Accepted Manuscript

Photochemical internalization (PCI) in cancer therapy; from bench towards

bedside medicine

Ole-Jacob Norum, Pål Kristian Selbo, Anette Weyergang, Karl-Erik Giercksky,

Kristian Berg

PII: S1011-1344(09)00067-0

DOI: 10.1016/j.jphotobiol.2009.04.012

Reference: JPB 8917

To appear in: Journal of Photochemistry and Photobiology B: Bi‐

ology

Received Date: 12 February 2009

Revised Date: 20 April 2009

Accepted Date: 23 April 2009

Please cite this article as: O-J. Norum, l.K. Selbo, A. Weyergang, K-E. Giercksky, K. Berg, Photochemical

internalization (PCI) in cancer therapy; from bench towards bedside medicine, Journal of Photochemistry and

Photobiology B: Biology (2009), doi: 10.1016/j.jphotobiol.2009.04.012

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Title Photochemical internalization (PCI) in cancer therapy; from bench towards bedside medicine. Authors Ole-Jacob Norum1+2

Pål Kristian Selbo1

Anette Weyergang1

Karl-Erik Giercksky 2 Kristian Berg 1

1Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. 2Department of Surgical Oncology, Norwegian Radium Hospital, and University of Oslo Corresponding author Kristian Berg, Department of Radiation Biology, The Norwegian Radium Hospital, N-0310 Oslo, Norway E-mail: kristian.berg@rr-research.no Phone: +47 40873549 / +47 22934260 Fax: +47 22934270

Abstract

PDT in cancer therapy has been reviewed several times recently and many published reports

have been showing promising results. The clinical approvals for PDT include curative

treatment of early or superficial cancers and palliative treatment of more advanced disease.

Still PDT has yet to become a widely used cancer treatment. This may partly be due to

limitations in current PDT regimens and partly due to effective alternative treatment

modalities. If the specificity and selectivity of PDT could be improved, PDT would probably

make substantial progress and comprise an even more competitive alternative in cancer

treatment.

The PCI technology is based on the same principles as PDT, the activation of a

photosensitizer by light and subsequently followed by formation of reactive oxygen species.

Unlike PDT, the photosensitizer used in PCI has to be located in the endocytic vesicles of the

targeted cells and will, upon activation of light, induce a release of endocytosed therapeutic

agents after a photochemically induced rupture of the endocytic vesicles. The endocytosed

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therapeutic agent will then be released and may reach their intracellular target of action before

being degraded in lysosomes. This site-specific drug delivery induced by PCI will take place

in addition to the well described cytotoxic, vascular and immunostimulatory effects of PDT.

PCI has been shown to facilitate intracellular delivery of a large variety of macromolecules

that do not otherwise readily penetrate the plasma membrane, including type I ribosome-

inactivating proteins (RIPs), RIP-based immunotoxins, genes and some chemotherapeutic

agents.

Several animal models have been used for in vivo documentation of the PCI principle and

more animal models of clinical relevance have recently been utilized for addressing clinical

issues. This review will focus on the possibilities and limitations offered by PCI to overcome

some of the challenges recognized in current PDT regimens in cancer treatment.

Key words Photochemical internalization, photodynamic therapy, phthalocyanine, bleomycin, cancer therapy. Abbreviations AlPcS2a Disulfonated aluminum phthalocyanine with the sulfonate groups on adjacent

phthalate rings AlPcS4 Tetrasulfonated aluminum phthalocyanine ALA 5-aminolevulinic acid BLM Bleomycin BPD-MA Benzoporphyrin derivate monoacid EGF(R) Epidermal growth factor (reseptor) CE-MRI Contrast-enhanced magnetic resonance imaging HpD Hematoporphyrin derivate m-THPC Tetra (m-hydroxyphenyl)chlorine PCI Photochemical internalization PDT Photodynamic therapy RIP Ribosome inactivating protein

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1. Introduction

Photodynamic therapy (PDT) is a treatment modality that takes advantage of the cytotoxic

effects induced by a photosensitizer and light in the presence of oxygen [1]. The therapeutic

effect of PDT may be due to direct cytotoxicity (i.e. damage of cell membranes), vascular

damage, and inflammatory and immunological responses [1,2]. The relative importance of

these pathways to therapeutic effects depends on the tissue oxygenation, photosensitizer

formulation, distribution and light dosimetry. In this review we will discuss the therapeutic

possibilities and limitations of PDT and the potentials in the photochemical internalization

(PCI) technology to overcome some of the limitations of PDT.

