy has been established to relieve the bone pain within 4–6 weeks. American

Society of Radiation Oncology suggests single fraction radiotherapy using a dose of 8Gy

to provide the palliation[64] and the repeated dosing is possible if pain recurs[65].

Radiopharmaceuticals like strontium-89, samarium-153, and radium-223 have been

approved by FDA as bone-targeted agents for pain control. These emitters serve as

substitutes, which will be uptake by osteoblast cells when they construct new bones in

prostate cancer (osteoblastic metastases)[66]. The radiopharmaceuticals were also

found to work well in combination of chemotherapy (taxane), and the toxicity was well

tolerated[67]. However, radiotherapy in general (except one study[68]) did not improve

the overall survival time in bone metastases[69].

20

1.5 CHEMO DRUG RESISTANCE

Drug resistance was a concept initially in anti-bacterial treatment, where the

bacteria become tolerant to antibiotics. The similar phenomenon is observed in cancer

treatment. Despite the various therapeutic agents based on the different mechanisms of

action, the tumor cells could always develop resistance during the therapy or after

relapsing. Due to the toxicity of anti-cancer drugs, the dose is limited, and thus the

development of resistance significantly hinders the therapeutic effect. With the advanced

analytical tools in genomic and proteomic researches, several mechanisms of the drug

resistance have been discovered, and strategies were explored to overcome the barriers.

Studies revealed that different tumors with characterization are sensitive/resistant to

different treatments. The concept of precision medicine was also proposed in this

context, that the optimal drug candidates could be identified based on the unique

biological fingerprint of different tumors. However, the question that how the cells

establish or acquire such resistance remains controversial and the approach to avoid

inducing the resistance is still unknown.

21

1.5.1 Mechanism of the drug resistance

A variety of molecular mechanisms have been revealed in drug resistance.

These could be summarized and classified into the following aspects: the accumulation

of the drug inside cancer cells, upstream action of the drugs, and downstream cellular

response.

The drug efflux is considered the main barrier in drug uptake and one of the well-

studies mechanisms of drug resistance. Tumor cells actively pump out the cytotoxic drug

to keep the intracellular concentration at a lower level and survive through the regular

dose. The ATP-binding cassette (ABC) transporter family proteins play a significant role

in this kind of resistance. ABCs are essential to maintaining the physiology condition.

Epithelia cells in liver and intestine express the transporters at a high level to avoid over

accumulation of toxins. In tumor cells, three transporters including multidrug resistance

protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer

resistance protein (BCRP) are accredited for resistance driven by drug efflux. These

transporters share a board range of substitutes specificity that overlaps with the

commonly used chemo drugs (taxane, anthracyclines and kinase inhibitors). Many of the

tumor (liver, kidney, and colon) were found to have higher expression of the transporter

proteins compared to the corresponding healthy tissue, whereas some of the tumors

were able to increase the expression upon chemo drug exposure. In the clinic, the

overexpression of MRP1 has been positively associated with chemoresistance and poor

outcome, in prostate, lung and breast cancer and the level of BCRP were used as a

predictive marker for potential drug response and survival rates in patients with small cell

22

lung cancer. The inherited resistance found in tumor stem cells was also suggested to

correlate with the expression of MDR and BCRP.

Another important upstream drug resistance is due to the drug inactivation.

Platinum-based drugs can be inactivated by thiol glutathione[70,71]. In other cases, the

prodrug cannot be metabolized into the active form, for example, capecitabine cannot be

converted into 5-FU by thymidine phosphorylase28 when the gene encoding thymidine

phosphorylase is inactivated by methylation[72]. Besides the alteration of

pharmacokinetics and pharmacodynamics, the drug’s target can also change to reduce

the efficacy. The change can be reflected as the different expression level or

composition alteration of targeted proteins. When using androgen deprivation agent, the

expression of androgen receptor was found to increase in the patients who showed

resistance to the therapy[73]. The increased quantities of the pharmacological targets

require more active inhibitors to show the treatment effect.

Even when the tumor cells are under stress by sufficient functional drugs that

successfully act on the target, the downstream survival response can still save the tumor

cells from dying. One typical mechanism is the DNA repairing, also known as DNA

damage response. The process is intrinsic in the normal cells and contributes to the

genome stability. The specific signal transduction pathways vary in the different type of

damage as reviewed by Alberto[74]. In the tumor, the nucleotide excision or mismatch

repair were recognized as the dominant mechanism that involved in resistance to

platinum-based DNA damaging agents[75]. However, tumors are frequently to have

23

mutations that cause the deficiency in one of the repairing pathways, and the actual

resistance relies on the redundant repairing pathways[76].

Apart from the repairing mechanism, the inhibition of apoptosis at the same time

plays an important role to allow tumor time to overcome the treatment effect. The

inhibition in cell death does not exclusively happen in tumors with DNA damage but also

in those treated with targeted inhibitors as well. Studies discovered that although

different chemo drugs may have distinguished mechanism of action, the intrinsic

apoptotic process is disrupted by the overexpression of BCL-2[77]. Extensive

researches have been done and identify a series of factors that dysregulate the

apoptotic function, including the BCL-2 family protein, p53, and inhibitors of apoptosis

proteins[78].

Related to the apoptosis inhibition, autophagy acts as a double-edged sword in

cancer resistance. Autophagy is considered a recycling process to increase the

threshold of the metabolic stress required for cellular apoptosis by eliminating the

malfunction organelles and pro-apoptotic signal transductors. Both apoptotic and

autophagic pathways share many signaling mediates in common, like p53 and BH3[79].

Evidence has shown that the pro-apoptotic factors can activate the autophagy, which is

a pro-survival activity[80]; while autophagy can end up with the completion of apoptotic

signaling transduction, which is the initiation of the cell death[81]. Thus, both inhibitors

and activators of autophagy have been proposed to overcome cancer resistance[82].

24

Epidermal growth factor receptor (EGFR) is another pro-survival factor that has

been intensely investigated. Activation of EGFR will subsequently trigger a series of

downstream signaling cascades, including the KRAS-BRAF-MEK-ERK pathway,

phosphoinositide 3-kinase (PI3K), phospholipase C gamma protein pathway, the anti-

apoptotic AKT kinase pathway, and the STAT signaling pathway, which tunes the

cellular activities like proliferation and survival[83]. Overactivation of EGFR was

commonly observed in many types of cancer and was considered as part of the

chemoresistance[84]. Small molecule inhibitors and monoclonal antibodies were

successfully developed and available for lung and colorectal cancer but yet failed to

prove the efficacy in patients with breast cancer[85].

25

1.5.2 The ways to acquire drug resistance

It has been widely acknowledged that the drug resistance can be driven by the

gene mutation. For example, cancer cells that resist to DNA damage were found to have

the mutation in p53[86], MLH1 and MSH2[87]; those resist to EGFR targeted inhibitors

showed gatekeeper residue mutations in EGFR[88] or mutations in the downstream

kinase, like ALK tyrosine kinase[89]. Indeed, the emerging of drug resistance has been

described as an evolution followed by Darwin's law. The genomic instability is one of the

hallmarks of cancer and contributes to a board spectrum of the tumor heterogeneity[90].

Chemotherapy holds the pressure of natural selection and allows the enrichment of the

subclonal populations that happen to have the resistant mutation[91] while increasing

the genomic instability to maintain the intratumoral heterogeneity[92]. A recent model

also supports the theory of evolution, where tumor cells were observed to gain the

amplification of an oncogene under the continuous treatment of ERK inhibitors[93].

However, the other study that focuses on the same oncogene but followed a different

route. The researchers observed profound variability in cellular dynamics with a small

population that had transient tolerance to the treatment, which ultimately converted to

the inheritable resistance via epigenetic reprogramming[94], suggesting the non-genetic

part of drug resistance. Another study about the multidrug resistance in HL60 leukemic

carried out the experiments to eliminate the selective effect and explained the

appearance of the resistance by Lamarckian induction, where tumor cells responded to

the stress with transcriptome changes and switched between resistant and non-resistant

phenotypes[95]. Similar phenotypical was found in breast cancer cells, and the

26

stochastic gene expression of CD24/CD44 was modeled to explain the chemotherapy-

induced resistance[96].

The nongenetic transition can be further interpreted under the concept of cell

attractors. Cells with the same genome can have multiple distinct phenotypes that are

considered to be stable, dictated by the "attractors." The relative stability of those cells

defines the popularity of each phenotype within the attractors, commonly creating a bell-

shaped cell distribution based on the gene expression level. Those who fall on the edge

of an attractor would have distinctive gene expression to the rest population. Cell

attractor can be redefined upon the exposure to chemo drugs, in other words, the

stability of the initial cell states can also change due to the treatment. If the state on the

edge of the attractor become more stable in the new condition, the subpopulation of

those cells will increase accordingly while the rest will decrease. Such phenotypical

transition shares the similar macrophenomenon as the natural selection but is based on

different mechanism. Qin analyzed the dynamics in a single cell resolution and proved

the transition continually happened within and between the attractors. Interestingly, the

study also found the cells tend to restore the original distribution after the loss a

subpopulation, suggesting that targeted elimination of drug-resistant or stem phenotypes

may not be a practical approach to overcome the resistance[97].

27

1.5.3 Overcome drug resistance

Many efforts have been made to overcome the drug resistance by developing

specific molecules that target the critical element on which the resistance relies. The

results were a mix of encouraging and disappointing.

Inhibiting the function of the overexpressed transporters was believed to enhance

the efficacy of cytotoxic molecules. Researches identified a few kinase inhibitors (e.g.,

Gefitinib) that can block the drug efflux and specific inhibitors with high affinity were also

developed into clinical stages (zosuquidar and tariquidar). However, the results of the

trial were negative, showing no improvement in overall survival or progression-free time.

The reason was suggested as "the high degree of functional redundancy” in transporter

family. Given the fact that chemo drug induces high expression of MDR, it could also be

speculated that the inhibition leads to "feedback response" in cells and results in more

production of the transporter to compensate the inhibitory effect.

For resistance to DNA damaging drugs, inhibition of the DNA repairing is a clear

approach. As the tumor cells commonly lack in one or more repairing pathway and rely

on the machinery that is redundant in normal cells, the synthetic lethality was

proposed[98]. The inhibitor of DNA damage response showed efficacy initially however

extra mutant was found to compensate the inhibition[99].

28

EFGR inhibitors are designed to cut off the pro-survival signaling and sensitize

the tumor cells to chemotherapy. However, the generation of secondary resistance is

almost inevitable. In an example of resistance against tyrosine kinase inhibitor, the

gatekeeper residue of BCR–ABL1 was mutated causing the inhibitor to lose the binding

affinity while maintaining the ontogenetical function[100]. Besides the alteration of drug

target, adaptive signaling bypass was discovered in the tumor that escaped from HER-

family tyrosine kinase inhibitors[101].

Such non-genetic adaptation is even more challenging to combat, especially

considering the complexity of the gene regulation network and the possible redundancy

in metabolic machinery. In the treatment against bacteria, people have found that the

resistance to one antibiotic may confer the susceptibility to another drug[102]. A similar

concept was proposed in cancer treatment that the resistance could be useful and

create the opportunity for “collateral sensitivity” as suggested by Mathew[103]. A

sequenced dosing regime was reported to overcome the adaptive resistance by inducing

the cell into the drug-tolerant state which is vulnerable to the secondary treatment[96].

Given the fact that the adaptive response could be universal and diverse to the stress

posed by the treatment, it is difficult to predict or avoid the actual resistance. Drug

combinations that affect different pathways at the same time is an option, as it could

diminish the pathway cross-talk[104] and decrease the possibility that tumor cells find an

effective way to transit into adaptation states.

29

1.6 NANOCARRIERS-BASED DRUG DELIVERY SYSTEM FOR CANCER THERAPY

Nanocarriers have been a versatile drug delivery platform for cancer therapy.

Various types of nanocarriers have been developed to encapsulate small molecules[105],

peptides[106], nucleic acids[107] and the combination of them. The chemical

composition and physical properties can be tuned to serve specific purposes, e.g.,

sustained release[108], stimuli-responsive behavior[109], tumor targeting[110],

prolonged circulation[111] etc. Systemically administration of the nanomedicine has

shown improved therapeutic outcome in the animal models as well as in the clinic[46].

30

1.6.1 Common types of nanocarriers and their general characteristics

Liposome:

The liposome is a sphere vesicle that consists of at least one lipid bilayer on as

an out shell and an aqueous phase in the middle. The lipid bilayer is usually

compromised by cholesterol and phospholipid. Depend on the number of the bilayers,

the liposome can be classified into multilamellar vesicles (multiple layers) and

unilamellar (single layers) vesicles. Two methods are frequently used to prepare drug-

loaded liposome, lipid film rehydration and reverse phase evaporation methods[112].

The former method produces small sized particles and the latter provide high

aqueous/lipid ratio and loading capacity. Due to the presence of aqueous phase and the

lipid bilayer, the liposome can encapsulate both hydrophilic and hydrophobic drugs

together and achieve better anticancer efficacy[113]. Even the carrier has the membrane

structure, it is still found to be treated as the foreigner when administered systematically

and is cleared by the mononuclear phagocyte system. PEGylation was reported to

effectively shield the liposome from being uptake and prolong the circulation time[114].

Liposome formulated chemo drugs have been successfully approved, and more clinical

candidates are under investigation[115].

31

Polymer-drug conjugate:

Polymer-drug conjugate is a macromolecule in which active pharmaceutical

ingredient is covalently attached to a water-soluble polymer via a labile linked in between.

The linker is supposed to undergo hydrolysis or be chopped off under the specific

condition, and the payload will then be released as active form. Various linker has been

designed to grant the conjugate with environmental responsive behavior to

pH(hydrazone), redox-condition(disulfide) and enzyme(cathepsin-sensitive

dipeptide)[116-118]. These linkers are chemically stable during the circulation and can

release the drug when being internalized into the tumor cells. N-(2-

hydroxypropyl)methacrylamide (HPMA) copolymers are one of the most widely used

polymers in drug conjugates. The hydroxy group in the side chain is ready to be modified

into suitable functional groups that can further attach the linkers and the drugs. Due to

the numerous side chain, it is accessible to conjugate drug combinations and to control

the ratio of individual drug in the same HPMA copolymer[119]. The dendrimer is another

popular format of the drug conjugates, a branched start-like polymer. Compared to

single strand copolymer (like HPMA), dendrimer had better-defined structure and well-

controlled molecular weight. Unlike HMPA-based conjugates, the dendrimer has the

drugs only attached to one end of the polymer, providing the ability to form secondary

amphiphilic micellar structures[120], but on the other reduce the loading capacity. Both

types of conjugates convert the small drug molecules into macromolecules which

prolongs the circulation time by avoiding renal filtration and limits the drug exposure to

healthy tissues[121]. Several polymer-drug conjugates have demonstrated the improved

antitumoral efficacy in animal models with different types of cancer. However no HPMA

or dendrimer based candidate so far had successfully clear the way to clinical

practice[122-124].

32

33

Antibody-drug conjugates:

The monoclonal antibody has a strong binding affinity and selectivity to the target

protein and is used to guide the small molecule drugs (warhead) to the tumor cells. Due

to the high specificity, the biodistribution of the warhead can be drastically improved,

which makes the drug candidate that is generally limited by off-target toxicity feasible

and well-tolerated in practice[125]. Each antibody typically comes with 2-3 warheads.

Similar to polymer-drug conjugates, the linker between antibody-drug conjugates and the

warhead is crucial in the successful design. The conjugation chemistry has evolved for

several generations. The attaching position has improved from random lysine residues

to the engineered cysteine group in the heavy chain, allowing better control of the

chemistry and quality. The labile linkers also changed from hydrazone-based to

protease-cleavable[126,127]. Thanks to the advanced understanding the fundamental

biology, the biparatopic antibody was adopted to enhance the receptor clustering and

facilitate the internalization process[128]. Antibody-drug conjugates have achieved great

success since their launch in the clinic[126] and many more candidates are entering the

clinical trials[129]. However, challenges remain in penetrating biological barriers and the

risk to cause fatal toxicity[130].

34

Polymeric micelles:

Micelles are self-assembling of the amphiphilic molecules (e.g., detergents) in

colloidal system. Similarly, polymers with both hydrophilic and hydrophobic residues can

form the micellar structure in the aqueous media. The polymeric micelles feature with of

high molecular weight, improved stability, and the potential to solubilize substantial

quantities of drugs. Most of the polymeric micelles reported were based on the block

copolymer and grafted polymers that comprised of a PEG shell and a hydrophobic core.

The length and the chemical composition of the polymers can be engineered to optimize

the micelles’ physicochemical properties, including size, surface charge, critic micellar

concentration, stability, drug loading capacity, stealth properties, targeting ability and

stimuli-responsive dissociation. The introduction of chemical functional groups (like

carboxy and primary amine) provided additional flexibility in loading drug with distinct

properties (hydrophilic drugs, nucleic acids drugs)[131] and in attaching tracers for

imaging purposes[132]. In particular, Swapnil used the carboxy group to crosslink the

micelles and further enhance the stability[131].

Moreover, the crosslinker can be replaced by labile ones to achieve stimuli-

responsive characteristic[133]. Drug-loaded polymeric micelles can be prepared by

several methods, film rehydration, dialysis, and nanoprecipitation[134]. A series of

studies have demonstrated the potential of polymeric micelles to improve the overall

survival in a mouse model of breast cancer, ovarian cancer, pancreatic cancer, and lung

cancer[131,135-137].

35

1.6.2 Passive tumor accumulation and active tumor targeting

One of the most important goals of formulating drugs into nanocarriers is to

improve the biodistribution and achieve accumulation selectively in tumor tissues.

Chemo drugs are generally toxic to the healthy tissue and may cause a severe adverse

effect, which limits the total dose in the treatment. As the on-site drug concentration is

directly related with the treatment efficacy, ideally the targeted delivery to the tumor can

increase the dose limit or enhance the effectiveness with the same dose, while keeping

the side effects at a minimum level.

Three main approaches were used to grant the nanocarriers with the tumor

targeting ability, either passively or actively. Tumor cells grow rapidly and commonly

have newly formed blood vessels that are formed by defective endothelial cells with

higher permeability than normal ones, while the lymphatic drainage is impaired. It is

believed that the leakage-retention would facilitate the diffusion of the small particles to

the tumor tissue during the circulation over the normal organs and lead to the

preferential drug accumulation(EPR effect)[138]. Since the born of the concept in 1986,

numerous studies have shown the evidence that supported the hypothesis and explored

the characteristics that determined whether the nanocarrier could take advantage of

EPR effect or not[139,140]. Size appeared to be one the most important factors and

particles with 50-100 nm diameter are generally considered to be effective[141-143].

