ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Characterizing the HER2/neu Status and Metastatic Potentialof Breast Cancer Stem/Progenitor Cells

SuEllen J. Pommier, PhD1, Glenda G. Quan, MD1, Dara Christante, MD1, Patrick Muller, BS1,

Amy E. H. Newell, PhD2, Susan B. Olson, PhD2, Brian Diggs, PhD1, Leslie Muldoon, PhD3,

Edward Neuwelt, MD3, and Rodney F. Pommier, MD1

1Division of Surgical Oncology, Department of General Surgery, Oregon Health & Science University, Portland, OR;2Cytogenetics Research Laboratory, Oregon Health & Science University, Portland, OR; 3Department of Neurology,

Blood Brain Barrier Institute, Oregon Health & Science University, Portland, OR

ABSTRACT

Introduction. Treatment resistance, long latency, and

high recurrence rates suggest that breast cancers arise from

defective breast stem cells.

Hypothesis. Within cancers, subpopulations of cells will

demonstrate differences in stem/progenitor potential,

HER2/neu amplification, and gene expression. Related

cells will be found in normal breast tissue.

Methods. ER-/PR-/HER2/neu ? breast cancer cells were

flow-sorted into subpopulations: (A) CD49f? CD24-,

(B) CD49f?CD24?, (C) CD49f CD24-, and (D) CD49f-

CD24?. Gel matrix cell invasion, fluorescence in situ

hybridization (FISH) HER2/neu amplification, and qRT-

PCR gene expression were measured in all groups. Cells

from sorted groups were implanted into rat brains. Resul-

tant tumors were analyzed by immunohistochemistry (IHC)

and FISH. Normal breast tissue was examined by IHC.

Results. Tumor development varied among sorted groups

(25–75%), but was highest in group A. Tumor cells were

mostly CD49f-CD24-, with variable fractions of other

stem/progenitor cells. Tumors showed HER2/neu amplifi-

cation, but fewer chromosome 17 per cell than inoculates.

Group A tumors exhibited cells with normal chromosome 17

copy number and near normal HER2/neu amplification. Cell

invasion was 61% higher in unsorted cells and 34–42% in

sorted groups compared with controls. Sorted groups

showed significantly different expression of development,

proliferation, and invasion associated genes. In normal breast

tissue, CD49f? cells were identified in CD14? CK19- basal

epithelial layers of mammary glands; these were 95%

CD24? and 60% CD44?.

Conclusions. Breast cancer stem/progenitor cell popula-

tions differ in tumor-initiating potential but are not solely

responsible for metastasis. Cancer stem/progenitor cells are

less polyploid than cancer cells in general and may not

be HER2/neu amplified. In normal breast tissue, breast

stem/progenitor cell-like populations are present.

Breast cancer is one of several cancers that is believed to

develop when acquired genetic damage results in the for-

mation of cancer stem cells.1–6 This transformation may

occur when normal breast stem cells develop mutations in

cancer-associated genes and produce a reservoir of self-

sustaining cells with the special ability to self-renew and

maintain tumor proliferation.7–10 Breast cancer demon-

strates a recurrence rate of greater than 60%, irrespective of

the use of chemotherapy or hormonal treatment, in both

ER- and ER? cancers.11 The biological behavior of

breast cancer stem cells suggests that among the population

of cells that make up a tumor, they may represent a treat-

ment-refractory population, dormant and unapproachable

with current targeted breast cancer therapies.12,13

IDENTIFYING NORMAL AND CANCER BREAST

STEM CELLS: FROM MOUSE TO HUMAN

Presently, normal stem cells of the breast are identified

by a limited number of cell surface markers. These mark-

ers, first established in mouse mammary tissue, identify

cells that are capable of reconstituting a complete

mammary gland in vivo, are self-renewing, contribute to

both luminal and myoepithelial lineages, and generate

� Society of Surgical Oncology 2009

First Received: 2 March 2009;