1.1. PDT in cancer treatment

PDT in cancer treatment has been presented in several recent reviews [1,3-6]. PDT has the

advantage of good cosmetic results [3], low mutagenic potential [7] low systemic toxicity

except for skin photosensitivity, no cumulative or long term toxicity [8] and not inducing

resistance to chemotherapy or radiotherapy [9]. PDT is used on a regular basis in treating non-

melanotic skin cancers like superficial basal cell carcinoma, actinic keratosis and Bowen’s

disease (squamous cell carcinoma in situ) using the photosensitizer precursors 5-

aminolevulinic acid and methyl aminolevulinate (Levulan® / Metvix®). The cure rate is high

and the side effects are favorable compared to alternative treatments (e.g. surgery) [3]. These

lesions are ideal for clinical PDT treatment since they are available for topical administration

of the photosensitizing compounds, resulting in no systemic phototoxicity, and they are early

cancers or precancerous lesions with little or no metastatic potential. Another superficial and

early cancer that may be suitable for standard PDT treatment is carcinoma in situ of the

esophagus with a reported 89% complete remission rate after PDT [10]. The clinical outcome

seems independent of the photosensitizer used (m-THPC, HpD and Photofrin II). Similar rates

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of complete remission were obtained with ALA-PDT [11], with no complications observed.

Fistulas, perforations and stenosis are reported with chemically synthesized photosensitizers

exposed to red light, although, this is mainly prevented by using green light [10]. The

treatment of Barrett’s esophagus and early esophageal cancers will have to compete with the

therapeutic safety and costs benefit analysis of endoscopic mucosal resection and

radiofrequency ablation reporting even higher cure rates than PDT [12]. There is a need for

randomized trials to define the preferred treatment method, or combination of methods, of the

patient population with these lesions. Similarly, there are unanswered questions concerning

the optimal treatment of early lung cancer [13]. Local treatment options like laser therapy,

electrocautery, brachytherapy and cryotherapy and PDT will all need to withstand the

comparison with surgery. PDT has the advantage of better specificity and lower complications

rates compared to the other modalities. PDT is a good alternative in patients not eligible to

surgery due to co-morbidity or previous contralateral lung resection. Currently there are

clinical PDT approvals for treatment of early lung and esophageal cancer, superficial gastric

cancer, carcinoma in situ of cervix and vulva and recurrent superficial bladder cancer based

on Porfimer Sodium (Photofrin®) as photosensitizer [3]. One of the drawbacks of utilizing

Photofrin, however, is the long lasting skin photosensitivity. PDT is clearly suitable for

precancerous and early non-invasive cancers where PDT may be expected to result in high

cure rates. However, accurate staging is a prerequisite for predicting the clinical outcome of

PDT as more advanced disease yields lower complete remission rates [10,11].

PDT has also proven its suitability in palliative cancer therapy, especially in recanalization of

near obliterated tubular anatomical structures accessible via endoscope like the bile ducts

[14], bronchi [15] and esophagus [4] and for palliative treatment of head and neck cancer with

temoporfin (m-THPC / Foscan®) [16]. PDT is also an alternative when the patient does not

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accept or tolerate surgery, ionizing radiation or chemotherapy due to anatomical

considerations, cumulative toxicity, comorbidity or when existing treatment modalities are

associated with mutilation or very high complication rates (e.g. surgery for head and neck

cancers or in case of esophagectomy). PDT has received attention as a potential treatment

modality for achieving local control in a variety of advanced malignant tumors of the

esophagus, bile ducts, pancreas, colon, rectum, carcinomatosis, bronchi, lung, pleura, head &

neck, connective tissue (sarcomas) and brain among others [17-24]. Many patients have been

treated in numerous small nonrandomized trials, but more data from trials with sufficient

statistical power demonstrating superiority to currently available treatment methods, is needed

to change clinical practice in larger scale, although there are some promising results from

smaller clinical studies [25,26]. The reason for PDT not being standard treatment in several

cancers may however be multifactorial; lack of coordinated randomized controlled trials, lack

of photosensitizer specificity and potency and practical challenges in optimizing the PDT

regime. The PDT regimens consist of a sequence of drug and light of which the individual

doses, administration form, irradiance and timing will have influence on the treatment effect

and hence the clinical outcome. Optimizing these parameters is difficult, expensive and time

consuming. PDT may also be limited by reduced penetration of light through the target tissue

and local acute phototoxicity to normal surrounding tissue.

PDT has been explored as an adjuvant treatment after debulking surgery with promising, but

not convincing results. Multiple Photofrin- and Foscan-based phase I and phase II clinical

trials with PDT after cytoreductive surgery [18-20,27-29] have been reported. In general,

maximum tolerated dose in these clinical trials is limited by treatment related side effects on

normal tissue. A problem using PDT after debulking surgery of peritoneal carcinomatosis has

been excessive fluid shift with volume overload has [19]. Moreover, PDT after surgical

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debulking of non-small cell lung cancer and malignant pleural mesothelioma in thoracic

surgery has resulted in esophageal perforations and bronchopulmonal fistula [17,18,29].

Regarding neurosurgery, the post treatment edema after PDT of brain tumors is potentially

limiting and has been challenging [20]. These clinical side effects have been tentatively

overcome by giving fluid resuscitation, anti-inflammatory drugs, adjusting the drug light

interval, and most of all by reducing the light and drug doses. In general, these clinical trials

have not been successful in achieving complete local responses. Many studies conclude that

there is a need for a more specific treatment effect either by adjusting current treatment

regimes, modifying the existing photosensitizers or developing new and more specific

photosensitizers. The reasons for treatment failure of PDT as an adjunct to surgery are

considered to be of critical importance and will be further discussed (see 4.2).