Much less has been understood in the metastatic cancers that whether the EPR effect

36

exists or not. Two studies suggested the EPR effect may still be valid in lung and bone

metastases[144,145].

Besides the passive targeting ability based on EPR effect, decorating

nanocarriers with the binding ligands or antibodies has been extensively studied to

provide more specific targeting efficacy. Tumor cells were found to overexpress various

cell markers (e.g., CD19 CD44), EGFR, HER-2, folic receptor and ανβ3-Integrins,

providing the useful anchors for the targeting moieties. The nanocarriers, on the other

hand, have versatile reaction residues which can be chemically conjugated with the

targeting ligands, typically through click reaction, amide formation, ester formation, thiol-

maleimide reaction, etc [146,147]. The potential of different ligands or antibodies has

been explored, among which hyaluronic acid, folic acid, RGD peptides, monoclonal

antibodies to EGFR and HER-2 are those commonly used and the nanocarriers with the

targeting moiety exhibit enrichment in tumor and decreased accumulation in the liver or

other visceral organs[148-153].

For bone metastases, the current options are the targeting ligands that bind to

the mineral compartment of the bone or the antibody for the RANK ligand[154].

Bisphosphonate is one of the most commonly used ligands in the bone-targeted delivery

system, e.g., zoledronate and alendronate. It showed high binding affinity to

hydroxyapatite (HAP) on both bone formation and bone resorptive surface. Acidic

oligopeptides like Asp8 also exhibit strong binding to the HAP preferentially on the bone

37

resorption surface, on the contrary (AspSerSer)6 demonstrate selective accumulation on

the bone formation surface[155]. Beside of focusing on the HAP binding, Denosumab is

a monoclonal antibody that selectively neutralizes RANK ligand in the bone

microenvironment and has been approved for bone metastases treatment, offering an

alternative to the current targeting strategies.

38

1.6.3 Combinational therapy

The efficacy of monotherapy is generally limited due to many pro-survival

mechanisms and drug resistance in cancer. Combination of two or more drugs has been

proved to be an effective way to improve the therapeutic outcome and has been widely

adopted in the clinic. For patients with bone metastases, combination therapy prolonged

the survival time for 1-4 month when compared to monotherapy, and the improvement

tended to be more remarkable when the patients were not chemo-naieve[156]. The

conventional modalities contain two first-line chemotherapy or one with the addition of

targeted therapy that blocks the ontogenetic signaling pathways, suppressing the

survival response from multiple nodes[157,158]. Challenges remain to select the proper

drug unit for the combination. Exerting synergistic effect is an attracting principle and

serves as the fundamental that underlines the identification of optimal drug combination.

Synergy describes a scenario of the combination is more effective than the additive

result of the corresponding monotherapies. The concept allows fast screening of

possible combinations in vitro and determination of the optimal drug ratio. However, the

result may not be translated successfully in clinical trials. One important reason is the

different pharmacokinetic and biodistribution of two drugs could not ensure the tumor

cells are exposed to both drugs at the same time[159], whereas in vitro cell-based test

does not reflect the hindrance. Careful planning with computation modeling on the dose

regimes is necessary to compensate such difference[160-162] and the accuracy is

limited. With the help of nanocarrier, it is possible to load drug combinations inside the

particles and alter the PK profile of each drug to the same. Upon the uptake of drug-

loaded carriers, tumor cells are exposed to individual drugs at the predetermined ratio.

39

Another challenge of the combinational therapy is the additive toxicity. Therefore, each

drug component is applied under the optimal dosage. The increased risk of adverse

effect in the conventional treatment could also be offset by the change of biodistribution

in the nanoformulation. The nano strategy has been reported in a study by Keren Miller

that use bisphosphonate bearing HPMA-paclitaxel conjugate to treat breast cancer bone

metastases. The HPMA polymer did not only serve as a carrier but also the

antiangiogenetic agent that exerted the antitumoral efficacy in the combination of

paclitaxel[163,164].

40

1.7 CONCLUSION

Despite the remarkable advance in cancer therapy, metastatic malignancy remains

the leading cause of cancer death. Metastasis to bone is unique and yet dissertating.

The bone serves as fertile soil, and the host cells cooperate with tumor cells through

ligand-receptor crosstalk, supporting their growth and survival. Such interaction leads to

bone degradation, causing skeleton complications of pain, fracture, spinal compression

and hypercalcemia that drastically decrease the quality of life. However, the current

clinic treatments have limited efficacy other than pain control. The expected life span can

be only a few months or up to 3 years. The challenge is due to the bone-tumor

microenvironment as well as the "resistant" nature of the tumor itself. The studies in

adaptive resistant phenotype transition revealed another fact that needs to be taken into

consideration when developing new treatment modalities. Nanocarriers have been

developed as an attracting platform for cancer treatment. With the ability of tumor

targeting, combinational delivering and reducing toxicity, the nanocarrier-based delivery

system has demonstrated the potential to improve the therapeutic outcome and emerges

as one of the most useful tools in the treatment for bone metastases.

41

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CHAPTER II

TARGETED POLYMERIC MICELLES FOR THE TREATMENT OF

BREAST CANCER BONE METASTASIS

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2.1 INTRODUCTION

Tumor metastases remain the primary cause of cancer-related mortality. Bone

has been widely recognized not only as one of the frequent destinations of metastatic

spread of multiple solid tumors, but also as an organ that can provide a favorable

microenvironment for cancer cell survival and further growth or dormancy of metastases.

The complex mechanism of bone metastasis involves a coordinating interplay of bone-

homing cancer cells, osteoclasts, osteoblasts and the immune cells in the bone

marrow[1]. Tumor cells secrete factors which stimulate osteoclast-mediated bone

destruction and negatively affect osteoblasts. The consequent release of numerous

factors immobilized within the bone matrix that act on cancer cells, promotes a more

aggressive tumor phenotype and potentiating cancer spread and bone destruction. In

addition, physical properties of bone, such as hypoxia, acidic pH, and a high

extracellular calcium concentration, also support tumor growth. In patients with breast

cancer, the skeleton is the most frequent site for metastases. It is estimated that up to

75% of late-stage breast cancer patients will eventually develop bone metastases[2].

These lesions lead to numerous skeletal complications, including osteolysis, bone pain,

hypercalcemia, pathologic fractures, and spinal cord and nerve compression

syndromes[3]. Such complications increase morbidity and diminish the quality of life in

these patients as well as often account for the poor prognosis[4]. While early diagnosis

and effective chemo- and radiotherapies have significantly reduced the mortality rate

from primary breast cancer, the current therapeutic modalities for advanced metastatic

breast cancer patients remain palliative. Radiation therapy continues to be a gold

standard for palliative care of painful bone metastases and suppression of local disease.

Osteoclast inhibitors, bisphosphonates (pamidronate, zoledronic acid) and denosumab,

57

an antibody targeting RANKL, are commonly prescribed therapeutics for management

the progression of the established disease and reducing the risk of occurrence of

skeletal-related events. However, the benefits of these agents on improved survival was

observed in only a small population of patients[5,6]. Another challenge in the treatment

of metastatic cancer is effective delivery of therapeutics to the tumor lesion due to poor

penetration of most drugs in the bone tissue. Moreover, these agents also affect non-

cancerous cells within the bone microenvironment, inhibiting normal bone healing

processes and potentially leading to serious adverse events. Thus, despite significant

advances in the care of these patients there is a need for more effective treatment

options to prolong the survival of patients with metastatic bone diseases.

During the last decades, substantial efforts have been made in targeting bone

metastases using various delivery systems such as polymeric nanoparticles, liposomes,

micelles, and polymer-drug conjugates. These systems aim to improve the

biodistribution and target site accumulation of drugs thus reducing side effects. To

further enhance targeting of nanoparticles to bone lesions, the bone-binding moieties,

such as bisphosphonates, were utilized as targeting ligands due to their affinity to the

mineral compartment in bone tissue[7,8]. Indeed, hydroxyapatite is exposed as bone

metastases progress providing a target for bisphosphonate attachment. In line with this,

detection of metastatic involvement of the skeleton using 99mTc - radiolabeled

bisphosphonates has been in clinical practice for many years[9]. A number of in vitro and

in vivo studies substantiated preferential and efficient accumulation of bone-targeted

drug carriers within the bone lesions. Interestingly, despite demonstrated improved drug

efficacy, decreased toxicity and delay of tumor progression in mouse models, no

significant survival improvement between targeted and non-targeted nanocarriers has

58

been reported[10-17]. Nonetheless, combining the benefits of drug delivery systems,

bisphosphonate bone targeting to manipulate the tumor cells and their microenvironment

may further enhance the capabilities of drug delivery to eliminate or ameliorate bone

metastases.

Herein, we engineered bone-homing polypeptide-based polymeric micelles

carrying the chemotherapeutic docetaxel for the targeted treatment of breast cancer

bone metastases. To achieve this, an amphiphilic block copolymer composed of

polyethylene glycol, polyglutamic acid and phenylalanine (PEG-PLE-PLF) was

functionalized with a bisphosphonate, alendronate (ALN). The phenylalanine moieties

facilitate self-assembly of block copolymers and formation of micelles and are essential

for solubilization of hydrophobic compounds. Using an immunocompetent mouse model

of breast cancer dissemination to the bone, this study demonstrates that targeted

micelle-based therapy significantly attenuated tumor burden and extended animal

survival, proving the effectiveness of this targeting approach for the delivery of

chemotherapeutics to the bone.

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2.2 MATERIALS AND METHODS

2.2.1 MATERIALS

α-Amino-ω-alkyne-poly(ethylene glycol) (ALK-PEG-NH2 , MW = 5,000 g mol-1, Mw / Mn

= 1.02) was purchased from JenKem Technology USA Inc. L-glutamic acid γ-benzyl

ester (BLE), L-phenylalanine (LF), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

hydrochloride (EDC), N-hydroxysuccinimide (NHS), tris(3-

hydroxypropyltriazolylmethyl)amine (THPTA), hydroxyapatite (HA, powder, 10μm, ≥100

m2/g), ethylenediaminetetraacetic acid (EDTA), 3-bromopropionic acid, sodium

ascorbate, copper (II) sulfate pentahydrate, Phosphate Colorimetric Kit (MAK030), Acid

phosphatase Leukocyte (TRAP) Kit, and other chemicals were purchased from Sigma-

Aldrich. Receptor activator of nuclear factor kappa-Β ligand (RANKL) was obtained from

R&D System. Docetaxel (DTX) was purchased from Selleck Chemicals LLC.

Alendronate sodium trihydrate (ALN) was purchased from Alfa Aesar. CF488A amine

was obtained from Biotium. Fetal bovine serum (FBS), RPMI 1640 medium, DMEM

medium, penicillin/streptomycin, and other chemicals were purchased from Invitrogen.

XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate was purchased from

PerkinElmer.

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2.2.2 METHODS

Synthesis of ALK-PEG-PLE10-PLF10 block copolymers

The BLE-NCA (γ-benzyl L-glutamate-N- carboxyanhydride) and LF-NCA (L-

phenylalanine -N- carboxyanhydride) monomers were prepared as described

previously[18]. First, ALK-PEG-PLE diblock copolymer was synthesized via ring –

opening polymerization of BLE-NCA using ALK-PEG-NH2 as a macroinitiator. ALK-

PEG-NH2 (150 mg, 0.03 mmol) was dissolved in 10 mL of anhydrous DMF in an oven-

dried flask under nitrogen atmosphere followed by addition of BLE-NCA (79.6 mg, 0.302

mmol) dissolved in 5 mL of DMF. The feed molar ratio of ALK-PEG-NH2 to BLE-NCA

was 1:10. The reaction was allowed to proceed at 40°C for 72 h. An aliquot of the

reaction mixture was precipitated using an excess of diethyl ether, purified by repeated

precipitation from DMF/diethyl ether and dried under vacuum. The composition of ALK-

PEG–PLE copolymer was determined by 1H NMR from the peak intensity ratios of the

methylene protons of PEG and -benzyl groups (400 MHz, DMSO-d6, ppm: 3.52 (s,

4H, -OCH2CH2-), 5.14 (m, 2H, -CH2C6H5)). After the completion of the reaction, LF-NCA

(145 mg, 0.26 mmol), previously dissolved in 5 mL of anhydrous 1,4-dioxane, was then

added and let to react for an additional 120 h. The final product was isolated by

precipitation in diethyl ether, purified by repeated precipitation from DMF/diethyl ether

and dried under vacuum. The residue was dissolved in DMF and added dropwise to 0.5

N NaOH to deprotect glutamate residues. The mixture was stirred overnight and then

dialyzed using a dialysis membrane (MWCO 2 kDa) against distilled water for 48 h. After

dialysis, the product was filtered (0.8 μm filter) and lyophilized.

61

62

Synthesis of azido-ALN

3-azidopropionic acid was synthesized as previously described[19]. To 3-

azidopropionic acid (40.8 mg, 0.3 mmol) dissolved in 1 mL of anhydrous CH2Cl2, EDC

(66 mg,0.3 mmol) and NHS (48.6 mg,0.4 mmol) were added and the reaction was stirred

at room temperature for 6 h. The reaction mixture was washed with brine and an organic

layer was evaporated to remove the solvent. ALN (12.3 mg, 0.04 mmol) alendronate was

dissolved in 0.5 mL of water (pH 8.0). The NHS activated 3-azidopropionic acid (14.6 mg)

dissolved in 0.2 mL of acetonitrile/water mixture (1:4 v/v) was then added dropwise in

ALN solution while adjusting pH to 8,0 after each drop. The reaction was continued

overnight at r.t., then precipitated in ethanol three times to give the pure product and

dried. The structure was confirmed by 1H NMR.

63

Synthesis of ALN-PEG-PLE-PLF

The azide-ALN (6.64 mg, 0.02 mmol) was reacted with ALK-PEG-PLE10-PLF10

(13.4 mg, 0.002 mmol) dissolved in 10 mL of water and methanol mixture (1:1 v/v) in the

presence of copper (II) sulfate pentahydrate (0.5 mg, 0.002 mmol), THPTA (1.06 mg,

0.002 mmol) and ascorbic acid (3.52 mg, 0.02 mmol) for two days at r.t under argon

atmosphere. Then, EDTA (6.7 mg, 0.02 mmol) was added to the solution followed by

dialysis against deionized water (MWCO 2 kDa) for another 48 h to remove unreacted

small molecules. The dialyzed solution was then acidified and lyophilized to produce

ALN-PEG-PLE-PLF.

64

Polymer characterization

The 1H NMR spectra for the monomers and copolymers were acquired in DMSO-

d6 using a Bruker 400 MHz spectrometer. Gel permeation chromatography (GPC)

measurements to determine the molecular weights and polydispersity of the copolymers

were carried out at 40°C using a Shimadzu liquid chromatography system equipped with

TSK-GEL® column (G4000HHR) connected to Shimadzu RI and UV/vis detectors.

DMF/LiBr (10 mmol) was used as mobile phase at a flow rate of 0.6 mL/ min.

Poly(ethylene glycol) standards (Agilent Technologies, USA) with a molecular weight

range of 282 – 34,890 were used to generate the standard curve. Self-assembly

behavior of ALK-PEG-PLE-PLF and ALN-PEG-PLE-PLF copolymer was examined

using pyrene as a hydrophobic fluorescence probe[20]. The ALN conjugation efficiency

was determined by phosphate assay as previously reported[21]. Briefly, 20 µL of ALN-

PEG-PLE-PLF solution (5 mg/mL) was mixed with 20 µL of H2O2 and 120 µL H2SO4 in

the ampule and the mixture was heated for 15 min at 200 0C. 1.38 mL of Na2S2O5 (3

mg/mL) was added to the ampule and samples were incubated at 100 0C for another 15

min. Finally, the sample was filtered through a 0.2 µm filter and 200 µL of the solution

were collected into a 96 well plate. A series of standards was prepared in the same plate

according to the manufacture’s instruction ranging from 2.5 to100 µM. Finally, 30 µL of

the phosphorus reagent was added to each well and incubated with mild shaking

overnight at r.t. The absorbance at 650 nm was measured using by Spectramax M5

plate reader.

65

Polymeric micelles preparation and characterization

The empty and drug-loaded micelles were prepared by nanoprecipitation method.

Briefly, the polymers without or with DTX (polymer/drug molar ratio was 4:1) were

dissolved in minimal amount of 80% ethanol and dispersed into an excessive amount of

ice-cold water or PBS solution (water: ethanol = 20: 1 v/v). The solutions were stirred for

an additional 5 min and ethanol was evaporated under vacuum. The unbound drug was

removed by centrifugation (1000 g, 5 min). The drug content in micellar formulations was

determined by high-performance liquid chromatography (HPLC) analysis under isocratic

conditions using an Agilent 1200 HPLC system and a diode array detector set at 227 nm.

A Nucleosil C18 column was used as stationary phase (250 mm × 4.6 mm), and mobile

phase comprised of acetonitrile/water mixture (55/45, v/v) at a flow rate of 1 mL/min. The

loading capacity (LC) was expressed as LC (%) = 𝑊𝐷𝑇𝑋

𝑊𝐷𝑇𝑋+𝑊𝑝𝑜𝑙𝑦𝑚𝑒𝑟× 100%, where wDTX

and wpolymer are the weight amount of solubilized drug and polymer excipients in solution.

Intensity-mean z-averaged particle diameter (Deff), polydispersity index (PDI) and ζ-

potential of the drug-loaded micelles were determined by dynamic light scattering (DLS)

using Nano ZS Zetasizer (Malvern Instruments, UK). All measurements were performed

in automatic mode at 25°C at a fixed 173° scattering angle. Software provided by

manufacturer was used to calculate the Deff, PDI, and ζ-potential. Data are presented as

mean values ± SD (n = 3).

66

Drug release studies

The drug release from the micelles was examined in PBS (pH 7.4) or 70% FBS

in PBS at 37 0C by dialysis method. Briefly, 1 mL of drug-loaded micelle formulation in

PBS (approximately 0.1 mg/mL of DTX) was transferred to a floatable dialysis tube

(Slide-A-Lyzer G2, MWCO 3.5 kDa) and suspended in 45 mL PBS solution. At

predetermined time points, samples of 100 µL were withdrawn from the dialysis device

and analyzed quantified by HPLC using the same conditions reported above.