Published Online: 17 October 2009

S. J. Pommier, PhD

e-mail: pommiers@ohsu.edu

Ann Surg Oncol (2010) 17:613–623

DOI 10.1245/s10434-009-0730-z

functional lobuloalveolar units.14,15 Characterization of

cell surface markers demonstrated that these cells were

positive for CD29 (beta-1 integrin), CD49f (alpha-6 inte-

grin), CD24, CD14, CD61 (beta-3 integrin), and Sca-

1.16–18 These cells also are positive for the myoepithelial

marker cytokeratin 14 (CK14).17 The frequency of these

cells has been measured at 0.02–1% of mouse mammary

gland cells.14,19,20 CD24? CD49f low cells are estimated at

an average frequency of 1.6% of mammary gland cells.14,15

In human breast tissue, CD49f? EpCAM progenitor

cells have been reported in keratin 19?, keratin

14? regions of the terminal ductal lobular unit (TDLU) of

the mammary gland, but the frequency is unknown. MUC1

is a cell-specific glycoprotein marker for mammary luminal

cells.17,21–23 ESA? MUC1-cells seem to be bipotent and

capable of producing both luminal and myoepithelial cells

as well as TDLUs in culture and tumors in vivo.24,25

IDENTIFYING BREAST CANCER STEM CELLS

Evidence of human breast cancer stem cells is supported

by serial transplantation experiments in which

CD44? CD24-/low ESA? mammary cancer cells gener-

ated tumors in recipient animals when as few as 102 tumor

cells were injected into mice.6 Furthermore, this subpop-

ulation of cells generated new tumors that contained

additional CD44? CD24-/low cells and a heterogeneous

population of non-tumorigenic supporting cells. This study

also showed that human CD44? CD24? cells were some-

times tumorigenic. This study did not detect tumor growth

from CD44- tumor cells.

The CD44? CD24-/low profile may not be a universal

cancer stem cell marker profile, however. In vitro experi-

ments have demonstrated that stem cell-generated structures,

such as mammospheres, can be formed from pleural effu-

sions that do not contain CD44? CD24-/low cells.26

Furthermore, whereas CD44 is known to be upregulated in

both in situ and invasive ductal carcinomas of the breast, the

clinical impact of CD44? CD24- cells in breast cancer is

controversial.27 They seem to be inversely associated with

lymph node metastasis and relapse-free survival and posi-

tively associated with distant metastasis and invasion, basal-

like tumors, and decreased survival.28–32 Of note, other data

indicate that tumors that are CD44-negative and CD24-

positive (CD44-; CD24?) are associated with decreased

disease-free survival and overall survival.28

BREAST CANCER STEM CELL SIGNALING

PATHWAYS AND ASSOCIATED GENES

The cancer stem cell theory suggests that during the

malignant transformation of normal tissue-specific adult

stem/progenitor cells, reactivation of distinct growth factor

signaling pathways may occur. The reactivation of sig-

naling pathways during malignant transformation, in which

specific genetic abnormalities are acquired in breast cancer

stem/progenitor cells, may explain the formation of dif-

ferent breast cancer subtypes.33 Several developmental

signaling pathways (notch, wnt/b-catenin, Bmi-1, and

hedgehog) are believed to be involved in normal breast

development and dysregulated in breast cancer.34–39 In

addition, several mutations (PTEN, BRCA1, HER2) have

been implicated in breast cancer stem cell prolifera-

tion.40–42 ALDH1 expression is observed in both normal

and cancer breast stem cells.43 Genes involved in self-

renewal and pluripotency maintenance, such as NANOG

and OCT4, are upregulated in breast cancer cells and show

stem cell-like properties.44 Recent work suggests that

CD44 interacts with the embryonic stem cell transcription

factor nanog in concert with stat-3 to promote tumor

proliferation.45

Overexpression of the HER2/neu protein is seen in

approximately 25–30% of breast cancers and is primarily

due to an amplification of the HER2 gene.46,47 Diagnostic

testing for HER2 amplification by fluorescence in situ

hybridization (FISH) is a crucial component of breast

cancer management and critical for treatment decision-

making regarding trastuzumab (Herceptin) therapy.48,49

Molecular testing often uses a dual-probe FISH approach,

which detects the HER2 gene and chromosome 17.50–52

This method is quantitative; results are presented as the

ratio of HER2 signals to the number of chromosome 17

signals. Normal cells will have two chromosome 17 signals

and two HER2 signals with a ratio of 1.0. For diagnostic

purposes, guidelines designate HER2 amplification as a

HER2/chromosome 17 ratio [2.0.50 Standard reporting,

which sets the upper limit of amplification of HER2/neu at

[10, can produce a low 4 amplification ratio when there

are multiple copies of chromosome 17 in a cell. Deviations

in the number of chromosome 17 per cell (aneusomy) in

breast cancer are common.53 Heterogeneity of HER2/neu

amplification within the tumor cell population also

occurs.54

This study was undertaken to determine whether sub-

populations with defined cell surface markers have

different tumorigenic and invasive potentials and exhibit

differences in gene expression and HER2/neu amplifica-

tion. To establish a biological link between normal breast

stem cells and cancer stem cells, the location and frequency

of cells expressing similar cell surface markers were

determined in normal breast tissue. If the cancer stem cell

theory is correct—that breast cancer stem cells initiate

from damaged normal breast stem cells—then normal

breast stem cells should express related cell surface

markers. Because breast carcinomas, including ductal

614 S. J. Pommier et al.

carcinoma in situ (DCIS), are believed to originate from

the epithelial cells in the TDLU and breast stem cells are

proposed as the source of breast cancer cells, then breast

stem cells may be located in the TDLU of breast tissue.55,56

MATERIALS AND METHODS

Fluorescence-Activated Cell Sorting of Cultured

HCC1954 Breast Cells

HCC1954 cells (ATCC, Manassas, VA) (ER-/PR-

HER2/neu amplified) human breast cancer cells were

grown in RPMI-1640 supplemented with 10% serum and

antibiotics. Cultured cells were resuspended at a concen-

tration of 105 cells/2.0 ll of primary antibody. They were

labeled with fluorochrome-conjugated monoclonal anti-

bodies against human CD24 (PE), CD49f or CD29 (PE-

Cy5), and CD44 (FITC) and then stained with DAPI to

measure cell viability during fluorescence-activated cell

sorting (FACS). Isotype controls included FITC, PE, and PE-

Cy5. Cells were analyzed on a Cytopeia Influx FACS using

Spigot. Cell subpopulations were separated based on surface

antibody labeling and collected by discriminatory gating.