In summary, PDT is ideal for treating superficial and early cancers with little or no metastatic

potential, accurate staging being a matter of utmost importance and in many cases the

therapeutic ratio needs to be increased. The aims of palliative treatment are relief of symptom

and local control, however sustained local tumor control has not been achieved for most of the

suggested PDT indications. We hypothesize that the PCI technology may improve the results

of many already approved indications for PDT and possibly be able to obtain significant

clinical results where PDT has been undeceive. In addition to being a “fortified PDT” version,

PCI is also functioning as a system for site-specific drug delivery, opening up for new and yet

unmet needs in targeted cancer therapy not optimally addressed today (e.g. delivery of

immunotoxins and nucleic acids for gene therapy).

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2. Photochemical internalization (PCI)

Photochemical internalization (PCI) is a technology based on the same principles as PDT and

sharing many fundamental characteristics [30]. With PCI we have found the same three main

effect mechanisms to eradicate tumor cell as reported for PDT: direct cytotoxic [31], vascular

starvation [32] and a possible activation of the immune system (OJ Norum, K Berg

unpublished results). In addition PCI is introducing a fourth novel effect [30]; photochemical

release of the content of the endocytic vesicles into the cytosol in a functionally active form as

described in more detail below.

2.1. Endocytosis of macromolecules and photosensitizers

Mammalian cells have several different ways of taking up and internalize extracellular

substances, one of them being endocytosis. Most mature cells exert endocytic activity (except

mature erythrocytes), but the rate of endocytosis varies. Endocytosis involves internalization

of the extracellular substances through invagination of the cell membrane, forming an

intracellular bud, resulting in an endocytic vesicle containing the originally extracellular

substances inside the vesicles. In this way, cells can take up substances that do not readily

pass the plasma membrane. In general, molecules taken up by endocytosis are rapidly

processed and directed to other organelles, if not destined otherwise they are subjected to

enzymatic degradation [33]. Therapeutic agents that target intracellular sites have to penetrate

the plasma membrane or escape from the endocytic vesicles after endocytosis to accomplish

their therapeutic effect. Membrane impermeable molecules of therapeutic interest without any

alternative escape mechanism are consequently trapped in the endocytic vesicles (endosomal /

lysosomal compartments) and degraded [34]. There is probably a number of compounds that

have been excluded from further clinical development due to inadequate penetration through

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the plasma membrane and consequently low cytosolic concentration, in early pharmaceutical

screening experiments.

Some photosensitizers are for various reasons unable to penetrate the plasma membrane and

will enter the cells through endocytosis (Fig. 1). The most hydrophilic photosensitizers, such

as AlPcS4 and uroporphyrin do not efficiently intrude into the plasma membrane and will be

taken up into the cells by pinocytosis, which is an uptake mechanism of low efficacy.

Amphiphilic photosensitizers intruding into the plasma membrane, but unable to penetrate

through the plasma membrane will enter the cells via adsorptive endocytosis, which is a

substantially more efficient uptake mechanism than pinocytosis. These photosensitizers may

be incorporated into the outer leaflet of the plasma membrane and consequently ending up in

the inner leaflet of the endocytic vesicles as indicated by the localization of the disulfonated

photosensitizer in Fig.2. Photosensitizers may also be taken up by endocytosis through

receptor-mediated endocytosis when linked to targeting moieties such as antibodies or

exogene ligands. In all these cases the photosensitizer may reach the endosomes and

lysosomes. An alternative route for photosensitizers to be localized in endocytic vesicles is

directly through the plasma membrane and the endocytic membranes if the photosensitizer

contains lysosomotropic weak base properties, such as acridine orange, that cause the

photosensitizer to be trapped in the acidic compartments of late endosomes and lysosomes

(Fig. 1).

2.2. Activation of photosensitizers in PCI

The most efficient photosensitizers for use in PCI have an amphiphilic structure (Fig. 2) with

a hydrophilic part inhibiting penetration through cellular membranes as illustrated in Fig. 3.

As described above the amphiphilic photosensitizers will accumulate in the inner leaflet of the

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endocytic vesicles. As for PDT, reactive oxygen species are formed when the photosensitizer

is exposed to light (Fig. 3). Singlet oxygen (1O2), assumed to be most important reactive

oxygen species in PDT, has a short range of action (20-100 nm,[35-38]) and is more efficient

in the membranes than in an aqueous environment. It should be noted that hydrophilic

photosensitizers located in the matrix of the endocytic vesicles are not able to induce any PCI-

effect, pointing to the importance of the intravesicular localization of the photosensitizer [39].