67

HA binding assay

To investigate the biomineral-binding ability of ALN-functionalized micelles, CF-

488 labeled copolymers were synthesized by EDC coupling (at a molar ratio of

[COOH]/[dye] = 10] and purified by dialysis to remove the uncoupled dye. The CF-488

labeled micelle solution in PBS (1.1 mg/mL, 1.5 mL) were incubated with HA powder (10

mg) for 4 hours at r.t. upon gentle shaking. HA was removed by centrifugation (2200 g,

for 1 min) and the fluorescence intensity of the supernatant (Isupernatant) was determined at

515 nm (ex=490 nm) and compared with the initial micelle solution (Io). The relative

binding affinity was calculated as (1 −𝐼𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝐼𝑜) × 100%. CF-488-labeled ALK-m were

used as controls. The binding test was performed in triplicate.

68

Cell culture and cytotoxicity studies

The murine 4T1 mammary adenocarcinoma cell line was purchased from the

American Type Culture Collection (ATCC). Cells were maintained in RPMI 1640

supplemented with 10% FBS, and antibiotics (streptomycin 100 U/mL and penicillin 100

U/mL) at 37 0C, 5% CO2. Cells were seeded in the 96-well plates at a density of 3500

cells per well and incubated for 12 h. The cells were then exposed to free DTX

(formulated as 10 mg DTX, 80 mg Tween 80, 648 mg PEG300, 275.9 mg ethanol 96%,

4 mg citric acid), DTX/ALK-m and DTX/ALN-m at serial concentrations for 36 h at 37 0C.

The cell viability was be evaluated by standard MTT assay[22] and the IC50 values were

calculated using GraphPad Prism software. To confirm the anticancer activity, 3D

clonogenic assay, cells were seeded at a density of 95 cells/cm2 in 24-well plates that

were pre-coated with Matrigel. Cells were treated with free DTX or DTX-loaded micelles

(0.1 µg/mL on DTX basis) for 36 h and allowed to recover in fresh medium for another

72 h. Colonies were stained with crystal violets and counted. The surviving fraction (SF)

was calculated as:

𝑆𝐹 =𝑁𝑜. 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑎𝑡 𝑡ℎ𝑒 𝑒𝑛𝑑 𝑝𝑜𝑖𝑛𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑢𝑑𝑦

𝑁𝑜. 𝑜𝑓 𝑠𝑒𝑒𝑑𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 × 𝑃𝐸

where plating efficiency (PE) is calculated as:

𝑃𝐸 =𝑁𝑜. 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑎𝑓𝑡𝑒𝑟 𝑠𝑒𝑒𝑑𝑖𝑛𝑔

𝑁𝑜. 𝑜𝑓 𝑠𝑒𝑒𝑑𝑒𝑑 𝑐𝑒𝑙𝑙𝑠

69

Inhibition of osteoclast differentiation and absorptive activity

The Raw264.7 monocyte/macrophage-like cells (ATCC, TIB-71) were cultured in

DMEM supplemented with10% FBS and 1% penicillin/streptomycin, in an atmosphere of

5% CO2 and 95% humidity at 37°C. Cells were harvested after reaching about 75%

confluency, seeded on 48-well plates (1.25×104 cells/well) and supplemented with

RANKL at the final concentration of 50 µg/mL. Cells were allowed to attach and then

were treated with empty ALN-decorated micelles or non-decorated micelles at serial

concentrations (0-100 µM on ALN basis) for one week. The culture medium containing

RANKL and corresponding treatment were exchanged every three days. Cells were then

fixed with 10% formaldehyde/PBS solution and stained with TRAP. The stain-positive

cells (osteoclasts) in each well were numerated and the IC50 were calculated using

GraphPad Prism. The bone resorption activity of osteoclasts derived from Raw264.7

cells was evaluated in 24-well Osteo Surface plates. Raw264.7 cells were continuously

cultured with RANKL (50 µg/mL) in the presence or absence of ALN-decorated micelles

or non-decorated micelles at two different concentrations (0.81 µM or 9 µM on ALN

basis) for 7 days. In another setting, Raw264.7 cells were cultured in the presence of

RANKL for 4 days and then exposed to the micelles for another 3 days. On day 8, cells

were bleached and the area/numbers of the pits (resulting from the absorption activity of

osteoclasts) appeared on the plate surface were quantified by using Image Pro software.

70

Macrophage migration assay

Cell migration assay was performed using Transwell permeable supports with 0.8

m pore membranes. Conditioned medium (CM) was collected from 4T1 culture

following 12 h of incubation in serum-free DMEM at 37°C and used as the

chemoattractant. 4T1 cells were seeded on 24-well plates (10,000 cells/well) and

cultured in serum-free DMEM for 12 h followed by replacement with CM and incubation

for an additional 8 h. Raw264.7 cells (1,000 per insert) were allowed to attach to the top

inserts and then were placed into the wells. The macrophages were allowed to migrate

to the underside of the top chamber for 24 hours in the presence of 4T1 cells and CM in

the bottom chamber. Where indicated, ALN-decorated or non-decorated micelles were

present in both top and bottom chambers throughout the assay. The wells without 4T1

cells and chemoattractant were used as negative controls. The nonmigratory cells on the

upper membrane surface were removed with cotton swabs and the migrated cells

attached to the bottom side of the membrane were fixed in 10% neutral buffered formalin

and then stained with 0.25% crystal violet aqueous solution. The numbers of migratory

cells were counted with an inverted microscope using a 20× objective. Each

determination represents the average of five random spots for each well (n = 3).

71

Pharmacokinetics studies in mice

All animal studies were performed in accordance with the guidelines of the

University of Nebraska Medical Center Institutional Animal Care and Use Committee

(IACUC). Pharmacokinetics of DTX and DTX-loaded micelles were determined in 8-

weeks old female Balb/C mice (Charles River Laboratories). The 18 mice were

randomly divided into three treatment groups of 6 mice and given either DTX (in Tween

80/ethanol/PEG300) or DTX-loaded ALN-micelles or DTX-loaded non-decorated

micelles at an equivalent dose of 10 mg DTX/kg body weight through intravenous tail

vein injection. At predetermined times, blood samples (100-150 μL) were withdrawn from

a submandibular vein into heparinized tubes, and then centrifuged at 10,000 g for 3 min.

DTX was extracted from plasma samples (10 μL) using excess (40 μL) of ice-cold

acetonitrile. All samples were sonicated for 1 min and then centrifuged at 10,000 g for 3

min. The clear supernatants were separated and used for analysis. Paclitaxel was used

as an internal standard at a final concentration of 625 ng/mL. Calibration curves were

built by spiking plasma samples from control animals to achieve the final concentration

of 5–2000 ng/mL DTX. These samples were analyzed on LC-MS/MS system using a

Nucleosil C18 column (250 mm × 4.6 mm) and a mobile phase of 80% acetonitrile in

water was applied under isocratic conditions at a flow rate of 0.5 mL/min. An LC-MS/MS

system equipped with triple quadrupole (QTRAP® 6500, Sciex) coupled to HPLC system

(Nexera × 2, Shimadzu) was used in multiple reaction monitoring (MRM) mode as

follows; m/z 853.995/876.25 → 286/308.25 for paclitaxel and 808.08/830.26 →

527/549.24 for DTX.

72

Evaluation of therapeutic efficacy

To establish the animal model of breast cancer bone metastases, 1×104 4T1/Luc

cells, constitutively expressing firefly luciferase, were suspended in 50 μL of sterile PBS

and injected into the left cardiac ventricle[23] of 6-week old female Balb/C mice under

isoflurane anesthesia. Disease progression and dissemination to the bone were followed

by noninvasive bioluminescence imaging (BLI) using IVIS Imaging System (Xenogen).

Total photon flux (photons/sec) was measured from fixed regions of interest (ROI) over

the entire mouse. On day 12 post-intracardiac injection, mice were randomized (4

treatment groups, n = 8) and treated with saline (control), DTX (in Tween

80/ethanol/PEG300), DTX-loaded ALN-decorated micelles and DTX-loaded non-

decorated micelles at an equivalent dose of 10 mg DTX/kg body weight. Treatments

were administered via tail vein injections daily for three consecutive days. At 48 h after

the last treatment, 3 mice per each treatment group were euthanized, tissues (liver,

spleen, kidney, bones) were dissected and fixed in 10% neutral buffered formalin. Bones

were decalcified using Regular Cal Immuno (BBC Biochemical) according to the

manufacturer’s protocol. After fixation, tissues were processed for routine embedding in

paraffin, preparation of 5 micron thick sections, and stained with hematoxylin and eosin.

The slides were evaluated with the pathologist blinded as to which group was being

evaluated. The rest of the animals were sacrificed at signs of paralysis or low body

condition score. We also used two additional groups of animals to elucidate the efficacy

of targeted treatment of bone metastases in combination with radiotherapy. Metastatic-

bearing mice (n = 6) received 3 i.v. injections on the daily basis of either saline or DTX-

loaded ALN-decorated micelles (10 mg/kg on DTX basis). 24 h following the last dose of

micelles or vehicle mice were irradiated using the TrueBeam Linear accelerator (Varian

73

Medical Systems, Inc. Milpitas, CA) at a dose rate of 3 Gy/min with the total effective

dose of 5 Gy in one fraction with Anterio-posterior/posterior-anterior opposing beams.

74

2.3 RESULTS AND DISCUSSION

Synthesis of alendronate-conjugated polymers.

Scheme 1 shows the synthesis of the parent ALK-PEG-PLE10-PLF10

copolymer and the subsequent modification of the distal end of the PEG block

with ALN moieties using azide-alkyne “click” chemistry. The unreacted small

molecules were removed by dialysis against deionized water (MWCO 2 kDa).

The chemical composition, molecular weights and polydispersity of synthesized

copolymers were determined by 1H NMR and GPC analyses (Supporting

information, Figure. S2.1 and S2.2, Table S2.1). The ALN conjugation efficiency

was determined using a phosphate assay21 and was ∼75%. We have previously

demonstrated that the incorporation of hydrophobic PLF block into triblock

copolymers confer amphiphilic properties and facilitates self-assembly of block

copolymers in an aqueous medium, which was ascribed to hydrophobic and π–π

stacking interactions of the phenylalanine units, and was essential for

solubilization of various hydrophobic compounds[18]. An association behavior of

PEG-PLE-PLF block copolymers was confirmed by fluorescence measurements

using pyrene as a probe (Figure S2.3). Using this approach, we determined that

the critical micelle concentration (CMC) for ALK-PEG-PLE-PLF was low (0.4

mg/L). The onset of aggregation of functionalized ALN-PEG-PLE-PLF chains

was shifted to higher concentrations with determined CMC value of 8.2 mg/L.

The observed increased CMC value is consistent with the presence of distal

75

charged ALN groups on the polymer chains that are leading to increased

repulsion between the copolymer chains upon formation of micellar aggregates.

This CMC value is still extremely low and comparable with those reported for

PEG-poly(lactide) micelles [24-25], which suggests that micelles formed from

these polymers should be expected to exhibit good stability. As was previously

demonstrated, the incorporation of aromatic units in the polymer chains forming

the hydrophobic micellar core can considerably improve the stability, loading

capacity and drug retention of taxane-loaded polymeric micelles[26]. Therefore,

we believe that PLF-based cores of the micelles may provide a suitable

environment for encapsulation of DTX.

76

Scheme 2.1: Synthesis of the bone-targeted copolymer.

Conditions: A). DMF, 40 0C, N2, Glu(z)-NCA, 72 h. B). DMF / 1,4-dioxane, 40

0C, N2, PhA-NCA, 120 h. C).

THF / 0.5N NaOH, RT, 24 h. D). MeOH / H2O (1:1, v/v), CuSO4 x 5H2O, THPTA, ascorbic acid, Ar, RT. E).

ACN, NaN3, reflux, 6 h; (F) DCM, EDC/NHS, RT, 6 h. (G) ACN/H2O (1:4, v/v), pH 8.0, RT, 24 h.

HO Br

O

HO N3

O

O N3

N

O

O

O

OP

NH

OH

HO

OHO

OO

NH

HN H

OO

114 10O

ONH

HN

OO

114 10

HN

O

H

10

OO

NH

HN

OHO

114 10

HN

O

H

10

PO

OHHO

OO

NH

HN

OHO

114 10

HN

O

H

10

NN

HN

OP

NH

OH

HO

OHO

PO

OHHO

N3

B

G

D

OP

NH2

OH

HO

OH

PO

OHHO

HN

OO

NH2114

O O

O

A

C

+

E

F

O

HN

O

HN

O

HN

O

HN

O

77

Figure S2.1: 1H-NMR spectra (400 MHz, DMSO-d6) of synthesized block-copolymers. A). ALK-PEG-

PBLE10. B. ALK-PEG-PBLE10-PLF10.

78

Table S2.1: Polymer characteristics determined by 1H NMR and GPC

copolymer Feed ratio,

Glu / PhA

Yield ratio, 1H-NMR Composition ratio, GPC

Glu / PhA Mn,

g/mole

Mw,

g/mole

Mn,

g/mole

Mw/Mn (Đ)

ALK-PEG-PLE(OBzl)10 10 10.0 7217 7655 6858 1.11

ALK-PEG-PLE(OBzl)10-PLF10 10 / 15 10.3 / 9.9 8806 10656 9726 1.10

79

Figure S2.2: GPC traces (DMF + 10 mM LiCl; 40 0C, 0.6 mL/min). A). ALK-PEG-NH2. B). ALK-PEG-

PBLE10. C). ALK-PEG-PBLE10-PLF10.

80

Figure S2.3: The plot of fluorescence intensities ratio of I3/I1 for aqueous pyrene solutions (110-6

M) as

a function of (A) ALK-PEG-PLE10-PLF10 and (B) ALN-PEG-PLE10-PLF10 concentration at 25oC.

A B

81

Preparation of docetaxel-loaded polymeric micelles and characterization of their

physicochemical properties

DTX, a taxane that inhibits cell replication by stabilizing the microtubule

cytoskeleton, is one of the chemotherapeutic agents approved for the first-line treatment

of metastatic breast cancer[27,28]. Despite its potent anticancer activity, DTX exhibits

serious dose-limiting toxicities partly due to the formulation excipients. Polyoxylated

surfactant, polysorbate 80, is utilized in Taxotere, a commercial formulation of DTX, and

has been implicated in observed hypersensitivity reactions, nonallergic anaphylaxis, and

injection- and infusion-site adverse events[29]. For these reasons, we selected DTX as

the model chemotherapeutic agent for solubilization into polymeric micelles. The micelle

formation and drug loading were accomplished in one single step using a method known

as nanoprecipitation. A polymer or a mixture of a polymer and drug were dissolved in a

minimal amount of ethanol and the resulting solution was injected into a 20 volume of

ice-cold DI water or PBS. Under these conditions we were able to obtain stable

dispersions of DTX-loaded micelles with a loading capacity of 22.4% as was determined

by HPLC and it was associated with high loading efficiency (about 97%). Targeted

micelles with varying ALN density were prepared by co-assembly of ALK-PEG-PLE-PLF

and ALN-PEG-PLE-PLF copolymers at various molar ratios. In this way, we prepared

ALN-m with the various proportion of ALN moieties ranging from 3.8% to 75%. Micelles

prepared from ALK-only copolymers (ALK-m) were used as a non-targeted control for

comparison. Of note, the modification with targeting ligand practically did not change the

size of the micelles: the average hydrodynamic diameters (Deff) of the ALN-only micelles

(ALN-m) and ALK-m in DI water at neutral pH were about 70 and 67 nm, respectively, as

measured by DLS. The DTX-loaded micelles had a greater size (82-84 nm) than that of

82

empty micelles and exhibited uniform size distribution (PDI < 0.2) (Table 2.1). Both types

of micelles displayed net negative charge which can be explained by the presence and

ionization of carboxylic acid groups of PLE chains in the shell of the micelles. As

expected, the ζ-potential of the ALN-m/DTX was further decreased to −32 ± 2 mV

compared to −17.0 ± 1 mV for the unmodified ALK-m/DTX (Table 2.1), which is

indicative of the presence of additional negatively charged ALN moieties on the surface

of ALN-m. One of the common metabolic complications of bone metastasized breast

cancer is hypercalcemia. It is characterized by elevated serum calcium level (about 3 -

3.5 mM for moderate cases) and indicates poor prognosis[30]. Therefore, we next

examine how the presence of calcium ions may affect the characteristics of DTX-loaded

micelle. When ALN-m/DTX were incubated in DI water in the presence of 3.4 mM Ca2+,

their size substantially decreased to ca. 57 nm and ζ-potential increased (Table 2.1).

Likewise, a decrease in size and the net negative charge was observed for ALK-m/DTX,

however, in the less pronounced manner. This behavior is consistent with effective

chelation of calcium ions by bisphosphonate groups as well as partial neutralization of

carboxylate groups of PLE chains leading to decrease of net charge, reduced repulsions

between the polymer chains in the shell of the micelles and collapse of the micelle.

Similarly, the size of DTX-loaded micelles was also affected by the ionic strength:

micelles prepared in PBS were substantially smaller compared to those in DI water

(Table 2.1) due to the masking of the electrostatic repulsions in the shell of the micelles

by the electrolyte (0.15 M NaCl). Interestingly, the addition of calcium ions to dispersions

of micelles in PBS did not lead to a further decrease in particle size. Based on these

results, we can expect that dimensions of ALN-m/DTX under physiological conditions will

be around 60 nm and thus will be suitable for bone targeting and extravasation since the

capillaries and sinusoids in the bone marrow have pores of approximately 80-100

nm[31]. Dispersions of DTX-loaded micelles remained stable in solution, and no

83

changes in size or PDI were detectable within at least 4 weeks of storage at ambient

conditions.

84

Table 2.1: Physicochemical characteristics of DTX-loaded micelles

Sample

DI water DI water with 3.4 mM Ca2+

PBS

Deffa

(nm)

PDIa

-potentiala

(mV)

LC

(%)

Deff

(nm)

PDI

-potential

(mV)

Deff

(nm)

PDI

ALK-m/DTX 82 ± 4 0.20 -17 ± 1 22.4 73 ± 1 0.17 -6 ± 1 60 0.16

ALN-m/DTXb 84 ± 5 0.18 -30 ± 2 22.4 57 ± 1 0.16 -12 ± 1 67 ± 2 0.16

a: Effective diameter (Deff), polydispersity index (PDI) and -potential were determined by DLS

(copolymer concentration 1 mg/mL; 250C). Data are expressed as mean ± SD (n = 3).

b: ALN-m/DTX corresponds to drug-loaded micelles prepared from ALN-PEG-PLE-PLF copolymers.