After selecting for CD44? cells, breast cancer cell suspen-

sions were sorted into CD49f?CD24-, CD49f?CD24?,

CD49f-CD24?, and CD49f-CD24- enriched subpopulations.

Cell Invasion Assays

Unsorted HCC1954, sorted FACS groups, and rat

fibroblasts (negative control) were grown overnight in

serum-free DMEM medium (Invitrogen, Carlsbad, CA)

and then harvested with 0.25% trypsin. A total of 125,000

cells were placed into each test-well insert. Five hundred

microliters of DMEM with 10% FBS (HyClone, Logan,

UT) (chemoattractant) was added to each well beneath the

insert. Samples were incubated for 72 h at 37�C. Cells

were dislodged and prepared for fluorescence measurement

as per the manufacturer (Millipore Corporation, Temecula,

CA).

Stereotactic Intracerebral Breast Cancer Cell

Implantation

Adult female athymic nude rats were anesthetized with a

mixture of intraperitoneal ketamine (60 mg/kg) and diaz-

epam (7.5 mg/kg), and a 2-mm burr hole was made in each

skull. After being prepared as described above, tumor cells

(10 ll, *103–105 cells, [90% viability by trypan blue

exclusion) were inoculated through stereotactic coordinates

for intracerebral localization in the right caudate putamen.

Inoculation was performed for a period of 5 min to prevent

backflow of tumor cells out of the brain tissue. All animal

subjects were weighed and monitored every other day for

changes in behavior to examine for signs of intracerebral

disease (weight loss, gait changes, inability to feed/drink,

inability to groom). When animals exhibited signs of

intracerebral disease, they were euthanized. Animals who

exhibited no signs were euthanized at day 90 after

implantation. After 2–3 days in 10% formalin, verification

of metastatic breast tissue in the sampled brain tissue was

made by immunostaining with anti-cytokeratin CAM-5.2-

conjugated with FITC (Becton–Dickinson, San Jose, CA)

or by H&E. This animal research had been approved by the

OHSU, IACUC.

Antibodies

Monoclonal and polyclonal anti-human antibodies

included CD24, CD44, p63, CD31, CK14, CK18, CK19

(Santa Cruz Biotechnology, Santa Cruz, CA), CD49f

(R&D System, Minneapolis, MN), CD29, and CD45

(Chemicon International, Temecola, CA). Fluorochrome-

conjugated secondary antibodies included anti-rabbit and

anti-mouse Ig-Alexafluors 488 or 594, as well as anti-rat

Ig-Alexafluors 594 (Invitrogen). CD29 and CD49f are both

anti-integrin antibodies and were used interchangeably in

this study. Formalin-fixed, paraffin-embedded blocks were

made from cultured cells from specific cell lines (ATCC,

Rockville, MD) and served as positive controls for the

antibodies as follows: chronic myelogenous leukemia

K562 cells for CD24; histiocytic lymphoma U937 cells for

CD29; and colorectal carcinoma cells DLD-1 for CD49f.

IHC Staining of Cell Cultures and Paraffin-Embedded

Tissue Sections

Cultured breast cancer cells were fixed in 100% meth-

anol at -20�C for 5 min and then permeabilized in 1%

triton X100 in PBS for 15 min. For normal breast tissue

and breast tumors 4-microns thick, formalin-fixed, paraffin-

embedded tissue sections were obtained from four normal

human breast tissue blocks. Histology sections of normal

tissue were selected for high numbers of ducts, lobules, and

intact glandular segments of terminal ductal lobular units.

Sections were deparaffinized with CitriSolv (Fisher Sci-

entific, USA). Epitope retrieval was performed by

incubation in 95�C 10 mM Na Citrate pH 6.0 solution for

20 min (Fisher Scientific). Tumor sections were addition-

ally placed in proteinase K for 30 min at 37�C and blocked

with goat or donkey serum (Sigma–Aldrich, USA). Tissue

sections were then incubated with primary antibodies at

concentrations of 1:100 (final concentration 1–2 lg/ml),

followed by incubation with biotinylated Ig-AlexaFluors

Breast Cancer Stem Cell HER2/neu Status 615

488 or 594 at concentrations of 1:500. Finally, slides were

stained with DAPI (Invitrogen, Molecular Probes).