The chemical reactions induced by 1O2 leads to disruption of the endocytic vesicle and release

of its contents (Fig. 4). Although the exact structure of the damaged vesicles has not been

revealed, results from previous studies indicate that relatively large particles can escape the

vesicles after PCI [40]. It was initially thought that the photosensitizer and macromolecules

to be released from the endocytic compartments had to be located in the same compartments

at the time of light exposure. However, it has been demonstrated that the photochemical

treatment can be performed up to 6-8 hours prior to the delivery without reducing the

synergistic effect of the combined treatment [40]. The assumed mechanistic basis for such an

observation is that newly formed vesicles are able to fuse with photochemically damaged

vesicles.

3. Possibilities and limitations of photochemical delivery of therapeutic agents

Therapeutic agents for photochemical delivery may be divided into four main groups: (I)

proteins, (II) nucleic acids, (III) synthetic polymers for delivery of chemotherapeutic agents

and (IV) other molecules that do not easily penetrate a cell membrane and therefore are

inhibited from entering the cellular cytosol. A prerequisite for PCI is that the molecule of

interest is accumulated in endocytic vesicles at some stage in the process. The environment of

the endocytic vesicles may set limits to the therapeutic effect of PCI in that the hydrolytic

enzymes in the endocytic vesicles may degrade the therapeutic agent. Furthermore, the

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reactive oxygen species formed by the photochemical treatment may inactivate the therapeutic

agent by an oxidizing process. The quantum yield for inhibition of the therapeutic activity of

the molecule will depend on its 3-D structure, i.e. the access of reactive oxygen species to

amino acids or guanine of importance for the biologic activity. In addition, the distance

between the photosensitizer and the molecule to be delivered, will have impact on the rate of

inactivation. Finally, the rate of endocytosis, and hence the uptake of the desired therapeutic

agent, varies between different cell types. It should be noted however that 60% horseradish

peroxidase taken up into endocytic vesicles has been released into the cytosol in a functionally

active form after PCI [41]. This indicates that photochemical inactivation of therapeutic

macromolecules may be of marginal importance.

In summary: the success of PCI in inducing release of functionally active remedies from an

endocytic vesicle may depend on (I) the hydrolytic activity of the endocytic vesicles, (II) the

sensitivity of the molecule to the photochemical treatment and (III) the endocytic activity of

the target cells. The present status on the use of PCI for increasing the biological activity of

various substances and the PCI effect observed in different cell lines will be reviewed.

3.1. PCI of proteins

Ribosome-inactivating protein toxins (RIPs) have extensively been evaluated for use in cancer

therapy and other diseases [42], and has been used in the PCI technology because of its

general therapeutic potential. RIPs kill cells by inhibiting protein synthesis through

inactivation of 28S ribosome. The 28S RIPs we have been using are plant toxins taken up by

endocytosis. PCI has been shown to release the type I RIPs (like saporin and gelonin, small

proteins of about 30 kDa) from endocytic vesicles and to activate their cytotoxic effect

[31,43,44]. Type I RIPs de novo has minimal therapeutic effect due to their low escape rate

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from the endocytic vesicles after endocytosis. With PCI of type I RIPs, a curative rate of 60-

80% has been obtained in animal models, while similar photochemical treatment without

protein toxin injection (i.e. PDT) induced only 0-10% complete response [31,45]. However,

these studies have not been optimized with respect to maximum therapeutic effect and even

higher cure rates may be obtained.

3.2. PCI of targeted proteins, increasing the specificity and cytotoxicity

Side effects of therapeutic agents often limit the treatment dose. It might be feasible to

minimize the adverse effects through drug delivery modalities that reduce the drug uptake by

non-target cells and selectively deliver the drugs to the cancer cells at low extracellular

concentrations. A current approach to site-specific drug delivery is to conjugate the

therapeutic agent to a carrier recognized only by the cells where the pharmacological action is

desired. This will provide additional specificity to the already exciting dual specificity of PDT

and PCI arising from the tumor cell retention of photosensitizer and their biological activities

restricted to illuminated areas.

The uptake and specificity of protein toxins, like type I RIPs, may be enhanced by means of

targeting moieties such as monoclonal antibodies (i.e. immunotoxin) or growth factors (e.g

epidermal growth factor, EGF). On the cellular level, enzymatic degradation of such targeting

toxins in the endocytic vesicles is a major hindrance for their therapeutic effect [46]. We have

used gelonin conjugated to the monoclonal antibody MOC31, which targets an epithelial

antigen (EpCAM/EGP-2) that is expressed on nearly all types of carcinoma cells. After PCI-

induced release of the immunotoxin MOC31-gelonin, a >10-fold increase in cytotoxicity as

compared to PCI of gelonin was observed [47]. PCI of MOC31-gelonin was efficacious

against several different carcinoma cell lines (lung (H-146), breast (T47D) and colon (WiDr

and KM20L2)), demonstrating that PCI of immunotoxins has the potential to become a potent

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anti-cancer application. However, it was later found, by means of confocal microscopy, that

most of Alexa488-labelled MOC31-gelonin was bound to the cell surface and was not

endocytosed (data not published) after overnight incubation. Due to the low penetration rate

of immunotoxins through solid tumours, it is of outermost importance to select targeting

ligands, which are efficiently endocytosed by the tumour cells. Epidermal growth factor

receptor (EGFR) is overexpressed in many different types of solid tumours and is associated

with metastasis and poor prognosis [48]. Thus, both antibodies and tyrosine kinase inhibitors

against EGFR are used in treatment of several cancers [49]. After activation by a ligand such

as EGF, the EGFR-complex is rapidly endocytosed and may therefore be a good candidate

target for efficient endocytosis of therapeutic agents. EGF linked to the type I RIP saporin,

has been evaluated for its therapeutic potential as an affinity toxin in combination with PCI.