85

Both DTX-loaded formulations displayed similar sustained release profiles

without burst release (Figure 2.1A). Under normal physiological conditions (PBS, pH 7.4)

the cumulative release of DTX from both types of micelles was about 25% within 12 h.

The slightly slower release rates were observed for ALN-m/DTX as compared to ALK-

m/DTX at longer periods of times. Furthermore, the presence of serum albumin in the

medium (70% FBS) did not appear to have a significant impact on DTX release from

ALN-m/DTX suggesting that limited drug leakage from the micelles can be expected in

the circulation, which is the first step of the successful delivery.

86

Figure 2.1: In vitro characterization of ALN-m and ALN-m/DTX micelles. (A) Drug release profiles for

DTX in targeted ALN-m/DTX and non-targeted ALK-m/DTX micelles under physiological conditions as

determined by HPLC method; (B) Binding of the ALN-m to the bone mineral HA as a function of degree of

modification of ALN-m; (C) Cytotoxicity of DTX and micelle formulations in 4T1 breast cancer cells

determined by clonogenic assay. Cells were treated with 0.1 µg/mL DTX equivalents for 36 h and allowed to

recover in fresh medium for another 72 h. Data are presented as mean SD (n = 3), *p < 0.05, NS - not

significant.

A B C

87

Bone mineral binding of ALN-decorated micelles.

Bone metastases cause structural damages in the skeleton, which ultimately lead

to exposure of bone mineral crystals within the metastatic lesions. These bone

resorption sites thus represent an ideal target for the design of osteotropic systems

using calcium chelating molecules such as bisphosphonates. The bone targeting and

retention capacity of ALN-m/DTX were evaluated using HA powder as a bone mineral

mimicking model[32]. To this end, we have screened a series of fluorescently-labeled

targeted micelles with various content of ALN moieties that was controlled by the molar

ratio of fluorescently labeled ALK-PEG-PLE-PLF and ALN-PEG-PLE-PLF copolymers.

The quantification of HA-associated fluorescence indicated that all bone-targeted

micelles displayed a good affinity to HA as a result of multivalent interactions and

anchoring via ALN moieties (Figure 2.1B). As expected, the binding of targeted ALN-m

exceeded that of the non-targeted ALK-m and became more efficient as the density of

targeting ligands increased. The bound fraction of ALN-m reached saturation level

(approximately 70%) for the ALN-m with 37% ALN moieties on their surfaces and a

further increase in ligand density did not provide statistically significant improvements in

HA binding. The observed ligand density effect can be related but not limited to improper

ligand presentation since ALN moieties attached to the flexible PEG chains may be

buried in the shell surrounding the core of the micelles or steric hindrance of the ligands

due to the size of the micelles. Importantly, the binding capacity of ALN-m was not

compromised by the presence of free calcium ions in the solution (Figure S2.4). This

would imply that binding of ALN-m to calcium ions present in the bloodstream upon

intravenous administration would not prevent their adsorption to the mineral matrix in

bone. In addition, since calcium-bound ALN-m had less negative ζ-potential (Table 2.1),

88

this observation also supports the argument that binding of ALN-m to HA is mainly

driven by specific interactions via ALN moieties rather than by the negative ζ-potential of

the micelles. In this context, it is also noteworthy that non-targeted ALK-m displayed

relatively high binding efficiency (ca. 20%, Figure 2.1B). It may be attributed to the

contribution of binding via PLE block in the outer shell of the micelles. Indeed, it is well

known that acidic oligopeptides of aspartic acid or glutamic acid have an affinity toward

HA. Their binding capacity increases when repeating sequences of amino acid residues

are present in the sequence such as in osteopontin and sialoprotein, non-collagenous

bone proteins[33]. Thus, it is likely that an incorporation of short hydrophilic PLE blocks

(10 units) into the shell of the ALN-m may be beneficial not only for stabilization of

micellar structures but also by enabling additional targeting capabilities. Overall, these

results suggest that ALN-m could be a promising drug delivery system to target

chemotherapeutics to the bone resorption sites. Besides being an excellent bone

targeting agent, ALN pharmacological mechanism of action also involves an inhibitory

effect on osteoclasts as well as beneficial effects on osteocytes and osteoblasts[32]. In

addition to anti-resorptive properties, experimental data suggest that bisphosphonates

may also have indirect effects on tumor cells[34]. To evaluate whether the design of our

delivery system can capitalize on the above-mentioned ALN properties, we choose ALN-

m with maximal content of conjugated ALN moieties (ca. 75%) for the further

experiments.

89

Figure S2.4: Binding of the ALN(37%)-m to the bone mineral HA in DI water and in the presence of

3.4 mM CaCl2 in the solution. The binding capacity of ALN-m was not compromised by the presence of

free calcium ions in the solution.

DI water DI water + 3.4mM CaCl2

0

20

40

60

80

HA

-bo

un

d A

LN

-m (

%)

90

In vitro cytotoxicity studies.

Murine 4T1 triple-negative breast cancer cell line is known to be highly invasive,

relatively resistant to chemotherapy and radiotherapy[35] and is widely used to establish

primary or metastatic tumor models in BALB/c mice. The cytotoxic effect of DTX loaded

into the polymeric micelles was first evaluated against 4T1 cells using MTT assay. As

summarized in Table 2.2, cytotoxicity of the DTX formulated into ALN-m was

substantially increased compared to ALK-m/DTX or conventional DTX formulation

(Tween 80/ethanol/PEG300): the IC50 value for ALN-m/DTX was 0.035 0.016 M, an

order of magnitude lower than those for other treatments. Interestingly, when the micelle

formulations were tested under the conditions mimicking hypercalcemia (3.4 mM Ca2+),

the IC50 values were further lowered. It could be explained by the neutralization of the

net surface charge of the micelles, which may facilitate their enhanced cellular uptake.

Of note, ALN alone at the concentration corresponding to the highest tested

concentration of ALN-m (about 30 M) was found not to be toxic to 4T1 cells (Figure

S2.5). To further validate these results, an additional study was conducted using a

clonogenic assay (Figure 2.1C). Reduction in the colony-forming potential of 4T1 cells

was observed following a 36 h exposure to all DTX formats. Consistent with MTT assay

results, ALN-m/DTX showed the most potent cytotoxicity and resulted in significantly

lower (p < 0.05) fraction of surviving cells. The mechanism explaining observed

enhanced cytotoxicity of ALN-m/DTX is not clear. One possibility is that ALN conjugation

may affect the cellular entry of the micelles and/or alter their cellular trafficking. The

Hammond group has previously reported that liposomes which were coated with

poly(acrylic acid) functionalized with ALN via a “layer-by-layer” method were more

effectively taken up by osteosarcoma cells compared to non-targeted liposomes[36].

91

These ALN-targeted liposomes loaded with doxorubicin also displayed higher levels of

cytotoxicity compared to the uncoated control. More studies are needed to understand

the observed cytotoxic effects of ALN-m/DTX.

92

Table 2.2: IC50 values of free DTX and DTX-loaded micelles in 4T1 breast cancer cell line.

Sample

IC50, µMa

RPMI RPMI (3.4 mM Ca2+

)

DTX 2.16 ± 0.82 -

ALK-m/DTX 1.39 ± 0.49 0.26 ± 0.05

ALN-m/DTX 0.035 ± 0.002 0.012 ± 0.020

a: The IC50 values represent the means SD (n = 3).

93

Figure S2.5: In vitro toxicity of free ALN in 4T1 cancer breast cells as determined by MTT assay.

100.5 101 101.5 102 102.50

50

100

150

ALN, µM

4T

1 c

ells v

ialb

ilit

y (

%)

94

Remodeling of the tumor-bone microenvironment.

As a therapeutic agent ALN was reported to inhibit osteoclast formation and limit

their resorptive activity. ALN was also reported to indirectly affects osteoclast precursors,

which are a source of resorption-stimulating cytokines[37]. We next investigated whether

ALN moieties tethered to the surface of the micelles could retain its inhibitory effect on

osteoclast formation in vitro. Murine monocyte/macrophage-like RAW264.7 cells that

maintain the capacity to differentiate into osteoclast-like cells in the presence of RANKL

were used in these studies. To this end, osteoclast precursor RAW264.7 cells were

stimulated by RANKL (50 ng/mL) with or without the presence of free ALN or ALN-m in

the culture medium. RAW-derived osteoclasts were confirmed through the staining of

TRAP enzyme, a key marker involved in osteoclastic bone resorption. As shown in

Figure 2.2A, treatment with ALN-m attenuated osteoclasts formation in a concentration-

dependent manner similarly to free ALN. The IC50 values for free ALN and ALN-m (6.4

2.5 M and 8.8 4.6 M, respectively) did not differ significantly from each other (p >

0.05). Importantly, treatment with ALK-m did not have any noticeable effect on

osteoclastogenesis (Figure 2.2B). The tumor cells are also known to play a critical role in

this cycle by cleaving anchored RANKL into soluble format via an elevated expression of

MMP-7[38]. Thus, we next performed osteoclastogenesis assay using the conditioned

medium (CM) from cultured 4T1 cells that contained secreted differentiation factors.

Consistent with the former results, ALN-m significantly repressed (p < 0.01) the

formation of mature RAW-derived osteoclasts at the ALN concentration corresponding to

its IC50 value (9 M) (Figure 2.2C). In contrast, multiple multinucleated TRAP-positive

osteoclasts were formed in 4T1-CM treated RAW264.7 cells in the presence of ALK-m at

95

the same polymer concentration. These results suggest that ALN-m provide substantial

inhibitory capacity against cancer-induced osteoclastogenesis.

96

Figure 2.2: Effects of ALN-m on osteoclastogenesis. RAW264.7 cells were stimulated with RANKL (50

g/mL) in the presence or absence of ALK-m or ALN-m at different concentrations (based on ALN

equivalents). TRAP staining was used to indicate mature multinucleated osteoclast cells. The results are

normalized to cells grown only in the presence of RANKL (control). (A) free ALN and ALN-m displayed

comparable inhibition effects on osteoclasts differentiation; (B) ALK-m did not show any effect on the

osteoclast differentiation; (C) RAW264.7 cells were stimulated with CM from 4T1 cells in the presence or

absence of ALN-m. Data are expressed as the mean ± SD (n = 3). **p < 0.01, NS - not significant.

A B C

97

In breast cancer bone metastasis microenvironment bi-directional interactions

between tumor cells and osteoclasts lead to both osteolysis and tumor growth[39].

Hence, it is also crucial to suppress the resorptive activity of the osteoclasts. To evaluate

the effects of ALN-m on osteoclast bone lytic activity, RAW264.7 cells were cultured on

plates coated with microcrystalline calcium phosphate and stimulated with RANKL in the

presence or absence of ALN-m. We selected two concentrations of ALN-m, 9 M and

0.81 M (on ALN basis) that were determined to suppress or not affect osteoclast

differentiation, respectively. The resorption activity was quantified as a total resorptive

area of “pits” formed by osteoclasts (Figure 3A). RANKL-stimulated cells formed a

number of pits, suggesting that the bone resorption activity of RANKL-treated cells made

them into functionally active state-resembling osteoclasts. As expected, non-targeted

ALN-m did not affect their resorption activity while treatment with ALN-m suppressed

formation of resorption pits. Treatment with 0.81 M ALN-m, the concentration that

ineffectively inhibited osteoclast differentiation, significantly reduced (p <0.01) overall

area of resorption pits compared with treatment with RANKL alone. Osteoclastic bone

resorption was inhibited practically completely by the treatment with 9 M ALN-m which

is in line with our observation that ALN-m at this concentration also significantly inhibit

osteoclast formation (Figure 2.2C). To exclude the direct effect of ALN-m on

osteoclastogenesis, in parallel experiment RAW264.7 cells were first pre-differentiated

into osteoclasts for 4 days and then were treated with ALN-m. As it seen in Figure 2.3B,

osteoclasts treatment with ALN-m significantly inhibited their resorption activity in a

concentration-independent manner. Together these results implicate that the ALN-m

have an inhibitory activity on both osteoclast differentiation and function and thus could

provide additional benefit in combating bone metastases by intervening the disease-

induced bone loss.

98

Figure 2.3: ALN-m inhibits the resorption activity of osteoclasts. (A) RAW264.7 cells were seeded on

Osteo Surface plates and stimulated by RANKL (50 g/mL) in the presence or absence of ALK or ALN-m as

indicated. Polymer concentration is expressed based on ALN equivalents. The total area of the resorbed

regions was quantified and normalized to the cells grown in the presence of only RANKL (control). (B)

RAW264.7 cells were first differentiated into osteoclasts on Osteo Surface plates in the presence of RANKL

(50 g/mL) for 4 days and then treated with ALK-m or ALN-m for next 3 days. Data are expressed as the

mean ± SD (n = 3). **p < 0.01, NS - not significant.

Contr

ol

[ALK

-m],

9µM

[ALN

-m],

9µM

[ALN

-m],

0.81µM

0

50

100

150

200

To

tal re

so

rpti

ve a

rea (

% t

o c

on

tro

l)

**

**

NS

Contr

ol

[ALK

-m],

9µM

[ALN

-m],

9µM

[ALN

-m],

0.81µM

0

50

100

150

200

To

tal re

so

rpti

ve a

rea (

% t

o c

on

tro

l)

**

**

NS

A B

99

In the osteolytic cycle in bone metastases, residential macrophages and

circulating monocytes stimulating by the cytokines secreted by the tumor can be

recruited and infiltrated into the tumor microenvironment[40,41]. The tumor-associated

macrophages are not only capable of osteoclast differentiation but also are considered

to play an important role in angiogenesis, immunosuppression and thus contribute to

disease progression and poor chemotherapy response. Here, we intended to determine

whether ALN- m were able to attenuate macrophage recruitment by cancer cells in vitro.

To this end, we used the Transwell method where the Raw264.7 cells were seeded on

the upper insert and allowed to migrate across the membrane to the lower chamber, on

which 4T1 cells were seeded (Figure 2.4A). In the 24 h period, a substantial number of

the macrophages were found to migrate across the membrane toward 4T1 cells, with

more than a 10-fold increase in a number of migrated cells compared to the negative

control (Figure 2.4, B and C). When treated with ALN-m, as low as 0.81 µM on ALN

base, migration ability of macrophages was suppressed to a practically negligible level

(p > 0.05 compared to negative control). Given that ALN-m at such concentration is non-

toxic to both types of cells, it could be assumed that the “cooperation” between tumor

and macrophage was inhibited.

100

Figure 2.4: Inhibition of tumor-induced macrophage migration by ALN-m. (A) Schematic representation

of the experiment. RAW264.7 cells (103 per insert) were added to the upper chamber with DMEM without

FBS (negative control) or 4T1 cells (105/well) and their conditioned media (CM) added to the lower well

(positive control). RAW264.7 cells were allowed to migrate for 24 h at 37°C prior to staining and quantitation

of chemotaxis. In experimental groups ALN-m were added to both chambers; (B) Representative images of

migrated RAW264.7 cells; (C) Quantification of migration of RAW264.7 cells treated with ALN-m. Data

represents the average ±SD for 5 randomly selected fields over 3 separate experiments. **p < 0.01, NS - not

significant.

9 µM ALN-m 0.81 µM ALN-m

Negative control Positive control 9 µM [ALN-m] 0.81 µM [ALN-m]

Neg

ativ

e

Posi

tive

[ALN

-m],

9µM

[ALN

-m],

0.81µM

0

5

10

15

Mig

rati

on

fo

ld c

han

ge t

o c

on

tro

l

**

**

NS

NS

A

B

C

101

In vivo therapeutic efficacy.

Before assessing the therapeutic efficacy of DTX-loaded micelles, we evaluated

the pharmacokinetics of both ALN-m/DTX and ALK-m/DTX in healthy BABL/C mice and

compared to “free” DTX (in Tween 80/ethanol/PEG300) at an equivalent dose of 10

mg/kg DTX. The PK parameters calculated using a non-compartment model are

presented in Supplementary Table S2.2. DTX incorporation into micelles resulted in a

modest increase in plasma half-life (8.9 h for ALK-m/DTX and 7.7 h for ALK-m/DTX vs.

6.2 h for free DTX). Moreover, DTX plasma exposure as measured by the area-under-

the-curve (AUC) was ~1.5 times higher for ALN-m/DTX than that for ALK-m/DTX. Both

the volume of distribution and clearance were decreased by 1.3-fold and 1,5-fold,

respectively, in the ALN-m/DTX treatment group compared with the ALK-m/DTX

treatment group. These data suggest that ALN-m can prolong circulation time of DTX

through reducing its rapid elimination and contributing to more DTX distributed into bone

tissue.

To test the therapeutic effect of ALN-m/DTX on established bone metastases, we used

syngeneic 4T1 breast cancer model[42]. The 4T1/Luc cells were inoculated into a left

cardiac ventricle of Balb/C mice to establish disseminated metastatic disease. Bone

metastatic seeding and burden was monitored by whole body BLI. The disease was

found to be very aggressive and associated with substantial weight loss: tumor burden

exclusively inside the skeleton was successfully established in 1-2 weeks after cell

injection and often resulted in mortality within 3 weeks. Due to the aggressiveness of the

disease, the treatments were administered daily for the first three days to assure that all

individual experimental subjects received the same total dose of the drug. In this model

of late treatment of aggressive bone metastasis, we detected a trend of delayed

102

progression of bone metastasis by either ALN-m/DTX or ALK-m/DTX treatment

compared with the control group (p < 0.05, Figure 2.5A and Figure S2.6). In addition,

both treatments attenuated animal weight loss (the effect of ALN-m/DTX was statistically

significant, p < 0.02 vs. control group) and can also be indicative of their therapeutic

potential (Figure 2.5B). This is in contrast with the treatment of DTX, which had a

negligible effect on reducing bone metastasis (p ≈ 0.28 versus the control group), which

can be explained by the aggressiveness of the disease model and the drug-resistant

nature of the 4T1 cell line. Of note, neither of the micelles-based formulations reached

statistical significance compared to free DTX treatment at the endpoint of the BLI

monitoring (18 days) when the mice in the control group succumbed to metastatic

cancer. However, the promising efficacy of DTX micellar formulations translated into

improved survival (Figure 2.5C). In line with BLI studies, the treatment with free DTX did

not provide any improvement in animal survival over the saline control. The non-targeted

ALK-m/DTX slightly extended the mean survival time from 18 to 21 days, albeit all mice

reached the endpoint before day 24. Remarkably, the ALN-m/DTX demonstrated the

best therapeutic outcome and significantly improved the survival time (p < 0.05)

compared to ALK-m/DTX group with 2 out of 5 long-term survivors who reached the end

point at day 35 (Figure 2.5C). Such a therapeutic response to ALN-m/DTX is far superior

to either ALK-m/DTX or DTX alone suggesting a benefit of bone targeting along with

chemotherapy in late-stage bone metastasis. Notably, no visceral organ toxicities were

observed as a result of the treatments as representatively assessed for liver, spleen and

kidney by tissue histopathology analysis (Figure 2.5D). Extramedullary hematopoiesis is

often reported as a consequence of bone metastases in this model[43]. In the control

group, strong extramedullary hematopoiesis was observed in the liver, while in the

treatment groups it was significantly reduced. Marrow toxicity indicated by decreased

cellularity was occasionally present in the bone sections, but was similar between

103

groups. These observations suggest a favorable toxicity profile for the DTX micellar

formulations.