Microscopy of Immunofluorescence Staining

Tissue sections were examined using the Nikon Eclipse

TE300 fluorescence microscope. Digital images were

captured with a mounted Nikon DC290 digital camera and

Kodak MI Molecular Imaging Software v4.0.3. Visualiza-

tion and correlation of multiple markers within the same

cell were achieved by using human primary antibodies

derived from multiple sources, such as mouse, rabbit, or

rat. To ensure that intact cells were being counted, only

cells whose nuclei stained positive for DAPI were assessed

for the presence of antibodies. For normal breast tissue

sections, the CD29 marker was assigned as the reference

marker to which the colocalization frequency of all other

antibody markers was compared. The frequencies of cells

containing various combinations of antibody markers were

tabulated, and their locations within the mammary gland

were identified. Results from all four normal individuals

were combined and averaged. In tumor sections, approxi-

mately 104 cells were scored for the presence of each of the

cell surface markers examined in this study.

HER2/neu FISH

HER2/neu FISH was performed on 4-lm paraffin sec-

tions and interphase nuclei from cultured cells dropped

onto slides with 3:1 methanol:acetic acid using the FDA-

approved PathVysion kit containing a probe for the HER2/

neu locus (17q12, SpectrumOrange) and using CEP17 as

the chromosome 17 control probe (D17Z1,17p11.1-q11.1,

SpectrumGreen). Deparaffinization for paraffin slides was

performed according to the manufacturer’s instructions.

Interphase nuclei were treated with 0.005% pepsin in

0.01 N HCl solution at 37�C for 10 min in place of

deparaffinization. After these treatments, hybridization,

washing, and counterstaining were performed according to

the manufacturer’s instructions. Fluorescence was visual-

ized on a CytoVysion image capture system (Applied

Imaging, San Jose, CA) with a Nikon E800 (Nikon, Mel-

ville, NY) microscope. HER2/neu and CEP17 signals were

enumerated both in 25 cells in paraffin sections and in all

cells found in cultured cell samples, and a total HER2/neu

to CEP17 ratio was calculated. For each probe, signal

counts[10 were considered innumerable and scored as 10.

Isolation of RNA and cDNA and Gene Expression

of FACS Cells

mRNA was isolated from HCC1954 cultured cells

and FACS cells per manufacturer protocol (Qiagen,

Germantown, MD). cDNA was produced by reverse tran-

scription using random hexamers, 4 ll of mRNA, and the

Superscript-III kit (Invitrogen, Carlsbad, CA). cDNA

obtained from HCC1954 cultured cells and from the

CD49f?CD24-, CD49f?CD24?, CD49f-CD24-, and

CD49-CD24? flow sorted cells groups were tested for the

expression of 11 stem cell-associated genes and for 17

cancer-associated genes by q-RT-PCR. The custom

Applied Biosystems TaqMan Express panel of tested genes

is shown in Table 1. Ten nanograms of cDNA and 20 ll of

TaqMan master mix solution were applied to single wells,

each containing gene-specific primers and probes, in a 96-

well TaqMan Express plate. This assay was performed on

the Applied Biosystems 7900HT RT-PCR system, and

results were provided in a quantitative format.

Statistical Analysis

Significant differences in the results of gel matrix cell

invasion assays were determined using ANOVA tests. For

TaqMan gene expression plate assays, sort A was desig-

nated as the reference sample. The fold change in gene

expression for a tested gene was calculated by 2-DDCt,

where DDCt is defined as: (the average Ct value for the

gene in sort A minus the average Ct value of the ribosomal

S18 gene in sort A) minus (the average Ct value of the gene

minus the average Ct value of the ribosomal S18 gene)

(Applied Biosystems User Bulletin No. 2 (P/N 4303859).57

Comparisons of fold changes in gene expression between

cell populations were done with Student’s t test.

TABLE 1 Applied Biosystems TaqMan express plate gene expres-

sion array genes associated with cell fate genes associated with

proliferation and cancer40,43,45,61–63,65–73

Notch-1 MUC1

Notch-2 FCYD3/MAT-8

Notch-3 S100P

Notch-4 VEGFR2

NUMB FAS

WNT3 ALCAM

BMI-1 c-MYC

PTEN c-MYB

ALDH1 EGFR

STK15

BARD1 STAT13

SURVIVIN

MMP1

Ki-67

CYCLIN D1

CYCLIN B1

p53

616 S. J. Pommier et al.

RESULTS

FACS and Implantation

Cells were sorted into CD29?CD24-, CD29?CD24?,

CD29-CD24-, CD29-CD24? groups and designated as

groups A, B, C, and D respectively. Approximately 5 9 105

cells were collected for groups A and B, whereas approxi-

mately 5 9 104 cells were collected for groups C and D. This

was a consistent finding over multiple FAC sorting proce-

dures and most likely reflected the percentage of these cells

within the total HCC1954 cell line (Fig. 1). To test the

tumorigenicity of the HCC1954 cell line, inoculates of 105

unsorted HCC1954 cells were implanted into eight female

nude rats; all developed tumors. After FACS, 103 cells from

each of the sorted groups were similarly transplanted into

different rats. Tumors developed in all groups, with the

highest efficiency in group A (Table 2).