The results indicate that EGF-saporin is efficiently endocytosed and that PCI of EGF-saporin

improves both efficacy and specificity as compared to PCI of saporin [50]. Improvement of

efficacy has also been obtained by linking Cetuximab to saporin. Cetuximab is a monoclonal

antibody recognizing the ligand binding extracellular domain of the EGFR [51].

Immunotoxins and affinity toxins like MOC31-gelonin, EGF-saporin and Cetuximab-saporin

represents highly toxic targeted molecules with good therapeutic potential in combination

with the PCI technology.

3.3. PCI of nucleic acids

PCI in gene delivery has recently been reviewed in detail [52] and will only briefly be

described here. Gene therapy is recognized as having the treatment potential for a wide range

of diseases [53]. With most gene delivery vectors, the therapeutic gene is taken into the cell

by endocytosis, and for many of these vectors the efficient mechanisms for translocating the

gene out of the endocytic vesicles turns out to constitute a major hindrance for realization of

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the therapeutic potential. Photochemical internalization has been studied as a gene delivery

technology mainly by using reporter genes, but has also been shown to induce the delivery of

therapeutic genes [54]. The photochemical translocation of nucleic acids into cytosol has been

shown to enhance the transgene activity both when non-viral, adenoviral and adeno-assisiated

viral vectors are used. PCI has also been shown to enhance gene silencing by means of

peptide-nucleic acids and siRNA [20,55,56]. As shown for PCI of protein toxins, PCI of

targeted gene delivery vectors improves the specificity and efficacy of the transgene delivery

[57].

3.4. PCI of chemotherapeutic agents

Some chemotherapeutic agents accumulate in endocytic vesicles probably due to their size

and/or charge. Bleomycin (molecular weight about 1,4 kDa) is a widely used

chemotherapeutic agent for several types of cancer (e.g. testicular cancers, lymphomas and

germinal cell tumor of children), and known to accumulate in endocytic vesicles [58]. PCI has

been shown to improve its cytotoxic effect both in vitro and in vivo [59], indicating that not

only large macromolecules like proteins and genes may profit from a PCI-based treatment, but

also smaller molecules unable to penetrate the biological membranes efficiently. PCI has the

potential to reduce the administered dose of bleomycin, and hence the dose dependent side

effects, without losing clinical efficacy. These results warrant evaluation of PCI of BLM in

future clinical PCI protocols. PCI may also reverse resistance to weak base chemotherapeutics

such as doxorubicin when resistance is caused by lowered pH in endocytic vesicles [60]. The

specificity of small molecular chemotherapeutic agents may be improved by linking to

polymers. PCI has been shown to enhance the therapeutic efficacy of such complexes [61,62].

3.5. The PCI effect on different cell lines

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In addition to the ability of activating a plethora of therapeutic molecules accumulating in the

endocytic vesicles, it should be noted that the PCI technology induces biological effects in a

variety of cells and tissue types – both malignant and non-malignant cells. So far PCI of

gelonin, saporin, genes and bleomycin has been demonstrated in more than 30 cell lines and

documented in vivo with the same macromolecules in xenografted nude mice and allografted

immuno-competent mice using 6 different cell lines (WiDr, TAX-1, HT1080, CT26, THX ,

DU145) [61,63,64]. Since all mammalian cells, except mature erythrocytes, exert endocytotic

activity, the potential of the PCI technology is not restricted to specific indications, but may

be utilized in all cancers suitable for PDT as well as non-malignant diseases.

4. PCI in translational research

The PCI technology and its diversity have been described above. PCI is currently approaching

bedside medicine and we will review recent studies of assumed clinical relevance and the

possible clinical implications in the following paragraphs.

4.1. PCI for treatment of invasive cancers and as adjunct to surgery

The infiltrative growth of many cancers frequently results in local recurrences and is therefore

a major challenge in cancer treatment. All the in vivo PCI reports reviewed above are based

on subcutaneously located tumors in mice. However, PCI was found equally efficient on a

orthotopic, deep and invasive HT1080 fibrosarcoma model relevant for evaluating the clinical

potential of the PCI technology [32]. As in the subcutaneous tumor models PCI of BLM was

found to retard tumor growth significantly more than PDT (Fig. 5) and acting in a synergistic

manner as compared to the expected sum of BLM and PDT. Contrast enhanced magnetic

resonance images (CE-MRI) obtained two hours after PDT and histological sections seven

days after PDT both indicated that the tumor periphery responds differently from the central