104

Table S2.2: PK parameters of DTX in plasma in tumor-free nude mice.a

Free DTX ALK-m/DTX ALN-m/DTX

t1/2 (h) 6.2 7.7 8.9

AUC (h*ng/mL) 16024 18824 27487

Cl (mL/h/kg) 624 531 364

Vd (mL/kg) 5589 5926 4674

MRT (h) 4.2 6.9 10.7

a: The PK parameters were calculated using a non-compartment model with Phoenix WinNonlin (NCA,

rsq > 0.95). AUC, area under the curve from time 0 to 24 h; Cl, clearance; Vd, volume of distribution; MRT mean residence time.

105

Figure 2.5:. Anti-metastatic efficacy of ALN-m/DTX in syngeneic 4T1 breast cancer bone metastasis

model. Treatment consisted of iv injections of PBS, DTX (in Tween 80/ethanol/PEG300), ALK-m/DTX, or

ALN-m/DTX at a dose of 10 mg/kg DTX equivalents. Arrows represent iv injections. (A) Quantification of

bone metastasis burden based on whole body BLI imaging; (B) Body weight; (C) Kaplan−Meier survival plot;

(D) Histopathological analysis of representative kidney, liver, and spleen of mice received different

treatments. Data presented as mean ± SEM (n = 8). *p < 0.05 by log-rank (Mantel-Cox) test.

12 14 16 180

10

20

30

40

50

Days post tumor inoculation

Rt/R

0

Control

DTX

ALK-m/DTX

ALN-m/DTX

*

20 30 400

50

100

Days post tumor inoculation

Perc

en

t su

rviv

al

Control

DTX

ALK-m/DTX

ALN-m/DTX

*

12 14 16 180.8

0.9

1.0

1.1

Days post tumor inoculation

Wt/W

0

Control

DTX

ALK-m/DTX

ALN-m/DTX

A

C

B

D Control DTX ALK-m/DTX ALN-m/DTX

Kidney

Liver

Spleen

106

Figure S2.6: BLI captures and individual mouse respose. (A) Representative BLI images of four mice

per group at day 0 and day 6 after the beginning of treatments. (B) Quantification of bone metastasis burden

in individual mice treated with different formulations based on whole body BLI imaging.

Control ALK-m/DTXDTX ALN-m/DTX

Day 0

Day 6

12 14 16 180

20

40

60

Days post tumor inoculation

Rt/R

0

Control

12 14 16 180

20

40

60

Days post tumor inoculation

Rt/R

0

ALK-m/DTX

12 14 16 180

20

40

60

Days post tumor inoculation

Rt/R

0

DTX

12 14 16 180

20

40

60

Days post tumor inoculation

Rt/R

0

ALN-m/DTX

A

B

107

Among all options treating metastatic breast cancer including chemotherapy,

hormonal therapy and targeted therapy, radiation therapy (RT) remains an essential

modality particularly for the palliative purpose. The most common utilities of palliative

RT are for pain control, improving ambulation, preventing fractures and controlling spinal

cord compression/neurologic deficits and preserving the quality of life particularly for

patients with bone metastases. During the past few decades, multiple randomized

controlled clinical trials have demonstrated equivalent short-term pain relief between a

single 8 Gray (Gy) fraction and multiple fraction regimens, including most commonly 30

Gy in 10 fractions, 24 Gy in 6 fractions and 20 Gy in 5 fractions[44-48]. While single

fraction treatment optimized convenience of patients and their caregivers and was

related with lower acute toxicity, retreatment rates to the same anatomic site due to

recurrent pain were higher in those who received single fractions compared to

fractionated treatment courses. It is important for further studies to explore the

possibilities of combined therapy to enhance the efficacy of single fraction RT. To test

this notion, we conducted a proof-of-concept experiment utilizing sequential

chemotherapy with ALN-m/DTX and RT. Two groups of animals with developed

metastasis received three i.v. injections daily of either saline or ALN-m/DTX (10 mg/kg

on DTX basis) and then, 24 h after the last treatment, mice received a total of 5 Gy of

radiation at a dose rate of 3 Gy/min. 5Gy in one fraction is known clinically as an

ineffective dose for tumor control and is lower than the threshold dose causing

gastroenteral radiation syndrome as in human studies when large area or whole body is

irradiated. The treatment of late-stage metastasis in our mouse model with a single 5

Gy RT was not surprisingly ineffective and did not delay the progression of the disease

(Figure 2.6A). In contrast, the administration of ALN-m/DTX followed by RT delayed

tumor growth and extended the animals median survival from 19 days (RT) to 30 days

(Figure 2.6B). Due to the large variations within the RT group (Figure S2.7), the

108

differences in tumor growth or survival between RT and combination group was close to

but not statistically significant (p = 0.06 vs. the RT-only treatment group). We did not

observe any episodes of diarrhea or hematochezia in the mice treated with RT. Further

detailed studies are needed to confirm the benefits of this combined treatment regimen.

Particularly, it might be interesting to test this combination therapy in another model of

bone metastasis, when breast cancer cells are injected directly into bone (such as tibia

or femur). While it is technically not a model of metastasis, it can replicate tumor-induced

changes in bone[42] and will permit to apply RT locally. Nevertheless, these results

suggest that treating cancer patients with bone-targeted chemotherapy combined with

radiotherapy could potentially be more effective and suppress the development and

dissemination of bone metastasis.

109

Figure 2.6:. Treatment efficiencies of sequential chemotherapy with ALN-m/DTX and radiotherapy

(RT). Treatment consisted of iv injections of PBS or ALN-m/DTX (10 mg/kg DTX) followed by 5 Gy of

radiation at a dose rate of 3 Gy/min 24 h after the last injection. Arrows represent iv injections. (A)

Quantification of bone metastasis burden based on whole body BLI imaging; (B) Kaplan−Meier survival plot.

Data presented as mean ± SEM (n = 6).

A B

110

Figure S2.7: Quantification of bone metastasis burden in individual mice treated with (A) only

radiotherapy (RT) or (B) ALN-m/DTX (10 mg/kg DTX) followed by RT.

12 14 160

10

20

30

40

Days post tumor inoculation

Rt/R

012 13 14 15 16 17

0

10

20

30

40

Days post tumor inoculation

Rt/R

0

A B

111

2.4 CONCLUSION

We engineered a dual-functional bone-homing drug carrier based on

biodegradable polypeptide­based micelles that can deliver the drug to the bone and

shows a propensity for simultaneous remodeling the tumor-bone microenvironment. The

drug-loaded micelles were prepared via a robust procedure that allowed obtainment of a

high loading of docetaxel using an alendronate-modified copolymer with a relatively

short length of the hydrophobic segment. Docetaxel, when encapsulated into the ALN-m

was found to retain its cytotoxicity and to be even more effective in 4T1 breast cancer

cell killing in the conditions mimicking hypercalcemia in patients with metastases. These

ALN-m demonstrated enhanced binding to the bone mineral analog and were effective in

inhibiting osteoclast differentiation and their resorption activity as well as attenuate

tumor-induced migration of macrophages in vitro. In line with the in vitro results, ALN-

m/DTX delayed disease progression and improved survival in a syngeneic murine model

of breast cancer bone metastasis. This model represents a useful tool for examining the

later stages of breast cancer bone metastasis, however, the skeletal sites of metastatic

deposition and number and size of the metastases are extremely variable. Nevertheless,

our work provides new insight into the fusion of ALN moieties with drug-loaded micelles

to maximize the therapeutic efficiency of chemotherapy by targeting bone metastases

and their microenvironment.

112

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CHAPTER III

COMBINATION THERAPEUTIC PLATFORM FOR BREAST CANCER

BONE METASTASIS TREATMENT

117

3.1 INTRODUCTION

Chemo drugs based anticancer therapy remains dominant in the clinic. The

resistance to the toxic molecules, including the microtubing inhibitors, DNA damaging

agents and targeted kinase inhibitors, can drastically limit the treatment outcomes[1].

The general process of the generation of the resistance follows Darwin principle in which

the drug-tolerant cells (with right mutation) are positively selected by repeated drug

exposure[2,3]. Apart from that, researchers have also identified the acquired resistance

that emerges based on the cellular dynamics without genetic alteration[4,5]. The clinic

also showed evidence that such resistance occurred soon after the initial treatment[6]

and was found to be a drug-induced effect[7]. Its molecular mechanism was understood

as stochastic dynamics inside and between cell attractors, which are defined by the

microenvironment[8]. Such a phenomenon is clinically important, as it implies that even

the chemo drug naïve patients can develop something quickly that subsequently

reduces the drug efficacy.

The adaptive resistance unlike the intrinsic one, does not specify a certain class

of drugs. It could potentially arise against any environmental stress, including those

cytotoxic molecules. Several strategies were developed either to avoid or overcome the

adaptive[9-12]. However, the introduction of the new drug would likely to induce a

secondary adaptive resistance and how to resolve the puzzle is still uncleared. A recent

study[13] just prove the potential of what Matthew had suggested that the resistance

could create the new “Acais heel” and lead to ‘‘collateral sensitivity’’, where the resistant

118

might be useful[14]. By further extending the idea, a pair of drugs whose adaptive

statuses are sensitive to the counterparts would close the loop of generating secondary

adaptive resistance.

Described by the multiple cell attractor theory, a position that on the edge of an

attractor is unstable and the viability is low[15]. In the context of adaptive resistance in

cancer, a phenotype that falls outsides of the attractor defined by the chemo stress is

more sensitive. So, drug combinations that define different cell attractors (ideally without

any overlap) are critical to achieving the proposed paradigm. In other words, the drugs

need to induce different (ideally opposite) adaptive dynamics. In this study, we first

validated the existence of the adaptive resistance in drug-treated 4T1 cells and their

potential survival under high-concentration drug exposure. The level of AMPK activation

was evaluated as an indicator of two different adapted statuses. DTX and Das happened

to have the opposite effects on the adaptive dynamics. Therefore, we hypothesized that

the drug combinations of DTX and Das could have enhanced antitumor efficacy.

The paradigm was considered to work best when cells are exposed to both drugs

at the same time. The polypeptide-based micellar system has demonstrated the

capability to transport two or more drug simultaneously[16]. So, we aimed to engineer

the micelles with that could encapsulate both drugs efficiently and release them at a

similar pace. The size of the carriers would have to be small enough to harness the EPR

effect and passively targeted to the tumor area. After developing the formulation, breast

and prostate cancer cells were used to test the combinational treatment in vitro. Given

119

that DTX and Das are known to have their independent cytotoxic mechanisms[17,18],

and when applied together, it would be expected naturally to have some synergy effect

due to pathway crosstalk inhibition[19]. To separate the trait of the anticancer efficacy

from the sensitizing effect and validate the involvement of AMPK activation level in

establishing the drug-tolerant status, AMPK activator and inhibitor at nontoxic

concentrations were employed in conjunction with the chemo drugs.

Bone metastases remain the leading cause of cancer death, and no effective

treatment was available other than pain control[20]. One of the reasons why it is so

challenging might be associated with the adaptive resistance. Tumor in the bone

appears to be highly vascularized and could be expected to have sufficient nutrients

supply. In vitro, we observed that frequently refreshing the growth medium helped the

survival in Docetaxel and regrowth of the cells. Here, we used the murine breast cancer

bone metastases as established in Chapter II and evaluated the effectiveness of the

micellar drug combinations in vivo.

120

3.2 MATERIALS AND METHODS

3.2.1 MATERIALS

Besides of the chemicals and reagents purchased in the 2.2.1, FxCycle

PI/RNase, RPMI 1640, FK-12, MEM-Alpha (without ascorbic acid) and FxCycle

PI/RNase Staining Solution were bought from Thermo Fisher Scientific. AMPK Alpha

Total and Phospho T172 In-Cell ELISA Kit were purchased from Abcam. Docetaxel

(DTX) and Dasatinib (Das) were purchased from the AdpoGen Life Science. The Cell

Counting Kit-8 was purchased from Dojindo Molecular technologies. Compound C and

AICAR were obtained by Dr. Robert Lewis’ group in the Eppley Institute at the University

of Nebraska Medical Center. MDA-MB-231 and PC-3 were originally purchased from

ATCC and were grown in the lab according to the ATCC’s instruction. MC3T3 were

obtained from Dr. Dong Wang in the Department of Pharmaceutical Sciences at the

University of Nebraska Medical Center.

121

3.2.2 METHODS

Preparation of adapted cells

4T1 and MDA-MB-231 cells were cultured in RPMI1640 that contained 10% fetal

bovine serum (FBS) and 100U/mL penicillin-streptomycin. PC-3 cells were cultured in F-

12 medium that also contained 10% FBS and 100U/mL penicillin-streptomycin. Adapted

cells were prepared freshly for all experiments. Cells were seeded on T75 flask with 10%

confluency, allowed to attach overnight and treated with free 10 µM DTX

(polysorbate/PEG300/95% ethanol) for three days. The complete growth medium that

contained the same concentration of the drugs was refreshed until the end point of the

adaptation whenever the phenol red became orange (the interval ranging from 8 to 24 h).

To demonstrate the generation of adaptive resistance at a clinically relevant

concentration, adapted 4T1 cells were also developed by treating with 20 nM DTX for 36

h.

122

Characterization of the polyploidy formation in the adapted cell population

The proportion of polyploidy population with different chromosome copies was

determined by flow cytometry, using propidium iodide (PI). Data were obtained on BD

FACSCalibur (BD Biosciences) (Ex: 488 nm, Em: 585/42 filter). The adapted cells and

the corresponded parent cells (used as the negative control) were harvested

(trypsinized), washed in ice-cold 1X PBS and fixed in 70% ethanol for 30 min at 4 0C.

Cells were then washed with deionized water and stained with FxCycle PI/RNase

Staining Solution for 30 min in the dark and analyzed by flow cytometry.

123

Quantification of AMPK protein concentration in parent and drug-adapted 4T1

cells

AMPK Alpha Total and Phospho T172 In-Cell ELISA Kit were used for measuring

the shift of p-AMPK level during the adaptation process. Cells 1) without treatment, 2)

treated with 10 µM / 20 nM DTX or 10 µM / 50 nM Das for 2 days, 3) treated with the first

drug (DTX or Das) for 2 days and then with the other one (Das or DTX) subsequently for

2 h (high dose) or 5 h (low dose), were washed with 1X PBS, fixed in situ with 4%

formaldehyde for 3 min and then permeabilized with methanol at -20 0C for 45 min. The

p-AMPK and total AMPK was measured according to the manufactory protocol.

Quantitative fluorescence intensity was read by LI-COR Odyssey CLx at both 700 nm

and 800 nm channels. The fluorescence intensity in each treated group was normalized

(dividing by the one in parent cells group). Change in the p-AMPK and total AMPK was

expressed as folds to control. Representative images were captured by Olympus IX73

Inverted Microscope with a xenon excitation source and an Olympus DP80 Digital

Camera and cellSens Dimension software.

124

Western Blot Analyses for p-AMPK and its downstream protein

Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 1% NP-40, 0.5%

Na deoxycholate, 0.1% Na dodecyl sulfate, 150 mM NaCl, 0.5 mM Na3VO4, 2 mM EDTA,

2 mM EGTA, 10 mM NaF, 10μg/mL aprotinin, 20 mM leupeptin, 2 mM PMSF) was used

to prepare whole cell lysate from collected cells. Promega BCA protein assay was

utilized to evaluate protein concentration. SDS-PAGE gel electrophoresis was completed,

proteins were transferred to nitrocellulose membranes, membranes were blocked for 45

minutes in PBS-based blocking buffer (LI-COR Biosciences, 927-40000), and incubated

in primary antibody at 4°C overnight. Secondary antibodies (LICOR IR-Dye 680LT and

800CW) were diluted 1:10,000 in 0.1% TBS-Tween. The LI-COR Odyssey was used to

image the western blots. Primary antibodies were used in the indicated dilutions: pACC

(#3661, Cell Signaling) 1:1000, tACC (#3676, Cell Signaling) 1:1000, pRAPTOR (#2083,

Cell Signaling) 1:1000, tRAPTOR (#2280, Cell Signaling) 1:1000, pAMPKα1α2 (#2531,

Cell Signaling) 1:1000, AMPKα1α2 (#2532, Cell Signaling) 1:1000, and B-Actin (C-4,

47778, Santa Cruz) 1:2000. Anti-mouse and anti-rabbit secondary antibodies conjugated

to IRDye800 and IRDye680LT were used at 1:5000-1:10,000 dilutions.

125

Synthesis of triblock copolymer Methoxy polyethyleneglycol-b-polyglutamic acid-

b-polyphenylalanine (mPEG5K-PLE-PLF)

The same synthesis scheme in 2.2.2 was used to produce the triblock copolymer,

using methoxy-PEG-NH2 (M.W.=5,000 Da) as the macroinitiator. The length of the PLE

block was designed as five monomers and the polyphenylalanine as 15 monomers. The

chemical structure of the diblock copolymers, triblock copolymers, deprotected triblock

copolymers was confirmed by 1H-NMR. Deuterated dimethyl sulfoxide (DMSO-d6) was

used for the benzyl protected block copolymers and deuterated trifluoroacetic acid (TFA-

d1) for the deprotected block copolymers. The polydispersity of the benzyl protected

block copolymers was determined by gel permeation chromatography as described in

2.2.2.

126

Preparation and characterization of drug-loaded micelles DTX/m, Das/m,

(DTX+Das)/m

DTX, Das or both drugs were mixed with the synthesized triblock copolymers and

dissolved in the minimum amount of 80% ethanol. The solution was dispersed dropwise

into ice-cold 1X PBS (>20X volume) upon quick stirring. Then the ethanol was removed

under vacuum for 10 min, and the formulation was sterilized via a 0.45 µm syringe filter

(Millex, SLHV033RS).