IHC of HCC1954 Cultured Cells and Resultant

HCC1954 Tumors

In the breast cancer cell line, the profile was

CK18? (99.5%), CK14-, CK19? (94%), CD44? (93%).

CD29 and CD49f are both anti-integrin antibodies and

were used interchangeably in this study. Most cells were

CD29med or CD49f med and CD24med. CD29hi or CD49fhi

cells made up 2% of the population. Of these, 9% were

CD24hi. All eight breast cancer tumors that formed in the

brain were examined by hematoxylin and eosin (H&E);

two were fully examined by IHC (Fig. 2). In the breast

cancer tumors that formed in the brain, the marker profile

was CK18? (50%), CK14- (100%), and CD44? (96%).

CK19? cells were found in one of two tumors (0.25%).

CD29hi or CD49fhi cells comprised 3% of the population.

Of these, 92% were CD24hi. Most cells in the tumor were

CD29low/- or CD49fCD24low/-. In both cell cultures and

tumors, CD29? and CD24? cells were 95% CD44?.

IHC of Resultant Tumors from FACS Groups

After selecting for CD44? cells, breast cancer cell sus-

pensions were sorted into enriched subpopulations and

referred to as group A: CD49f?CD24-, group B:

CD49f?CD24?, group C: CD49f-CD24-, and group D:

CD49f-CD24?. Differences were observed between

tumors that developed from inoculates of the different

104

103

102

101

100 104

*PE - CD24103101 102

*PE-Cy5 - CD49F104

103

102

101

100 104

*PE - CD24

4.48 4.71

4.94 2.51

103101 102

*PE-Cy5 - CD49F

FIG. 1 Representative flow sort dot plots of breast cancer cells.

HCC1954 cells were labeled with fluorochrome-conjugated mono-

clonal antibodies against human CD24 (PE), CD49f or CD29 (PE-Cy

5), or CD44 (FITC) and stained with DAPI to measure cell viability

during FACS. Left: Isotype controls included FITC, PE, and PE-Cy5.

Right: The various cell subpopulations were separated based on

surface antibody labeling and collected by discriminatory gating.

Collected groups consisted of\5% of the total cell population. After

selecting for CD44? cells, breast cancer cell suspensions were

collected as: A: CD49f?CD24-; B: CD49f?CD24?; C: CD49f-

CD24-; D: CD49fCD24? enriched subpopulations

TABLE 2 Tumor growth after intracerebral inoculation of rats with

breast cancer cells

Cell

group

Inoculations

performed (n)

Dead from

other causes

Tumor

growth (n)

No tumor

(n)

% with

tumor

growth

Control 8 0 8 0 100

A 6 2 3 1 75

B 4 0 1 3 25

C 3 1 1 1 50

D 3 0 1 2 33

Breast Cancer Stem Cell HER2/neu Status 617

sorted groups in CK18 and CK19 expression. Groups A

and B showed patches of CK18? cells in approximately

50% of the tumor. The group C tumor had few CK18? cells

(11%), and group D was CK18-negative. One group A

tumor had five CK19? cells (0.05%). The tumor from

group B had two CK19? cells (0.02%). Tumors resulting

from all groups were CK14-negative. All cells appeared to

be CD44? in groups B and D tumors. Group C tumor cells

were CD44med, and group A tumor cells were CD44low/-. All

tumors, regardless of the cell sorted group from which they

arose, were primarily made up of CD49f low/-CD24 low/-

cells. The same result was obtained when CD29 was used

instead of CD49f. All tumors developing from sorted groups

had from one to four CD49fhiCD24hi cells (0.01–0.04%).

CD49fCD24hi cells were seen only in group A tumors

(0.02%). CD49f hiCD24- cells were seen only in the group D

tumor.

HER2/neu FISH

The amplification ratio for the unsorted HCC1954 cell

inoculates before implantation was 1.8. For sorted groups,

the ratios were as follows: A = 1.6; B = 1.9; C = 1.6;

D = 1.7. For all groups, the number of chromosome 17

signals per cell fell within the range of 1 to 15. Most cells

that were scored had an amplified HER2/neu signal that

was visually [30 in number (Fig. 3). HER2/neu conven-

tional scoring limits the uppermost score for HER2/neu at

[10. Therefore, due to the large numbers of chromosome

17 in these cells, the ratios do not accurately convey that

these groups were amplified for HER2/neu. One cell

(0.5%) in the HCC1954 cell line (Fig. 3, top left) and four

cells (4%) in group B were observed with normal chro-

mosome 17 copy numbers and no amplification.