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region after PDT in accordance with recent report on BPD-MA-based PDT [65]. The CE-MRI

indicated that PDT induced inhibition of perfusion throughout the tumor, except in the tumor

periphery where the tumor regrowth was confirmed by histology. The link between these

observations has not been unequivocally confirmed, but surgical removal of the central tumor

indicates low sensitivity of the tumor periphery to PDT (OJ Norum, K berg, unpublished

results). In contrast, PCI of BLM in the cavity after inadequate (R1) surgical resection of the

tumor induced a strong delay in tumor growth (Fig.6). Our results indicate that the limited

success of PDT as an adjuvant to surgery may be due to lower PDT sensitivity of the

infiltrative transitional zone left after cytoreductive surgery. We therefore anticipate that the

synergistic effect of AlPcS2a-PDT and BLM (i.e PCI of BLM) after cytoreductive surgery is

due to the internalization of BLM and that PCI in this study acts as an effective drug delivery

system in which BLM is released to the cytosol where it can reach its target and inhibit tumor

growth. The results also indicate that the photosensitizer is taken up by the remaining tumor

cells after surgery, but for reasons that are not fully understood is not able to induce detectable

photocytotoxicity. It is however not clear whether the enhanced treatment effect observed by

PCI is due to targeting of the parenchyma or endothelial cells of the tumor. The results may

indicate that PCI offers a new possibility as an adjuvant to surgery more effective than PDT.

4.2. PCI as adjunct to radiotherapy

Ionizing radiation has continuously been refined through many decades and is a generally

accepted standard in both curative and palliative therapy for a variety of cancers. In general

the therapeutic effect of ionizing radiation is via DNA damage while PDT mainly affects the

cellular membranes [8]. Previously published in vitro and in vivo studies on the interaction of

ionizing radiation and PDT have shown contradictory results, ranging from synergistic to

antagonistic effects [66-71]. In an athymic mouse model using a transplantable fibrosarcoma

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TAX-1, PDT and PCI were found to interact in an additive to antagonistic manner when

combined with ionizing radiation (OJ Norum, ØS Bruland, K Berg unpublished results).

However, PCI as neoadjuvant treatment to ionizing radiation induced a prolonged time to

progression as compared to PDT (Fig 7). The effect of BLM in combination with ionizing

radiation would be increased by higher cumulative doses of both fractionated BLM and

ionizing radiation [72,73]. However, there are animal and clinical reports showing that

increasing the BLM or ionizing radiation doses in radiochemotherapy will increase the side

effects to unacceptable levels [72-75]. PCI of BLM will increase the therapeutic effect of

BLM and hence lower the needed drug dose. When the PCI of BLM and ionizing radiation

were separated in time by one week, no side effects were observed. Thus, PCI of BLM

appears as a safe neoadjuvant to ionizing radiation exerting therapeutic advantages and may

be considered as a treatment modality to preceed a standard radiotherapy regimen.

4.3. PCI and the immunological response

While radiotherapy, chemotherapy and major surgery [76] predominantly evoke an

immunosuppressive response in the patients, PDT is reported to cause a strong local

inflammation leading to activation of the systemic immune system in the host [77]. The

immune response seems important for both the immediate tumor cell eradication and for the

long term tumor control [78]. Knowledge on PDT and immunity is manly obtained from

preclinical studies, however a few clinical reports [79,80] provide indications that the

immunostimulatory effects initiated by PDT may also influence on the clinical outcome of

PDT. Still much need to be done to close in on the optimal PDT regimen [81] and the

development of a clinically useful PDT tumor specific whole-cell cancer vaccine. One

possible great advantage of the potential of a PDT generated vaccine is that no immuno-

adjuvants are needed and that the vaccine may be developed in situ [82].

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In experimental models PDT has been proven to evoke an acute inflammation and activating

the immune system in immuno-competent mice to a degree that facilitates tumor specific

resistance if a re-challenge with the same cell line is administrated [82-84]. The vaccination

ability of PDT appears independent of the photosensitizer [83]. Accordingly, AlPcS2a-PDT

has been found to induce a similar inhibition of growth of CT26.CL25 mouse colon

carsinoma cells when immuno-competent BALB/c mice previously had been subjected to

curative PDT. PCI of BLM appears equally efficient in inhibiting regrowth of CT26.CL25

tumor cells as PDT (Fig. 8). Thus, PCI does not seem to be a hindrance for the immunological

activation and development of a cancer vaccine (unpublished, OJ Norum, K. Berg). A

possible vaccinating response to PDT and PCI would develop these treatment modalities from

aids to local cancer control to a curative endpoint. However, clinical documentation is still

sparse and in the case of PCI the influence of the macromolecule to be delivered on the

immune response among others need further evaluation.

5. Future perspectives

In this review, we focused on PCI as a potential treatment modality in cancer care. PDT is

today approved for treating superficial, precancerous or early malignancies with little or no

metastatic potential, but despite promising and impressive results from many clinical trials

PDT is only regarded as standard therapy within a limited number of malignancies.