Loaded drugs were confirmed by high-performance liquid chromatography

(HPLC) analysis under isocratic conditions using an Agilent 1200 HPLC system and a

diode array detector set at 227 nm for DTX and 325 nm for Das. A nucleosil C18 column

was used as stationary phase (250 mm × 4.6 mm), and mobile phase comprised of

acetonitrile/water mixture with 0.1% formic acid (60/40, v/v) at a flow rate of 1 mL/min.

The feed ratios were expressed as 𝑊𝐷𝑇𝑋+𝑊𝐷𝑎𝑠

𝑊𝐷𝑇𝑋+𝐷𝑎𝑠+𝑃𝑜𝑙𝑦𝑚𝑒𝑟𝑠%. The final yield after the filtration

was measured by the same HPLC method and calculated as

𝐷𝑟𝑢𝑔 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑎𝑓𝑡𝑒𝑟 𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝐷𝑟𝑢𝑔 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑏𝑒𝑓𝑜𝑟𝑒 𝑓𝑖𝑙𝑡𝑟𝑎𝑡𝑖𝑜𝑛% . All samples for HPLC analysis were prepared by

lyophilizing the 100 µl micelle solution and re-dissolving the solid residues in the same

amount of acetonitrile with 0.1% formic acid. The mixture was sonicated to extract the

drugs into the supernatant, and the rest insoluble contents were removed by

centrifugation.

127

Effective hydrodynamic diameters (Deff) of all micelles were determined at 25 0C

in 1X PBS solution by dynamic light scattering (DLS) using a Zetasizer Nano ZS

(Malvern Instruments Ltd., Malvern, UK). The measurements were performed per

manufacturer instruction to obtain the size distribution and polydispersity index (PDI).

The concentration of the polymers in measured samples was controlled in the range of

0.2-0.4 mg/mL. To test the stability of the drug-loaded micelles, (DTX+Das)/m was kept

at room temperature continuously for ten weeks and analyzed by DLS for potential

changes in size distribution. The experiments were repeated three times.

In vitro Drug release kinetics was studied by the dialysis method mentioned in

2.2.2 using 1X PBS as the releasing medium. 0.7 mL (DTX+Das)/m, DTX/m and Das/m

suspension in PBS that contained about 0.05 mg DTX and 0.03 mg Das were

transferred into the dialysis tubes (Slide-A-Lyzer G2, MWCO 3.5 kDa) and the tubes

were suspended in 50 mL PBS solution (sink condition). The drug release proceeded at

37 0C on the plate shaker. At predetermined time points (0, 4, 12, 24, 48, 72 h), an

aliquot of the 100 µL solution was sampled from each of the dialysis tubes. The

remaining drugs quantities in the aliquots were analyzed by the HPLC method in the

same manner as described above. The cumulative release was expressed as % of the

initial concentration. The experiments were repeated three times.

128

In vitro cell growth inhibition assays

Cells were seeded on 96-well plates, 10,000 cells per well, allowed to attach for 6

h (parent cells) or 12 h (DTX adapted cells) and treated with DTX, Das or the

combination of both drugs in the micellar formulation for 24 h. Viable cells were

quantified by using Cell Counting Kit-8 according to the manufactory’s protocol. The

absorbance (Abs) of the assay solution was read by SpectraMax M5 at 450 nm. All

reading was subtracted by the blank wells and normalized as % to the negative control.

The absolute IC50 was calculated in GraphPad Prism by fitting the dose-response data

with the non-linear function and interpolating the X values(concentration) when setting

Y(viability)=50%. Relative DTX sensitivity of adapted 4T1 to the parent cells was

calculated as 𝑉𝑖𝑎𝑙𝑏𝑖𝑙𝑖𝑡𝑦%𝑝𝑎𝑟𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠

𝑉𝑖𝑎𝑙𝑏𝑖𝑙𝑖𝑡𝑦%𝐷𝑇𝑋 𝑎𝑑𝑎𝑝𝑡𝑒𝑑 𝑟𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠. The experiments were repeated three times.

129

Sensitization of 4T1 cells to DTX or Das with 5-Aminoimidazole-4-carboxamide

ribonucleotide (AICAR) or Compound C

100 µM AICAR and 1 µM Compound C were first tested in 4T1 cell line for

potential growth inhibitory effect. Their sensitization effect was then evaluated in the 4T1

cell line. 4T1 cells were treated with two combinations DTX+100 µM AICAR and 1 µM

Compound C+Das for 24 h. Both tests used the same method in the growth inhibition

assay described above. The experiments were repeated three times.

130

Chemotaxis of tumor cells toward pre-osteoblast

MC3T3 cells were maintained in alpha-MEM (10% FBS and 100U/mL penicillin-

streptomycin, no ascorbic acid) at 80% confluency. MC3T3 conditioned medium (preOB-

CM) was prepared by incubating the cells with the same medium but without the FBS for

12 h. After incubation the medium was collected, sterilized by 0.2 µm syringe filter. 24-

well transwell permeable support (0.8 µm pore) was used for studying the migration. In

the experimental groups, MC3T3 cells with alpha MEM (10%FBS, no ascorbic acid)

were seeded on the receiver well and allowed to attach for 12 h. The complete growth

medium was then replaced by preOB-CM. MC3T3 with the PreOB-CM together served

as the chemoattractant. In the positive control group, the MEM medium (no ascorbic acid)

with 10% FBS was used as the chemoattractant. In the negative control group, only the

base MEM medium (no ascorbic acid) was used as a mock attractant. After setting up

the receiver, the inserts holding 15,000 cells (4T1/MDA-MB-231) with different

treatments were placed on the top of the receiver. Tumor cells were allowed to migrate

for 12 h, then fixed in 10% NBF and stained by crystal violet. The cells that did not travel

through (upper surface of the insert membrane) were wiped out gently by cotton swabs,

leaving the migrated cells on the lower surface. Migrated cells were counted under the

inverted light microscope (20X), in five random spots per well. The experiment was

repeated three times.

Osteoclast differentiation

131

10,000 Raw 264.7 cells were seeded in 24-well plates and incubated with the

complete growth medium (RMPI 1640 + 10%FBS + 100U/mL penicillin-streptomycin)

that contained additional 50 ng/mL of RANKL and Das/m treatment for 7 days. The

medium with exact RANKL and the drug were refreshed every three days. The cells

were fixed by 10% NBF for 60 sec, and the osteoclast was stained by the Acid

Phosphatase, Leukocyte (TRAP) Kit according to the manufactory protocol. The number

of multinucleated and positive stained cells in each treatment group was counted under

the inverted light microscope (20X). The experiment was repeated three times.

132

Establishment of murine bone metastatic model

Luciferase-expressing tumor cells (4T1/Luc) cells were cultured and selected in

the RPMI 1640 + 10%FBS as described in the in vitro experiment with the

supplementary of 15 µg/ mg of Blasticidin HCl for two days and without Blasticidin HCl

for another two days. Cells were sub-cultured when reaching 40% confluency. Cells

were trypsinized and washed thoroughly with ice-cold 1X DPBS. The suspension was

diluted to 200,000 cells/mL and was kept on ice for less than 20 min before being

injected. Eighty 5-week Balb/C mice were purchased from Charles River Laboratories.

For the convenience, the hair on the chest was removed before injection by Veet hair

removal gel. Mice were anesthetized by isoflurane and inoculated with the prepared cells

through the left ventricle injection, 10,000 cells/mouse in 50 µL DPBS (day0).

133

In vivo efficacy and survival study

Bioluminescence of the 4T1/Luc was measured by PerkinElmer In vivo Imaging

System (IVIS) starting from seven days post tumor injection to monitor the tumor growth.

Luciferin (15 mg/mL) was injected intraperitoneally (i.p.) for each mouse 10 min before

the imaging. On day9, mice were evenly distributed into five groups/10 mice per group

based on the bioluminescence intensity and randomly assigned to receive the

treatments 1) DPBS, 2) free DTX, 3) free DTX + free Das, 4) DTX/m + Das/m, 5)

(DTX+Das)/m respectively. The treatments that started on day10 were given via tail vein

injection with equiv. 10 mg/kg DTX, 6 mg/kg Das or both (based on the animal body

weight) on a daily basis and continuously for four days. The resulting cumulative dose

was 40 mg/kg DTX, 24 mg/kg Das or both. Body weight was monitored every day, and

euthanasia was performed when the body weight loss reached or exceeded more than

20% of the initial values or if the animals met other euthanasia criteria outlined by

(IACUC).

Twenty-four hours after the final treatments, three mice from each treatment

group were sacrificed. Whole blood was collected for blood cell counting and blood

chemistry analysis. Soft organs (the liver, kidney, spleen) and tibia/femur were collected

and fixed in 10% NBF for 24 h at room temperature. The tibias/femurs were further

decalcified in 14% EDTA-NH3H2O (pH=7.2) buffer for three weeks. Each piece of bone

tissue was incubated with the buffer (20-times volume) at room temperature with mild

shaking. The buffer was exchanged every day. The kidneys, livers, spleens and

134

decalcified tibias/femurs were processed subsequently in UNMC tissue facilities for

embedding, sectioning and H&E staining. The blood was again collected via maxillary

vein five days after the final treatment and counted for blood cell numbers. The blood

smear was also prepared to identify the unmatured neutrophils in the mice of the control

group (tumor-bearing).

The survival of the animals was recorded for 30 days from the first treatment.

The data was presented as Kaplan-Meier curves, and the Log-rank (Mantel-Cox) test

was employed to analyze the significance between the treatment groups. Animal death

was recorded when the mouse was found dead, the body weight dropped more than 20%

or suffered from stress that did not comply with IACUC.

135

3.3 RESULT AND DISCUSSION

Adaptive resistance in 4T1 cells

Cancer cells were reported previously to undergo phenotypical transition and

acquired adaptive resistance quickly when exposed to a variety of chemo drugs at

subtoxic concentration[6,21-23]. The adapted cancer cells are insensitive to the chemo

drugs and able to continue the proliferation after removal of the treatment[24]. In our

study, murine breast cancer cell line 4T1 was observed to transit into dormant status and

giant in size with the appearance of multiple nuclei. In both condition of 10 µM DTX and

10 µM Das, the morphology change started quickly after treatment for 24 h, however the

morphology was drastically different (Figure 3.1). Part of the cells was able to survive

through a 3-days treatment of 10 µM DTX with frequently refreshed culture medium

(containing the same drugs), which indicated the acquisition of the drug resistance. After

the removal of DTX, the dormant cells were found to produce daughter cells by budding,

similar to what was reported in work by Karuna[24].

136

Figure 3.1: Representative image of the morphology change in DTX and Das treated 4T1 cells. Upper

parent control, middle 10 µM DTX, lower 10 µM Das. Images were captured at 36 h of the treatment.

10µM Das

10µM DTX

Parent

137

To further confirm that the rise of such resistance was due to the adaptive

change rather than the selection of the subpopulation with intrinsic resistance (based on

genetic mutation), 4T1 cells were pre-treated with a lower dose of DTX (20 nM) for only

36 h. The treatment should not affect cell viability (Table 2.3). After the short drug

exposure, their sensitivity to DTX was further tested by growth inhibition assay for 24 h.

The IC50 increased more than three folds to ≈ 100 µM (Figure 3.2) compared to the

parent cells without the pre-treatment (Table 3.4). The drug exposure was also

considered as clinically relevant based on the pharmacokinetic data and the actual dose

regimen in pharmacy practice[25].

138

Figure 3.2: Dose-responsive profile of DTX to 20 nM 36 h pre-treated 4T1. Cells acquired the resistance

to DTX with the increased IC50 (≈100 µM).

12.5

0

25.0

0

50.0

0

100.

00

0

50

100

150

[DTX], µM

Via

lbilit

y %

to

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ol

139

Polyploid formation in the adapted cells

Based on visual examination under the microscope, the adaptive cells were giant

(10 times larger than parent cells) and multinucleated. Flow cytometry was used to

analyze the DNA quantities to confirm the visual changes. Compare to the parent cells

which are diploid (2N), DTX adapted 4T1 showed a large population of polyploid cells

(4N-8N). Similar results were also found for human breast cancer cell line MDA-MB-231

and human prostate cancer cell line PC-3 (Figure 3.3). These cell lines are all known to

be metastatic, especially to the bone. Such kind of reaction could be explained as the

cell cycle was arrested due to the DTX’s inhibition in microtubing disassembly[26], while

the duplication of DNA and the synthesis of cellular protein continued. This observation

together with the fact of cell survival matched with the observation that breast cancer cell

could exit the cell cycle and stays in a sub G1 phase[24]. Also, chronic autophagy that

did not proceed to the apoptosis was considered to take place during this process and to

be part of the adaptive resistance acquisition[27,28]. On the contrary, the Das did not

show such an effect, even at a higher concentration of 100 µM (Figure 3.4). After

removing the DTX treatment, cells were recovered the proliferation gradually, and the

whole population trended to restore the initial diploidy condition (Figure 3.5).

140

Figure:3.3: Adapted response to DTX treatment. Parent cells(left), DTX treated (right). The substantial

increase in the polyploid population after 10 µM DTX treatment for three days was confirmed by flow

cytometry for all the three cell lines.

141

Figure 3.4: Adaptive response to Das. Das treatment up to 100 µM did not induce 4T1 transitioning to

polyploidies.

142

Figure 3.5: Recovery of DTX adapted 4T1 cells. The population was shifting back to diploidy (2N) (left

panel), analyzed by flow cytometry four weeks after the removal of the treatment (right panel).

143

p-AMPK protein level as a potential marker to determine the direction of adaptive

change in drug-treated cells

p-AMPK/AMPK known as the energy sensor is a key fundamental protein to

regulate the cell metabolic system[29,30]. Studies have revealed the complexity of its

functions, and some of them are closely related to cancer therapy, e.g., cell

proliferation/cell cycle regulation, autophagy, fatty acid synthesis, abnormal glutamic

metabolic, etc[31-33]. Inhibition on its downstream pathway (fatty acid synthesis) was

able to enhance the anticancer efficacy of the chemo drugs[34]. It is also reported that p-

AMPK level is crucial for the survival of the tetraploid cells which have the potential to

become cancer cells and the chemoprevention efficacy of aspirin was also attributed to

the capability of selective elimination of tetraploid cells[35].

These evidence make the p-AMPK an appealing target marker to classify the

adaptive response. Two studies were designed to determine the change in pAMPK level

after adapting to the DTX or Das. One was that 4T1 cells were exposed to 20 nM DTX

for 48 h and then to 50 nM Das for 5 h separately after removing the DTX; in contrast,

the other was that cells were exposed to Das for 48 h and then to DTX (Figure 3.6).

Because the doses were well below the IC50 level (Figure 3.4), few cells were observed

to be dead during the 48 h treatment. Therefore, the process did not select a

subpopulation cell that expressed a different level of p-AMPK. The parent control, the

cells with the primary treatment and with the addition of secondary treatment were

harvested for western blot analysis. The results showed decreased p-AMPK in DTX

144

adapted cells, but the increased level in Das group. After the subsequent treatment of

the counter drug, the trend of such changes reversed (Figure 3.7), which demonstrated

the different cellular responses to the two drugs separately. The results were also

implying that the change is dynamic, and cells can continuously answer to the chemo

stress even after the initial adaptation. To further clarify the p-AMPK level shift was the

part of the necessary rearrangement made by the cells to survive rather than the part of

the cytotoxic mechanism, the concentration of DTX and Das was both increased to 10

µM. In this case, since the dose was around IC50, a small proportion of the cells was

killed during the first treatment, and thus the cells collected for p-AMPK level analysis

were the survivors. The results (normalized to housekeeping protein) showed the same

trend of p-AMPK shifting as well as the reversal behavior just as what was observed in

lower dose sets. And the higher dose set seemed to lead to more considerable deviation

from the base level. Given the fact that either DTX or Das was not reported to have

direct interaction with p-AMPK/AMPK, it is reasonable to speculate the opposite p-AMPK

shift could be the consequences of distinguished adaptive response to the drugs.

Besides, the previous study already uncovered that the Das resistant CLL (chronic

lymphocytic leukemia) have a much higher base level of p-AMPK compared to the non-

resistant subtype[36], whereas DTX insensitive cell line (4T1) has a much lower base

level. The evidence in addition proved possible relation between p-AMPK level and the

adaptive resistance.

145

Figure 3.6: The regimen of different drug treatments for the analysis of the corresponded adaptive

responses. 10 µM DTX, 10 µM Das for high dose set; 20 nM DTX, 50 nM Das for low dose set. Cells at

each stage were analyzed for AMPK activation level.

Parent 4T1

DTX treatment2 days

Das treatment2 days

Parent 4T1

Das treatmentHigh dose (2h), low dose (5h)

DTX treatmentHigh dose (2h), low dose (5h)

DTX adapted

Das adapted

146

Figure 3.7: Adaptive response of p-AMPK level to the drug treatments. In both dose set (high and low),

cell adapted to downregulated p-AMPK-level upon DTX, while upregulated upon Das.

Pare

nt

DTX

DTX

+ D

as

Das

Das

+ D

TX

p-AMPK 1.00 0.54 0.81 1.46 1.10

p-AMPK 1.00 0.70 0.87 1.27 1.14

High dose (10 µM DTX, 10 µM Das)

Low dose (20 nM DTX, 50 nM Das)

Pare

nt

DTX

DTX

+ D

as

Das

Das

+ D

TX

p-AMPK

p-AMPK

β-actin

β-actin

147

Quantification of AMPK protein level shift

The adaptive change in p-AMPK level observed by western blot was double-

checked by a different method In-Cell ELISA. The similar design as used in western blot

studies was adopted, using the high-dose treatment set (10 µM DTX, 10 µM Das, 48 h

for the primary treatment, 2 h for the secondary Das treatment, but 12 h for DTX). Cells

at each of the three stages were fixed in situ and remained intact throughout the ELISA

assay. After incubating with the fluorophore label antibodies, the wells were scan in Licor

Odyssey at 680 nm for the total AMPK (showed in green) and 800 nm for p-AMPK

(showed in red). For both primary and secondary treatments, the color shifted toward

greenish (total AMPK) upon DTX exposure, and it turned to yellowish (total AMPK + p-

AMPK) upon Das exposure. The minimum amount of unspecified binding could be seen

in the negative control that omitted the primary antibody (Figure 3.8). Representative

fluorescent images were also captured to reflect the detailed change in the cellular level

(Figure 3.8). Quantified data were read out by Image Studio Software (Licor), then

normalized by the total quantities of the cells (Janus Green B) and expressed as folds to

the parent control (Figure 3.9). Similar to the western blot results, the DTX treatment

was confirmed to be associated with p-AMPK downregulation, Das with upregulation

instead; and the dynamic trend was also reversed upon secondary treatment. Compared

to DTX, Das had a quicker and more substantial effect on the cells that had been treated

with the counter drugs. In contrast to the activated AMPK, the total AMPK level remained

relatively stable upon all treatments.