The amplification ratios for breast cancer tumors that

resulted from implantation of breast cancer unsorted and

FIG. 2 Resultant tumor from HCC1954 unsorted breast cancer cell

inoculation and cell surface marker profile. Top panel shows H&E

staining of metastatic breast tissue in the rat brain tissue. Lower

panels show examples of cell populations observed in the tumor. All

cells were CD44?. Resultant tumors also contained cells, indicated by

arrows, which were: left CD29?CD24?; middle CD29?CD44?; rightCD24-CD44?

FIG. 3 HER2/neu FISH of

breast cancer interphase nuclei.

Signals were visualized with the

PathVysion kit containing a

probe for the Her2/neu locus

(17q12, SpectrumOrange) and

CEP17 as the chromosome 17

control probe (D17Z1,17p11.1-

q11.1, SpectrumGreen). Leftpanels of images of cultured

cancer cells before intracerebral

injection. Right panels represent

cells from resultant tumors

(paraffin embedded). HER2/neu

amplification ratios for left

panels are: unsorted-1.8, A-1.6,

B-1.9, C-1.6, D-1.7. HER2/neu

amplification ratios for tumors

in the panels on the right are:

unsorted-3.9, A-2.5, B-1.8,

C-2.9, D-2.3. All samples

appear amplified visually,

although ratios may be low due

to chromosome 17 polysomy

618 S. J. Pommier et al.

sorted cells was as follows: from unsorted HCC1954 cells,

3.9; group A, 2.5; B, 1.8; C, 2.9; and D, 2.3. All samples

were amplified for HER2/neu. The increase in amplifica-

tion ratio compared with that of the inoculate was due to a

decrease in the numbers of chromosome 17 per cell. All

tumors showed an increase in the number of cells with

normal chromosome 17 copy numbers compared with

those of the inoculate. Fifty percent of tumor cells that

resulted from the unsorted inoculate had normal chromo-

some 17 copy numbers. In tumors resulting from groups B,

C, and D, 12% of cells had normal chromosome copy

numbers. In group A tumors, 32% of cells had normal

chromosome 17 copy numbers and 8% with near normal

amplification of HER2/neu (Fig. 3).

Cell Invasion of Gel Matrix

The sorted and unsorted populations were assessed for

their ability to invade a gel matrix. In unsorted HCC1954

cells, cell invasion was 61% greater than negative rat

fibroblast. Invasion results were similar among the

subpopulations CD49f? CD24-, CD49f? CD24? , CD49f

CD24?, and CD49fCD24- at 34–42% greater than control

cells.

Gene Expression

Significant gene expression differences were observed

between sorted groups A, B, C, and D (p \ 0.05) in 7 of

the 29 genes tested. Groups B and D were most similar in

their gene expression. Groups A and C were the most

divergent. The differences in gene expression are shown in

Fig. 4.

Cells with Stem/Progenitor Cell Marker Profiles

Were Identified In Normal Breast

A total of 5,727 cells from of 116 histology sections were

examined from normal human breast tissue samples (Fig. 5).

For each pair of markers tested for colocalization, an average

of 955 cells (range, 829–1220) was examined. In normal

breast tissue 2,616 cells were identified that were positive for

the reference marker CD29. These cells localized to regions

surrounding both the ducts and the lobules, with the greatest

intensity of staining in cells in the basal epithelial layer that

lined the mammary gland. It was not possible to determine by

this technique whether individual CD29? cells also were

FIG. 4 Gene expression by

q-RT-PCR. Significant changes

in gene expression of the sorted

groups are listed in boxes, with

fold changes denoted to the

right of the gene name. The

arrows denote a significant

increase in the fold change in

the population immediately to

the right. The fold change

expression is calculated by

2-AACt. Each of the sorted

groups demonstrated a unique

gene expression profile

FIG. 5 Structures in the terminal ductal lobular unit. Left normal

breast tissue section demonstrating structures in the terminal ductal

lobular unit (TDLU) at 9100 magnification. Ductal and lobular

structures are shown explicitly. Right Enlarged image illustrating

immunohistochemical staining of a portion of TDLU in normal

mammary tissue. This tissue section was treated with anti-CD29 and

anti-CD24 antibodies followed by fluorochrome-conjugated second-

ary antibodies. CD29 was conjugated with Ig-Alexafluor 594 (red),

and CD24 was conjugated with Ig-Alexafluor 488 (green). The pinkarrow shows CD29?CD24- cells and the yellow arrow indicates a

salmon-colored cell expressing both CD29? and CD24? markers

Breast Cancer Stem Cell HER2/neu Status 619

CK19? or CK14?, but it is the case that CD29? cells resided

between CK19? and CK14? zones.

Of the 861 CD29? cells examined for the coexpression

of CD24, 822 (95.5%) cells were CD29?CD24med. Five

cells (0.5%) were identified as CD29-CD24? . Thirty-four

(4%) cells were CD29?CD24-, three of which were

located in the basal epithelium at the distal tip of the

mammary duct in the TDLU. The remaining 31 cells were

identified in the basal epithelial layer of the lobular com-

ponent of the TDLU.