PDT is challenged by alternative treatment methods (open or endoscopic surgery,

radiofrequency ablation, cryotherapy, radiotherapy, chemotherapy among others) and for PDT

to succeed, PCI may contribute with increased therapeutic efficacy and specificity to the

photochemical treatment. It has been demonstrated that PCI is able to increase the depth of

necrosis and that the tumor periphery seems to be more susceptible to PCI treatment than

PDT. Thus, PCI may be more effective than PDT and by combination with appropriately

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targeted therapeutic agents it should be possible to enhance specificity and lowering the total

PDT dose, but still increasing the therapeutic efficacy.

In palliative cancer care aiming at prolonged local control, PDT has achieved some

impressive results (e.g. cholangiocarcinoma). PCI should be applicable for the same

indications assumed for PDT and warrant further studies. In case of PCI in combination with

surgery, our study demonstrates that the potential of synergistic achievements by means of

PCI may be superior to PDT.

It is important to emphasize that the current phase I study on PCI in humans is carried out

with bleomycin. Although PCI of BLM has shown promising synergistic effects, it is to be

expected that PCI of targeted therapeutics will further enhance the specificity and efficacy of

this treatment modality. The PCI technology may be regarded as a “fortified PDT”, but

perhaps more important is that the PCI technology is able to activate a plethora of potentially

biological active molecules that are today not in clinical use because they have intracellular

targets and do not readily pass the plasma membrane. With an ideal therapeutic agent, having

low biological activity without PCI, and high toxicity once released in cytosol, the PCI

technology may function as an efficient drug delivery system. In addition to the well-known

PDT-related cytotoxic mechanisms, this may be utilized to obtain better clinical results.

Acknowledgements

This study has been financially supported by the Norwegian Radium Hospital Research

Foundation, Norwegian Research Council and the Norwegian Cancer Society.

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Figure legends

Fig 1. Intracellular transport pathways for photosensitizers to endocytic vesicles.

Photosensitizers (P) may enter endocytic vesicles through 1) pinocytosis (Pp), e.g. in case of

hydrophilic photosensitizers and photosensitizers linked to hydrophilic moieties; 2) adsorptive

endocytosis (Pa), e.g. in case of amphiphilic photosensitizers; 3) receptor-mediated

endocytosis (P-λ) when the photosensitizer is linked to a ligand with affinity for a plasma

membrane receptor or 4) trapped in acidic vesicles when the photosensitizer exerts

lysosomotropic weak base properties (P-H+).

Fig 2. Proposed localization of sulphonated photosensitizers used in the endocytic

vesicle.

The figure shows a schematic drawing of an endocytic vesicle and the main localization of

tetrasulfonated (tetra/4-sulfonatophenyl)phorphine, TPPS4) and two amphiphilic disulfonated

photosensitizers (tetraphenylporphine disulfonate, TPPS2a) and aluminium phthalocyanine

disulfonate (AlPcS2a). The drawings are not in scale. AlPcS2a and TPPS2a are amphifilic

photosensitizers and are first adsorbed into the plasma membrane. By endocytosis the

photosensitizer is then transported into the cell and kept localized in the membrane of the

endocytic vesicle. TPPS4 is not localized in the membrane, but in the matrix and taken up by

pinocytosis.

Fig 3. Schematic representation of photochemical internalization (PCI).

The figure illustrates an endocytic vesicle. In photochemical internalization (PCI),

photosensitizer molecules and a therapeutic agent (a macromolecule) accumulate in the

endocytic vesicles as indicated. The most efficient photosensitizers for PCI are mainly located

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in the membranes (a) of the vesicle as illustrated. Upon illumination (b) reactive oxygen

species, mainly singlet oxygen, are formed. Constituents of the membranes are destroyed and

the passage of the therapeutic agent to cytosol becomes possible (c).

Fig 4. Schematic illustration of the PCI process.

The figure illustrates the photosensitizer (S) and the therapeutic agent (D) is endocytosed by

the cell. Endocytosis is illustrated with an invagination of the plasma membrane where both

compounds end up in the endocytic vesicle named endosome. In absences of light the

endocytic vesicle will degrade or recycle its content in the lysosomes. In the presence of light,

the membranes will rupture and release the therapeutic agent to cytosol where it can reach its

target.

Fig 5. Growth curves of HT1080 fibrosarcoma xenograft in mice subjected to PDT or

PCI of bleomycin.

The tumor was located intramuscular in the lateral gastrocnemius muscle. The various

treatments are indicated on the figure; i.e. BLM, bleomycin only; PDT, AlPcS2a and light;

PCI, AlPcS2a, BLM and light. The photosensitizer AlPcS2a (10 mg/kg) and bleomycin (1500

IU) was administrated intraperitoneally 48 hours and 30 minutes prior to light exposure

respectively. The tumors were illuminated with a diode laser emitting at 670 nm (90 mW/cm2

and 30 J/cm2). The tumors were 80-150 mm3 at the day of illumination. The tumor size was

measured five times per week and the tumor volume (V) was calculated using the following

formula: V = (W · W · L) ⁄ 2 where W (width) is the shorter and L (length) is the longer of two

perpendicular diameters, measured with a caliper. The figure shows the synergistic effect of

PCI on tumor growth as compared to the individual treatments, i.e. BLM and PDT.