148

Figure 3.8: Parent 4T1 and drug adapted 4T1 in 96-well scanned by Licor Odyssey. Greenish (680 nm

total AMPK) and yellowish (680 nm total AMPK and 800 nm p-AMPK) color shift was found in DTX treated

and Das treated respectively.

149

Figure 3.9: Representative fluorescent images parent 4T1 and drug adapted 4T1. Colocalization of

green (680 nm total AMPK) and red (800 nm p-AMPK) after Das treatment suggested the adaptive response

of AMPK activation. The red color deceased after DTX treatment indicated the opposite way of the adaptive

response.

p-AMPK Total AMPK Merge

Parent control

DTX adapted 48hr

DTX adapted 48hr + Das 2hr

Parent control

Das adapted 2hr

Das adapted 48hr

Das adapted 48hr + DTX 2hr

150

Figure 3.10: Quantification of p-AMPK level/total AMPK change in DTX and Das adapted cells by In-

Cell ELISA. A) DTX lowered the p-AMPK level in the drug-adapted cells and followed the treatment of Das

significantly bring up the level whereas Das significantly bring up the p-AMPK level (P<0.05). C-D) No

significant change could be observed in total AMPK across all treatment. E-F) Trend remains after

normalizing p-AMPK by total AMPK. Data was presented by mean ± SD and analyzed by t-tests (*, P<0.05;

**, P<0.01; ***, P<0.001).

Paren

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DTX

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Das

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**

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***

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Das

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Das

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TX 1

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A B

C D F

E

151

Rational of designing a micellar based formulation that co-delivers both DTX and

Das to overcome the adaptive resistance

Based on the results in western blot and In-Cell ELISA, it is clear that p-AMPK is

involved in the response of 4T1 cells to the DTX and Das treatment and the directions

were just opposite: one downregulated, and the other upregulated the p-AMPK. Also, the

mechanism of the drugs is known not directly connected the AMPK; the differences in

base levels of p-AMPK between drug sensitive and resistant cell lines are correlated with

the response trends observed in this study: it is reasonable to consider the response is

the adaptation for survival, belonging to “the way to resist to stress" rather than “the way

how cancer cells are killed".

Unlike the intrinsic resistance by drug selection or acquired resistance by a gene

mutation which was well defined by specific mechanism and takes time to develop, the

adaptive one is more dynamic, with the abilities to quickly enter a drug-tolerant status.

Given the variety of metabolic modes that cell could use potentially, it is difficult or nearly

impossible to inhibit one or two pathways to block the cell survival completely.

One strategy we proposed here is to use two drugs that can lead to the opposite

adaptive dynamics, like DTX and Das. The p-AMPK/AMPK serves as an “infrastructure”

element to support the proper cellular functions. And the same it is in drug adapted

cancer cells. An adaptive status (insensitive to the treatment) requires an appropriate p-

152

AMPK/AMPK level. To be specific, the survival of DTX treated cell relied on the low p-

AMPK level whereas Das adapted cells demanded the high p-AMPK level. Since there

would be only two conditions regarding the p-AMPK level (either low or high), cells would

not be able to locate at a proper status that can survive under both condition: any

adaptive effort that comes with a high level of p-AMPK would sensitize the cell to DTX,

while anyone ends with low level would be effectively killed by Das. Distinguished from

the classic approach that inhibits the pathways that cells use to survive, such a strategy

does not impede the adaption process directly, but making the drug adapted status more

sensitive to the counterpart treatment.

153

Synthesis of triblock copolymer methoxy-polyethyleneglycol-b-polyglutamic acid-

b-polyphenylalanine (mPEG5K-PLE5-PLF15)

The combination treatment of Docetaxel and Dasatinib has been explored and

tested for bone metastases in the clinical trials in 2013. However, apart from the well-

tolerated toxicity profile, the results were not encouraging. No significant improvement

was reported in term of the overall survival (OS) or bone associated biologic markers[37].

One reason for the inconsistency between in vitro and in vivo outcome might be the

discrepancy in pharmacokinetic profiles of both drugs[38], making it's challenging to

achieve the simultaneous drug exposure in the tumors.

In the proposed approach to enhance the anticancer efficacy, it is critical to

delivering both of the drugs simultaneously to achieve the best synergistic effect.

Polypeptide-based micellular nanoparticles have proved their capability to co-

encapsulate drug combination and transported them to the cancer cells at the same time.

To serve the purpose, mPEG5K-PLE5-PLF15 were synthesized. The polyphenylalanine

block was longer compared to the previous polymer described in Chapter II to

encapsulate DTX and Das together better, which are both hydrophobic and insoluble in

water. The chemical structures of the synthesized diblock copolymers, triblock

copolymers, and deprotected polymer were confirmed by 1H-NMR (Figure. 3.11, 3.12).

The polydispersity of the benzyl-protected polymers was characterized via GPC as

shown in (Table 3.1). The results suggested that the indicated polymers were

successfully synthesized with a low polydispersity within 1.05.

154

155

Figure 3.11: 1H-NMR (400 MHz, DMSO-d6) of synthesized block-copolymers. A) mPEG5K-PLE(OBzl)5. B)

mPEG5K-PLE(OBzl)5-PLF.

B

A

156

Figure 3.12: 1H-NMR (400 MHz, TFA-d1) of synthesized block-copolymers. A) mPEG5K-PLE(OBzl)5-PLF.

B) mPEG5K-b-PLE5-b-PLF.

B

A

157

MWw (Da) PDI

mPEG5K

-NH2 4839 1.03

mPEG5K

-PLE(OBzl) 5434 1.04

mPEG5K

-PLE(OBzl)-PLF 10542 1.05

Table 3.1: Polydispersity of synthesized polymers by gel permeation chromatography.

158

Preparation and characterization of drug-loaded micelles

The drug was loaded into the micelles through the nanoprecipitation method in

the ice-cold bath to achieve the best loading capacity as well as the narrow size

distribution. DTX-loaded micelles (DTX/m), Das-loaded micelles (Das/m), and two-in-one

combination (DTX+Das)/m were prepared separately by controlling the initial feed. The

micellar cocktail (DTX/m+Das/m) was obtained by mixing prepared DTX/m and Das/m

directly. No drug precipitation was observed during the loading procedure or after

centrifuging the formulation for 2 min at 1000g, which indicating an encapsulation

efficiency close to 100%. A small proportion (≈10% of the loaded drug) of the micelles

were found lost in the filtration through the 0.45 µm syringe filter (Table 3.2) by

comparing the quantity of drug concentration before and after the filtration. Hence the

total yield of the preparation was around 90%. Due to the high encapsulation efficiency,

the combination ratio between DTX and Das in the micelles can be precisely controlled.

Here, a 1:1 molar ratio was selected, and the total loading capacity of the (DTX+Das)/m

was 25%, consists of 15% DTX and 10% Das.

159

Drug feed per 1 mg polymer Loading capacity Filtration yield

Das DTX Das DTX

DTX/m - 0.2 mg 17% - 88%

Das/m 0.12 mg - 11% 93% -

(DTX + Das)/m 0.12 mg 0.2 mg 9% Das 15% DTX 92% 87%

Table 3.2: Yield of preparing drug-loaded micelles with DTX, Das and the combinations.

160

As summarized in Table 3.3, all drug-loaded micelles were mono-distributed with

the polydispersity index (PDI) below 0.2 and were around 35-40 nm on average. The

(DTX+Das)/m were found to be stable, and the size remained similar after being stored

at r.t. for ten weeks. Compared with the micelles synthesized in 2.2.2, the average sizes

were smaller, with the cutoff of the size distribution below 110 nm. The smaller size and

the lower cut off suggest most of the particles will be within the range that can harness

the EPR effect (discussed in 2.3).

161

Deff

mean ± SD nm PDI

Blank micelle 43 ± 1 0.19

DTX/m 34 ± 1 0.17

Das/m 42 ± 1 0.13

DTX/m + Das/m 36 ± 1 0.15

(DTX + Das)/m 35 ± 2 0.13

(DTX + Das)/m r.t. 10 weeks 42 0.16

Table 3.3: Size, PDI and distribution modal of the drug-loaded micelles.

162

As discussed previously, the simultaneous delivery of DTX and Das is the key to

success in the proposed approach. It requires a slow release during the circulation to

retain the drugs before reaching the cancer cells, and the similar release speed for both

drugs. The in vitro drug release studies in 1X PBS showed a moderate and steady

release profile that continued for more than three days, and the release rate of DTX and

Das were similar in single-drug-loaded micelles and double-drug-loaded micelles (Figure

3.13). Thus, it could be expected that the synthesized micelles have the potential to

serve our purpose of delivering both drugs at the same time.

163

Figure 3.13: In vitro release profile of DTX/m, Das/m and (DTX+Das)/m. The release continued steadily

for more than three days. Both drugs in single-drug-loaded micelles and double-drug-loaded micelles were

released at a similar rate. The cumulative release in % was calculated by normalizing the quantities of

released drugs to the total amount of the loaded drugs.

0 20 40 60 800%

50%

100%

Time (h)

Das, (DTX+Das)/m

DTX, (DTX+Das)/m

DTX, DTX/m

Das, Das/m

164

In vitro cytotoxicity of the drug-loaded micelles

The anticancer efficacy of drug-loaded micelles was evaluated in the cultured

murine breast cancer cells 4T1 in vitro. Both parent cell and DTX adapted one (pre-

treated with 10 µM DTX for three days) were used for the studies. The treatment

exposure time was limited to 24 h because it had been observed that cell might die after

exhausting the nutrients (refreshment of the medium with the same drug lead to cell

survival, 3.2.2). As shown in Table 3.4, the DTX and Das in the micelles had IC50 of 31

µM and 11 µM respectively; when given in combination, the drugs’ IC50 level of both

drugs decreased drastically to sub-micromolar concentration. The calculated

combination index (CI) was 0.107, indicating a strong synergistic effect. Not surprisingly,

the adapted cells that had been treated with 10 µM DTX developed the resistance, and

we could not observe any cell death even at 100 µM of DTX (data not shown). However,

the adapted cells became much more sensitive to the Das treatment, with one

magnitude lower IC50 (2.4 µM) in the single drug treatment group. Specifically, the

sensitivity to Das after the adaptation increased one-fold compared to the parent

counterpart (Figure 3.14). This was expected based on the studies of p-AMPK level shift

that the DTX treatment resulted in low p-AMPK response and such change made the

cell more vulnerable to Das. If we take a closer look and compare the IC50 between Das

and the combination in the adapted cells, it is noticeable that the addition of DTX did

provide an extra benefit, although in the single DTX treatment seemed already to lose all

the efficacy. It also reflects part of the hypothesis that DTX-adapted cell might be trying

to re-adapt to the secondary treatment (Das), and the existence of DTX made an

attempt unhelpful. Another point worthy to mention is that the IC50 of the combination

165

treatment remained comparable for both parent and adapted cells, which suggested the

combination might work very well not only for the patients who are chemo naïve but also

for the patients who have been treated with DTX.

166

DTX/m Das/m (DTX+Das)/m

Parent 4T1 31± 6 11 ± 0.3 0.87 ± 0.27

DTX adapted 4T1 >>100 2.4 ± 0.3 0.53 ± 0.04

Table 3.4: In vitro tumor growth inhibition of drug-loaded micelles. The absolute IC50 (µM) values were

the drug concentration to achieve 50% suppression of the tumor growth to the control. All treatment lasted

for 24 h.

167

Figure 3.14: Relative sensitivity to Das. The sensitivity increased two times after the adaptation at the

initial IC50 level.

-1 0 1 20

2

4

log [Das], µM

Rela

tive e

nsit

ivit

y

168

To test if this approach was only limited in 4T1 or could apply to other cell lines or

different types of cancer. The same experiments were repeated on human breast cancer

cell line MDA-MB-231 and human prostate cancer cell line PC-3. The results

demonstrated the same trend as well as the relation between the IC50 of combination

and single drug-treated group (Table 3.5). Both cell lines showed clear evidence of

strong synergistic effects (CI<0.1) and the retained the similar efficacy in DTX adapted

cells.

169

DTX/m Das/m (DTX+Das)/m

Parent MDA-MB-231 6.2 ± 0.7 3.2 ± 3.0 0.11 ± 0.02

DTX adapted MDA-MB-231 >>100 0.47 ± 0.23 0.14 ± 0.12

Parent PC-3 1.7 ± 1.5 12 ± 8 0.12 ± 0.18

DTX adapted PC-3 >>100 3.3 ± 0.3 0.53 ± 0.28

Table 3.5: Growth inhibition in MDA-MB-231 and PC-3 IC50 (µM). Strong synergistic effect (CI<0.1) of the

combination was demonstrated, and the same level of efficacy was retained in DTX adapted cells.

170

Sensitization of 4T1 cells to DTX or Das with 5-Aminoimidazole-4-carboxamide

ribonucleotide (AICAR) or Compound C

To clarify that the strong synergy found between DTX and Das was indeed

associated with the adaptive dynamics represented by p-AMPK level shift and was not

only due to the cytotoxic effects via two independent targets by the combinations as

reported frequently in other drug combinations. However, in this case, the cytotoxic

stress was the foundation of the adaptive dynamics, and the adaptive dynamics was

responsive to the synergy. It would not be possible to lower the concentration of the drug

to a level that does not have a cytotoxic effect but retains the ability to induce the

adaptive response and deliver synergy based on that. Thus, to separate the trait of

drugs’ cytotoxicity from the trait of the sensitization via opposite adaptive dynamics, an

AMPK activator (AICAR) and an inhibitor (Compound C) was used as the analog to Das

and DTX respectively[39]. Neither of the activator or inhibitor could inhibit the growth of

4T1 in the working concentration of 100 µM and 1 µM respectively (Figure 3.15).

171

Figure 3.15: Growth inhibition of AICAR and Compound C. Neither 100 µM AICAR (left) nor 1 µM

Compound C (right) of the molecules inhibited 4T1 growth in 24 h treatment compared to non-treated control.

Contr

ol1µ

M Com

pound C

0

50

100

150

Via

lbilit

y%

Contr

ol

100µM

AIC

AR

0

50

100

150

Via

lbilit

y%

172

The AMPK activator AICAR upregulates the AMPK level and hence to provide

the similar effect as the Das could lead to in adapted cells, whereas the inhibitor

Compound C downregulate p-AMPK and accordingly to represent the DTX. Therefore,

the AICAR was used in combination with DTX while Compound C was with Das to test

their sensitization effect on 4T1 cells for 24 h. Just as expected, the reversal of the

adaptive dynamics (AMPK activation in DTX treated cells, AMPK inhibition in Das treat

cells) remarkably increase the cells sensitivity and brought down the IC50 to or even

exceeded the comparable levels that were achieved by the drug combinations (Table

3.6). So, it is clear that the p-AMPK level shift did play a critical role in the synergistic

effect between DTX and Das.

173

DTX/m Das/m (DTX : Das)/m

Parent 4T1 31 ± 6 µM 11 ± 0.3 µM 0.87 ± 0.27 µM

Parent 4T1 + 100 µM AICAR 7.3 ± 0.9 nM - -

Parent 4T1 + 1 µM Compound C - 1.3 ± 0.2 µM -

DTX adapted 4T1 - 2.4 ± 0.3 µM 0.53 ± 0.40 µM

Table 3.6: Growth inhibition of the combination with AICAR and Compound C, IC50 µM. The sensitizing

effect was confirmed for both molecules, and the IC50 of Das and DTX reached a comparable level (or even

lower) to the combinational treatment.

174

Chemotaxis of tumor cells toward pre-osteoblast

As reviewed by Maggague, Obenauf, and Weilbaecher, the preference of tumor

growth in bone may closely related to the stem-like niches that permit the homing of the

disseminated cancer cells and provides the support for their growth[40,41]. One of the

niches where cancer cells invade to has been hypothesized to be the endosteal one

which is filled up of osteoblasts. And evidence from in vivo studies also implies that the

niche could be one of the factors that determine if the cells will populate into the

metastatic lesion or remain sleepy[42]. Dasatinib has been reported to inhibit the

migration of human sarcoma cell lines[43]. In this study, we developed a simplified

model and aimed to demonstrate the inhibitory effect of the Dasatinib in the combination

could potentially stop the tumor's invasion into the osteoblast niches. The model

comprised by a trans-well system where the pre-osteoblast cells MC3T3 with their

conditioned medium (preOB-CM) were on the lower chamber to serve as the

chemoattractant and the tumor cells 4T1 were in the upper insert with the treatments.

The preOB-CM was found more effective than the 10% FBS which is typically

considered as a robust chemoattractant. However, MDA-MB-231 did not migrate under

the same condition (Figure 3.19). It might be some intercellular reaction that was species

specific.

When treated with just 10 nM or 20 nM Das/m, the migration was substantially

inhibited, and the number of migrated cells were close to the negative (Figure 3.17). As

the dose did not affect the cell viability (Figure 3.16), the inhibitory effect was not part of

175

the growth inhibition. Representative images of the migrated cells on the downside of the

membrane were shown in Figure 3.18. The results proved that the Das/m were potent in

inhibiting the tumor chemotaxis induced by the pre-osteoblast and therefore

demonstrated the potential to benefit the anticancer therapy by interfering the

association of osteoblast niches and tumors cells.

176

Figure 3.16: Cell viability after 24 h treatment of Das/m. The concentration of Das/m used in migration

study was 10 nM and 20 nM, and the duration of the drug exposure was 12 h. Hence, they would not affect

the viability of the cells throughout the study.

0.3125 0.6250

50

100

150

[Das], µM

Via

bilit

y %

to

co

ntr

ol

177

Figure 3.17: 4T1 migration assay. Conditioned medium from MC3T3 showed substantial attractiveness to

the tumor cells, even stronger compared with the standard chemoattractant 10% FBS. Das/m demonstrated

the high potency of inhibiting the chemotaxis in a dose-dependent manner. Data was presented by mean ±

SD and analyzed by t-tests (*, P<0.05; ***, P<0.001).