Of the 926 cells examined for coexpression of CD29 and

CD44, 561 (60.5%) cells were identified as CD29?CD44?

and 364 (39.5%) cells were CD29?CD44-. No cells were

found to be CD29CD44?. The average frequency of

CD29? cells that also were CD44? was 65%. However,

unlike any of the other markers tested, which did not vary

significantly between individual tissue samples, the aver-

age frequency of CD29? that were CD44? cells varied

considerably (45%, 59%, and 84%) among the three nor-

mal samples of breast tissue. CD44? cells did not

colocalize with CK19; rather, they were located in the

basal epithelial layer of ducts and lobules. The number of

CD44? cells that coexpressed CD24 also was investigated.

Of the 857 cells that were identified as CD44?, 634

(73.9%) also were CD24? and 223 (26.1%) were CD24-.

These cells were located in the basal epithelial layer sur-

rounding ducts and lobules. CD31 did not colocalize with

any other antibody tested. CD31? cells were located in

vascular structures of breast tissue.

DISCUSSION

A cure for breast cancer will necessarily involve the

eradication of cancer cells that have the potential to

metastasize and initiate tumors in new sites. Breast cancer

stem cell theory suggests that stem/progenitor cells are

responsible for this function and that effective cancer

therapy will selectively target cancer stem/progenitor cells

while leaving normal stem/progenitor cells unaffected. To

achieve this goal, a better understanding is required of the

prevalence of stem/progenitor cell subpopulations within

breast cancers, as well as their contribution to tumorigen-

esis, invasion, metastases, and poor treatment response. To

this end, this study identified discrete subpopulations of

cancer stem/progenitor cells within a breast cancer cell

line. All cells exhibited invasive ability but differed from

one another in their tumor-initiating capacities and gene

expression. HER2/neu amplification differences gave

insight into the timing of HER2/neu amplification during

tumorigenesis, as well as into the way in which differences

in HER2/neu expression between primary tumors and

metastases may occur.

When cells were sorted and inoculates were reduced to

103 cells, inoculates from all four groups produced tumors

but at different frequencies from one another. Cells from

group A showed the highest level of tumorigenicity,

producing tumors in three of four animals inoculated;

histopathologic examination of the fourth brain indicated

that the cells did not successfully implant. This indicates

that all four groups were enriched for cells that were

capable of initiating tumors. However, group A was most

enriched for tumorigenic cells.

Although inoculates contained different groups of cells

enriched for specific cell surface markers, resultant tumors

in all animals primarily consisted of CD49fCD24low cells.

Concordance was observed between the unsorted and sor-

ted groups for the number of CD49f? CD24? cells that

were found in the tumors. The tumors differed in their

expression of the luminal keratin marker CK18, and CK19

was only observed in sort A tumors. Only the tumors

resulting from inoculation of group A cells contained all

the cells that were seen in the unsorted parent population,

indicating the multipotent capability of the CD49f?

CD24- cells. Study findings did not support the concept

that CD44 expression is a required intrinsic component of

cancer stem/progenitor cells. Taken together, the results

of this study support the concept that the subpopulations of

stem/progenitor cells vary in their ability to produce other

cell lineages within the tumor.

This study suggests that HER2/neu amplification may

occur progressively during the natural history of the tumor.

Cancer stem cells may not be HER2/neu amplified in

tumors that are heterogeneous for HER2/neu amplification

or those in which the metastatic lesions differ from the

primary tumor.54 It may be that HER2/neu amplification

occurs in the later progenitor cells of the cancer stem cell

hierarchy.

Latta and colleagues suggest that in breast cancer cases

in which the DCIS component is HER2/neu amplified and

the invasive component is not, the invasive component may

have arisen from a clone of tumor cells that developed

invasive abilities in the absence of HER2/neu expression.58

When applying this principle to cancer stem cells, it is

reasonable to deduce that the stem cell will not be HER2/

neu-positive in tumors that show no HER2/neu overex-

pression; however, this also may be true for tumors that are

heterogenic for HER2/neu overexpression. Both the Latta

study and ours suggest there is reduction of or selection

against HER2/neu amplification during tumorigenesis or

transition to the invasive state. Both bodies of work suggest

that HER2/neu-negative clones are enriched in the transi-

tion. During the recapitulation of tumorigenesis by breast

cancer stem/progenitor cells examined in this study, the

initiation process was accompanied by an increase in

the number of cells with near normal chromosome 17

620 S. J. Pommier et al.

complement and reduced HER2/neu amplification. The

findings that group A was the most tumorigenic and

exhibited the greatest increase in disomy 17 with an

accompanying decrease in HER2/neu amplification support

the concept that cancer stem/progenitor cells have less

polyploidy than their progeny that make up the bulk of the

tumor.