Fig 6. The treatment effect of HT1080 Fibrosarcoma xenograft in mice subjected to PDT

or PCI of bleomycin in combination with surgery. The tumor was located intramuscular in

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the lateral aspect of the femoral quadriceps muscle and the various treatments are indicated on

the figure. The photosensitizer AlPcS2a and bleomycin was administrated intraperitoneally

respectively 48 hours and 30 minutes prior to light exposure. The tumor bed was illuminated

with a diode laser emitting at 670 nm (90 mW/cm2 and 15 J/cm2) immediately after marginal

surgical resection of the tumor. The bars represent mean tumor volume and standard error on

day 20 after treatment. The tumors were 80-150 mm3 at the day of illumination. The tumor

size was measured five times per week and the tumor volume (V) was calculated using the

following formula: V = (W · W · L) ⁄ 2 where W (width) is the shorter and L (length) is the

longer of two perpendicular diameters, measured with a caliper. The figure shows the lack of

effect of PDT as an adjuvant to surgery in contrast to PCI resulting in a significant growth

delay in animals subjected to surgery.

Fig 7. The treatment effect of combining PDT and PCI with ionizing radiation.

The effect of combining PDT and PCI with ionizing radiation was assessed in TAX-1

sarcoma xenograft by determining the time for the tumors to double in volume after treatment.

The individual treatments are indicated on the figure. IR indicates ionizing radiation.

Photochemical treatment was carried out with AlPcS2a (10 mg/kg) and a diode laser emitting

at 670 nm, (90 mW/cm2 and 20 J/cm2) with or without administration of 1500 IU of

bleomycin 30 min prior to the light exposure. PCI + IR and PDT + IR indicate ionizing

radiation 7 days after the photochemical treatment. In all treatment regimens, except for IR

only, the cure rate was 10-20%. These animals are excluded from the figure. The figure

illustrates the additional treatment effect gained with neoadjuvant PCI before ionizing

radiation. The bars, with standard error, represent mean time to reach double tumor size.

Fig 8. The treatment effect of PDT and PCI of bleomycin on the CT26.CL25 tumors in

thymic and athymic mice.

PDT and PCI were performed in Balb/c nude/nude (athymic) and Balb/c (thymic) mice and

the treatment effects assessed 20 days after light exposure. The individual treatments are

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indicated on the figure. The bars represent the number of animals that did not reach the

endpoint of 1000 mm3 at day 20. PDT 30 indicates a treatment regimen with photodynamic

therapy using the photosensitizer AlPcS2a (10 mg/kg) and a diode laser emitting at 670 nm,

(90 mW/cm2 and 30 J/cm2). PDT 15 similarly represents photodynamic therapy with a total

light dose of 15 J/cm2. The figure illustrated the enhanced therapeutic effect of both PDT and

PCI in thymic mice compared to the same treatment performed in athymic mice.

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Endosomes

Lysosomes

Trans Golgi network

Golgi

ER

Secretoryvesicles

P-H+

P

P P-H+

P

P P-H+

PLDL

P

Pp

Pa

PaUncoated pit

Clathrin coated pit

H+Pa

Pp

Fig 1

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Fig 2

InsideSO3- SO3

-

Endosomes/lysosomes

SO3-

SO3-

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NN

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SO3-

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NN

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-

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Al

TPPS2a AlPcS2a

TPPS4SO3

-

SO3-

ACCEPTED MANUSCRIPT

Fig 3

N

NN

N

NN

N

N

SO 3-

A l

SO 3-

1O2

Macromolecules

Endocytic vesicle ~0.5-1 µm

Membrane~10 nm

AlPcS2a

a b c

ACCEPTED MANUSCRIPT

Extracellularspace

Cytosol

D

DD

DDDD

DD

D

DD D

DD

DDDD

D

Lysosome

Endosome

Drug

PhotosensitizerS

S

S S

SSS

SS S

S SS

D DD

DDDS

S

S

SSLight

DD

D

Fig 4

S

ACCEPTED MANUSCRIPT

Fig 5

Time after light exposure (days)0 5 10 15 20 25

Rel

ativ

e tu

mor

siz

e

0

2

4

6

8

10

12Control BLM PDT PCI

ACCEPTED MANUSCRIPT

Control PDT PCI

Surgery alone

PDT + Surgery

PCI + Surgery

Rel

ativ

e tu

mor

siz

e da

y 5

0

2

4

6

8

10

Fig 6

ACCEPTED MANUSCRIPT

Fig 7

Control PDT PCIIR only

PDT + IRPCI + IR

Tim

e to

2x

tum

or s

ize

(day

s)

0

10

20

30

40

ACCEPTED MANUSCRIPT

Fig 8

PDT 30 athymic

PCI 30 athymic

PDT 30 thymic

PDT 15 thymic

PCI 15 thymicTum

ors

< 10

00 m

m3

(%) o

n da

y 20

00

20

40

60

80

100