MEM

MEM

+10%

FBS

preOB-C

M

Das

/m 2

0nM

Das

/m 1

0nM

0

100

200

300N

um

ber

of

mig

rate

d c

ells

***

***

*

0.3125 0.6250

50

100

150

[Das], µM

Via

bilit

y %

to

co

ntr

ol

178

Figure 3.18: 4T1 migrated through the transwell membrane.

MEM MEM+10%FBS preOB-CM

Das/m 20nM Das/m 10nM

179

Figure 3.19: Migrated MDA-MB-231 using preOB-CM. No migrated cells could be seen on the membrane.

MDA-231 with preOB-CM

180

Inhibition of the osteoclast differentiation

In Chapter II, alendronate was attached to the micelles and served as not only

the targeting ligand but also a potential therapeutic agent who showed the efficacy of

amending the "vicious cycle" by inhibiting osteoclast differentiation and bone resorption

in vitro. However, the total dose of the alendronate administered in vivo was far below

the level used in the clinic, limiting the overall outcome. Dasatinib was also reported to

impede the osteoclast formation, and the potency was much higher compared to the

bisphosphonates[44,45].

Similar to the previous experiment in 2.2.2, the Raw cells were differentiated to

osteoclast cells with the RANKL. Polymer control, free Das (30 nM) and Das/m (30 nM,

10 nM) were given to the Raw cells from the beginning of the experiment. Compared to

the positive control, the number of differentiated osteoclasts was found to be remarkably

lower in Das treated groups but remain the same in polymer only group (Figure 3.20,

3.21). And loading Das into the micellar form seemed not to comprise the potency (no

significant difference was observed between free drug and micellar group). As expected,

the inhibitory effect was dose-dependent, and the IC50 was around the 10 µM level.

These results demonstrated that Das/m were more potent than alendronate to

suppress the osteoclast differentiation and thus could be a better option for treating the

pathological bone resorption in metastases.

181

182

Figure 3.20: Das inhibited osteoclast at nanomolar concentration. Both free Das and Das loaded in

micelles significantly decreased the amount of the osteoclast. Data was presented by mean ± SD and

analyzed by t-test (*, P<0.05; **, P<0.01).

Neg

a Ctrl

Posi

Ctrl

Poly

mer

s

Fres

Das

30n

M

Das

/m 3

0nM

Das

/m 1

0nM

0

20

40

60

80

100N

um

ber

of

oste

ocla

st

*

**

**

NS

**

0.3125 0.6250

50

100

150

[Das], µM

Via

bilit

y %

to

co

ntr

ol

183

Figure 3.21: Representative image of the differentiated osteoclast under different treatment groups.

Free Das and Das/m 30 nM showed substantial cytotoxicity to the Raw cells over five days. Although Das/m

10 nM had no noticeable impact on the cell density compared to the two control groups (positive and

polymer), it still retained some inhibitory effect.

184

Anticancer efficacy in vivo

The combination micellar formulation (DTX+Das)/m and DTX/m+Das/m were

tested in the murine breast cancer bone metastases model, and their efficacy was

compared with free drug combinations as well as free DTX. As seen in the 2.3 the

disease model was aggressive. To ensure each mouse receive the same dosage of the

drugs, the treatments started as early as the bioluminescence appeared and was given

on a daily basis for four days in total. Starting from the day (day 0) before the first

treatment, the bioluminescence of the implanted tumor was monitor continuously for

seven days. Relative tumor growth (bioluminescent intensity normalized to the initial

read) of the individual mouse was showed in Figure 3.22, and the average for each

treatment group was summarized in Figure 3.23. Based on one-way ANOVA analysis

and multiple comparisons by Prism GraphPad, the free drug combinations did not

demonstrate any improvement over the single drug, however when encapsulated in

micelles, the combination significantly delayed the tumor progression. No statistical

difference could be observed between the DTX/m+Das/m and the (DTX+Das)/m groups.

Since both treatment groups shared a similar drug release profile and the particles size,

it could be reasonable to speculate the pharmacokinetics were close, which could

explain the similarity of the therapeutic outcome.

185

Figure 3.22: Tumor growth in each mouse.

0 2 4 6 80

50

100

150

200

Days

Rt/R

0

DTX/m+Das/m

(Das+DTX)/m

Free DTX + Free Das

Free DTX

Control

186

Figure 3.23: Average tumor growth of mice with different treatment. Significant growth difference was

seen at Day 7 between any of the control, free DTX, free drug combinations and any of the micellar groups.

Data was presented by mean ± SEM and analyzed by ANOVA with multiple comparison at day 7: DTX/m +

Das/m vs Free DTX + Free Das*; DTX/m + Das/m vs Free DTX***; DTX/m+Das/m vs Control8***; DTX/m +

Das/m vs Control**; (Das + DTX)/m vs Free DTX + Free Das**; (Das + DTX)/m vs Free DTX****; (Das +

DTX)/m vs Control*** (*, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001).

0 2 4 6 80

20

40

60

80

100

Days

Rt/R

0

Control

Free DTX

Free DTX + Free Das

DTX/m+Das/m

(Das+DTX)/m

187

As the disease progressed, substantial body weight loss happened for all tested

groups (Figure 3.24). Some of the mice were anesthetized to comply to IACUC protocol

and recorded as the death event. However, the weight loss seemed not associated with

the toxicity of the treatment. The cumulative dose of the DTX was 40 mg/kg, and Das

was 24 mg/kg. As the maximum tolerant doses (MTD) are 80 mg/kg for DTX and 70

mg/kg for Das, the toxicity was expected to be limited. One day after the final treatment,

three mice from each group were sacrificed, and the blood, spleen, kidney, liver and

femur/tibia were collected for histological analysis (Figure 3.25). No obvious damaged

were observed for any of the visceral organs. Apart from the toxicology, several

secondary metastases were found in the kidneys in the control group and both free drug

groups, while no metastases were found in any of the organs in the two micellar groups.

188

Figure 3.24: Body weight loss along with tumor growth. Data was presented by Mean ± SD.

0 2 4 6 8 1070%

80%

90%

100%

110%

Days

Wt/W

0

(Das+DTX)/m

DTX/m+Das/m

Free DTX + Free Das

Control

Free DTX

189

Figure 3.25: Acute toxicity study based on H&E staining of the major organs collected 24 h after the

final treatment. Pathologically, all the organs were in normal condition, indicating the treatments were

tolerated.

Kidney

Liver

Spleen

Control Free DTX DTX/m + Das/mFree DTX + Das (DTX + Das)/m

190

The survival of the mice was monitored up to thirty days. Apart from the

individuals that died naturally due to the tumor growth, some of the mice were

euthanized due to the weight loss > 20% or suffered from severe pain according to the

IACUC guidelines. Correlated with the tumor growth found previously, the survival of the

free drug-treated group barely showed any improvement over the control, while both

DTX/m+Das/m and (DTX+Das)/m demonstrate significantly prolonged survival time

compared to either control or the free drug combinations (Figure 3.26).

191

Figure 3.26: Kaplan-Meier survival curves of tumor-bearing mice following the treatments. The

significant difference was detected between free drug combinations and any of the micellar drug groups.

The median survival days were 9, 12, 8, 14, 16 for the Control, Free DTX, Free DTX + Free Das,

DTX/m+Das/m, (Das+DTX)/m respectively.

0 10 20 300

50

100

Days

Perc

en

t su

rviv

al

Control

Free DTX

Free DTX + Free Das

DTX/m+Das/m

(Das+DTX)/m

192

The bone metastases model we used, in general, has a broad distribution of the

initial tumor burden volume. The mice were evenly distributed into the ten groups and

randomly assigned to the five different treatments to mitigate any potential bias caused

by the initial tumor burden. The possible correlation between the survival time and the

initial tumor burden was also analyzed within each treatment groups. Data were fitted by

linear regression, and the slopes of the trend lines were not deviated (P>0.1, F test) from

zero and the correlation did not exist in this study (Figure 3.27). This result implies the

potential effect of uneven distribution of the animal with different initial tumor burden was

limited. After completing four-times treatment, the survival time was found to be

correlated with the tumor progression recorded at Day 5, explaining the consistency

between tumor growth and survival data. The mice with lower Rt/R0 ratio tended to

survive longer in general.

193

Figure 3.27: Correlation between initial tumor burden (bioluminescence) and the survival time within

each treatment groups. A) Trend lines were fitted by linear regression. However, none of the slopes

significantly deviated from zero. B) Data included all survived mice on Day 5. A linear correlation was

observed between survival time and the tumor growth rate (Rt/R0). The slope significantly deviates from zero

(P=0.01).

0 10 20 30 400.0

5.0×106

1.0×107

1.5×107

2.0×107

Survial time (Days)

Init

ial tu

mo

r in

ten

sit

y (

p/s

)

Control

Free DTX

Free DTX + Free Das

DTX/m+Das/m

(Das+DTX)/m

0 10 20 30 400

20

40

60

Survial time (Days)

Rt/R

0 a

t D

ay 5

A

B

194

Blood cells were counted one day and five days after the final treatment. As

listed in Figure 3.28, the tumor-bearing mice displayed a much higher level of white

blood cells (WBC) than normal healthy mice and spleen enlargement[46]. The blood

smear revealed that substantial amount (≈50%) of the unmatured neutrophils were in the

blood (Figure 3.29). Upon treatment of DTX or DTX+Das, the WBC seemed to be

eliminated drastically. However, after resting for five days without any treatment, the

number of WBCs recovered partially back to the control level. On the contrary to the

substantial change found in WBCs, the red blood cells (RBC) remained relatively stable

in all the treated groups.

195

Figure 3.28: Blood count 24 h and five days after the final treatment. WBC was considerably higher in

the tumor-bearing mice compared the standard control (STD). In drug-treated mice, the WBCs were

eliminated substantialy and partially recovered after five days.

WBC

LYM

MO

NNEU

0

5

10

40

80

Cell c

ou

nt

10

9/L

24 h after treatments

Control

Free DTX

Free DTX + Free Das

DTX/m + Das/m

(DTX+Das)/m

Healthy

WBC

LYM

MO

NNEU

0

20

40

60

80

100

Cell c

ou

nt

10

9/L

Recovery

Control

Free DTX

Free DTX + Free Das

DTX/m + Das/m

(DTX+Das)/m

Healthy

196

Figure 3.29: Representative image of the blood smear from the tumor-bearing mice. About half of the

neutrophils were unmatured.

Imm

ature

mat

ure

0

20

40

60

80

Fra

cti

on

% o

f to

tal W

BC

197

3.4 CONCLUSION

In this study, the 4T1 cells were observed to become drug-tolerant upon limited

exposure to Docetaxel. The adapted response was characterized by the exit of the cell

cycle and the formation of the polyploid cells. The adapted 4T1 showed the metabolic

dynamics of the p-AMPK that deviated substantially from the base level. Different drugs

were found to result in the different direction of the adaptive dynamics. Here, we

demonstrated that DTX-adapted cell was featured by low p-AMPK, whereas Das-

adapted by high p-AMPK. In other words, the new metabolic pattern that cells use to

survive under the chemo stress demands the p-AMPK to be low for DTX and to be high

for Das. The mechanism was further confirmed in the later study where the AMPK

activator and inhibitor restored the sensitivity to DTX and Das respectively.

Because of the opposite direction of cellular dynamics induced by DTX and Das,

the combinations of both could be a potential solution for overcoming the adaptive

resistance. The adaptation to DTX will lead to the sensitization to Das or vice versa.

And given the essentialness of the AMPK and only two possibilities of the activation level,

cells are not expected to develop anything outside of the box. The enhanced growth

inhibitory effect and strong synergy of the combination were validated in both parent and

DTX-adapted cell in vitro for all 4T1, MDA-MB-231 and PC-3.

Forty-nanometers sized micelles were synthesized to encapsulate both DTX and

Das with high loading capacity and sustained release profile. The micellar formulations

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were tested in the model of murine breast cancer bone metastases, showing significantly

improved therapeutic compared to the free DTX and free drug combinations, while

keeping the toxicity at a tolerable level.

In the previous clinical trials, the combinations were considered no more effective

than the single DTX regimen, which might be due to the pharmacokinetic difference

between the two drugs. Our study reevaluated the potential of the combinational strategy

by developing a two-in-one micellar formulation that can deliver both drugs

simultaneously. The design achieved remarkable improvement and overcame the

adaptive resistance by inducing mirrored adaptive dynamics.

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CHAPTER IV

SUMMARY AND FUTURE STUDY

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4.1 SUMMARY

Metastases in bone are the major obstacle in cancer therapy and decrease the 5-

years survival rate. No effective treatment is available in the clinic other than pain control.

The conventional monotherapy and combinational therapy only gained limited survival

advantage. As reviewed in Chapter I, the nanocarrier-based drug delivery system

provides an attracting opportunity in the treatment of bone metastases, with its versatility

in loading drug combination and tumor-targeting ability. Bisphosphonates have a long

history in osteoporosis treatments and are known to have the high binding affinity to the

skeleton, which makes it as the potential targeting ligand that can drive the chemo drug

to the bone-tumor site. Moreover, bisphosphonates exert the antiresorptive effect by

being cytotoxic to the osteoclasts and suppress their differentiation, which turns it into a

dual-functional molecule that can also strike on the tumor-bone microenvironment. As

the tumor-induced bone degradation serves a critical role in the propagation of the

"vicious cycle," the intervention of osteoclast activity would be beneficial.

In Chapter II, one of the most widely used bisphosphonate, alendronate, was

chemically linked on the surface of polypeptides-based micelles to shape the bone-

targeting carrier. DTX, one of the first-line chemo drugs in breast cancer, was

successfully loaded into the micelles with almost 100% efficiency by nanoprecipitation.

The preparation method was robust to yield high-concentrated formulation (2 mg/mL

DTX) without comprising the loading ability. The drug-loaded micelles had an average

size of 70 nm in PBS solution, small enough to be accumulated to the tumor tissue via

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EPR effect. The formulation exhibited strong binding efficacy to the HAP which is the

mineral composition of the bone tissue. Comprehensive in vitro evaluation was made

demonstrating the stability, sustained drug release profile, retained anticancer efficacy

as well as the potential to reduce the formation of the osteoclasts and suppress the

macrophage recruitment by tumor cells. In mice, DTX showed prolonged circulation time

and increased AUC when encapsulated in the ALN-m. These characteristics were

translated into the treatment for the bone metastases murine model, where the targeted

micellar formulation was injected systematically through the tail vein and significantly

increased the animals' survival time compared without compromising the safety.

Das is a tyrosine kinase inhibitor that inhibits Src family proteins and was

reported to block the EGFR receptor that is frequently overexpressed in breast cancer

cells. As EGFR serves as the initiator of the pro-survival signaling in tumor cells, the

combination of Das and DTX could be an effective strategy to improve the therapeutic

outcome in bone metastases. However, clinical trials of such combination failed in

castration-resistance prostate bone metastases patients, which could be due to the

pharmacokinetic difference between two drugs. In Chapter III, we co-encapsulated the

Das and DTX at 1:1 molar ratio in a single micelle with a high loading capacity of 35% in

total. Both drugs were released at the same rate in vitro, suggesting the similar

pharmacokinetics profile. The combination showed substantial synergist effect (CI<0.1)

in breast and prostate cancer cell lines, with the IC50 that decreased in two magnitudes

compared to the monotherapy. Chemotherapies were discovered to induce the adaptive

drug tolerance in tumor cells rapidly and non-genetically, which is clinically relevant and

vital, as it may drastically impair the treatment efficacy. Notably, the chemo stress

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imposed by Das and DTX resulted in opposite phenotype dynamics in tested cell lines.

Das-tolerant cells had a higher level of pAMPK, while DTX-tolerant cells had a lower

level. The appropriate level of pAMPK was demonstrated to be necessary for

maintaining the specific drug-tolerant status. The finding explained the observed synergy

between Das and DTX, as the tumor cells could not develop the resistance phenotype

that demands both up and downregulation of pAMPK when exposed to Das and DTX

simultaneously. Indeed, the DTX-adaptive cells which completely did not respond to the

continuous DTX exposure have the comparable sensitivity to the Das/DTX combination

as their parent cells. Besides the enhanced anticancer efficacy, Das also inhibited the

tumor migration and scavenged osteoclast in vitro. Subsequent in vivo studies further

revealed the potential of the drug combinational in micelles to delay the progression of

the disease and prolong the survival time, while keeping the regimen well tolerated.

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4.2 FUTURE STUDY

1. The alendronate binds to the bone surface of formation and resorption without

preferential. In breast cancer bone metastases, tumor cells induce osteolytic lesion

that is mainly associated with bone resorption. Whereas prostate cancer originated

bone metastases are osteoblastic, and the bone formation is dominant. More specific

ligands can be chosen to target the surface of interest selectively. For example, Asp8

was reported to bind to resorption surface, and the (AspSerSer)6 showed specificity

to bone formation area[1]. Moreover, Denosumab[2] as a monoclonal antibody is

considered as a promising candidate that has not been investigated yet. It not only

shoots to RANKL with strong affinity but also cuts off the signaling of tumor-induced

osteoclast differentiation and activity, exhibiting both targeting and therapeutic

potential.

2. Using the two chemo agents that induce mirrored adaptive response is an intriguing

strategy to develop drug combination with high synergy. However, more detailed

investigation in the transcriptional or phosphorylated level of resistant phenotypical

transition is still needed. Due to the complication in the gene regulatory network, the

direction of the adaptive dynamic is described by a massive matrix of hyper

dimension. Proteomics and mRNA profile could be adopted to help identify individual

drug candidates that induce the distinctive adaptive response in tumor cells to the

maximum extent and thus to select the most effective combination.

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3. In this project, the murine model of bone metastases was established by left-ventricle

injection. During the circulation, injected tumor cells are believed to seed them self to

the skeletons via tumor-bone interaction, mimicking the real situation. The model

allows the development of multiple-site bone metastases in the lower skeletons, skull,

ribs, spine). However, the model is highly aggressive, and the lifespan of the animals

was generally less than a month, making it difficult to arrange the dosing and

demonstrate the potential advantage of the treatments. Another model was recently

developed by Takahiro Kuchimaru[3]. Cancer cells were injected through caudal

arteries and trapped in the lower skeletons to form the bone metastases. The model

was more producible with a longer survival time of the animals and easier to handle

than the previous one achieved by intracardiac injection. Last but not least, the new

model opened up a new road to test the nanocarrier-based delivery system for bone

metastases.

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