The cell line that was used for this study was obtained

from a stage IIA, grade 3, HER2/neu-amplified tumor. This

may explain why no HER2/neu nonamplified stem/pro-

genitor cell was found. Whereas this study did not

demonstrate a progenitor cell without HER2/neu amplifi-

cation, this does not preclude the possibility that there are

cancer stem cells that are not HER2/neu-amplified. Cancer

stem/progenitor cells may in fact exhibit normal HER2/neu

expression, even within HER2/neu-amplified tumors. This

finding has important clinical implications. Improved

response rates and improved disease-free and overall sur-

vival with trastuzumab therapy may be due to the effects of

trastuzumab on HER2/neu-amplified progenitor and com-

mon tumor cells. Recurrences with a long latency period

may still be seen among HER2/neu-amplified cases, how-

ever, if the original cancer stem cells do not overexpress

HER2/neu and are therefore not affected by trastuzumab

therapy.

All subpopulations exhibited similar levels of increased

capacity for cellular invasion compared with that of the

negative control, but these levels were not as high as

observed for the parent population from which they were

derived. This suggests that stem/progenitor cells evaluated

in this study may work in concert with other tumor cells to

coordinate tissue invasion and metastasis processes.

The gene expression differences between the flow sorted

cells suggest that even though they all appear to be stem/

progenitor cells, each group may have different functions.

Differences were observed in gene expression levels for

NOTCH, BARD1, EGFR, MUC1, CCNB1, and TP53. The

gene expression differences between the various cell pop-

ulations suggest that their presence may give tumors

different functional characteristics. In this study, groups B

and D (CD29? CD24? and CD29-CD24?) were the most

closely related by gene expression, whereas A and C were

the least. For all genes showing expression level differ-

ences, levels were higher than those in group A. The lowest

level of MUC1 expression in group A may indicate that

cells in this group are less differentiated than those in

groups B, C, or D. These findings are consistent with the

observation that group A cells were the most tumorigenic.

The expression of NOTCH and/or MUC1 in all sorted

groups indicates that they are enriched for stem/progenitor

cells. These genes are associated with breast development

and differentiation. The Notch signaling pathway, through

its four receptors (Notch 1-4), is thought to play a role in

stem cell self-renewal, cell fate, apoptosis, proliferation,

and migration.59 MUC1, discussed earlier, is important for

mammary cell type differentiation.

Differences in gene expression defects in genes associ-

ated with breast cancer also were observed between the

various sorted stem/progenitor groups. These genes inclu-

ded BARD1, EGFR, CCNB1, and TP530. BRCA1 is a

tumor-suppressor gene involved in DNA repair. A stable

complex of BARD1/BRCA1 seems to be required for

normal BRCA1 function.60 TP53, CCNB1, and EGFR are

responsible for apoptosis, cell cycle fidelity, and prolifer-

ative regulation.61–63 In all other groups there was

increased expression of TP53, CCNB1, BARD1, and

EGFR compared with that of group A. If group A, which is

enriched for CD29? CD24- cells, represents a less-dif-

ferentiated cancer stem cell than the other groups, this

would support the concept that cancer stem cells have

fewer gene changes and that damage is progressive and

cumulative in the cancer stem cell lineage.

Unsorted HCC1954 cells and sorted subpopulations

expressed barely measurable levels of ALDH1. That

HCC1954 cells express low levels of ALDH1 has been

reported.64 However, the subpopulations in this study that

did not overexpress ALDH1 exhibited stem/progenitor

behavior, indicating that ALDH1 expression may be nei-

ther a consistent finding nor a necessary function among all

cancer stem/progenitor cells.

This study successfully identified a population of cells in

normal human breast tissue, within TDLU structures, that

expressed cell markers that are known to be associated with

mouse and human breast stem and early progenitor cells.

Of the CD29? cells that were examined in the mammary

gland, the majority coexpressed CD24. CD29 and CD24

cell surface marker expression appeared as a gradient in

normal tissue. Some expression was observed, associated

with the luminal cells within the acini of lobules and

among epithelial cells of ducts. The expression was higher,

however, in CD29? cells that were found in the CK19- ,

CK14? basal epithelial layer that surrounded both the

ducts and lobules of the mammary gland.

The results of this study help to explain some of the

unusual behaviors that are associated with breast cancers. It

provided evidence for the existence of discrete subpopu-

lations of stem/progenitor cells within breast cancers that

express genes responsible for breast development and

cellular proliferation. It showed that these cells also har-

bored genetic abnormalities in cancer genes. Thus, while

retaining the functions imbued to them as stem or

progenitor cells, such as self-renewal, dormancy, and plu-

ripotency, they in addition acquired damage that leads to

malignant function. The result is that within tumors there

are cancer stem cells with tremendous generative and

proliferative ability. The resistance of breast cancer to

Breast Cancer Stem Cell HER2/neu Status 621

conventional therapies and the high relapse rates may be

explained by the presence of cancer stem cells. Any ther-

apeutic regimen designed to cure will necessarily involve

eradication of cancer stem/progenitor cells.

ACKNOWLEDGMENT The authors thank Christopher Corless,

M.D, Ph.D. and Megan Troxall, M.D. for their interpretation of and

technical assistance with breast histology sections.

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