Introduction
Breast cancer is the most common neoplastic disease in women and affects approximately 1 out of 10 females. Human breast cancer is a heterogeneous disease with varied clinical course [
1,
2]. Up to 5% breast cancer cases are attributable to inherited mutations in the
BRCA1 or
BRCA2 genes [
3]. Inherited mutations of
BRCA1 (chromosome 17q21) have been linked to increased risk for breast and ovarian cancers [
4]. The BRCA1 tumor suppressor plays a major role in DNA damage, signaling, repair and cell cycle control. BRCA1 is also a co-regulator of steroid hormone receptors and modifies steroid hormone action [
5]. Carriers of the mutant gene also have significantly higher risk for developing other tumor types, including ovarian, uterine, cervical, and prostate cancers [
6]. Breast cancer patients with
BRCA1 mutations are more likely to be estrogen receptor (ER) and HER-2 negative, and have mutant or deleted p53 [
7]. In spite of advances in detection and clinical management for patients with familiar BRCA1 mutant breast cancer, there has been no significant improvement in therapies and overall survival for these patients [
8].
Microarray analysis has been useful in identifying gene expression signatures that characterize qualities important for biological and clinical classification [
9,
10]. Phenotypic characterization and microarray profiling of breast tumors reveals distinct subtypes of breast carcinoma that are associated with survival. Five major groups of invasive breast carcinomas have been identified: luminal A, luminal B, HER2 +/ ER -, basal-like, and normal breast-like [
10]. The basal-like tumors are typically ER and HER2-negative, have high proliferative rate, and typically show a poor clinical outcome [
10].
BRCA1 and BRCA2 are essential for maintenance of genomic integrity by promoting repair of double-stranded DNA breaks. Following DNA damage, BRCA1 is phosphorylated by the ATM/ATR kinases and recruits multiple factors to the break site that actively participate in repair [
11]. BRCA1 associates and co-localizes with RAD51 nuclear foci in mitotic cells along with BRCA2 and BARD1, a BRCA1 binding protein [
12]. BRCA1 deficiency leads to impaired double-strand break repair and to enhanced sensitivity to ionizing radiation and genomic instability [
11,
13]. However, only limited studies are reported using human cells, most of them derived from analysis of one human cell line that is null for BRCA1, HCC1937 [
14]. Although BRCA1 replacement increased the resistance to Vinorelbine and Cisplatin, it did not change sensitivity to other agents, such as Docetaxel [
14] suggesting that multiple mechanisms may be associated with drug resistance in this cell line.
Studies of tumor biology and development of novel therapies for tumors associated with BRCA1 deficiency are hampered by the lack of readily available material for
in vitro and
in vivo studies. Genetically engineered mouse tumors are an excellent tool for studying cancer biology and can potentially improve preclinical studies. Development of multiple mouse models with deletion or mutations in
Brca1 targeted to the mammary gland has provided an opportunity to examine the biology and therapeutic implications of BRCA1 loss in breast cancer [
15] and some studies find similarities between the mouse and human tumors [
11,
16,
17]. In particular, correlations with human basal-like tumors have been reported in
Brca1 mouse models [
17,
18]. However, using
Brca1 mouse mammary models for preclinical development has been limited due to complicated breeding schemes, variable penetrance, and prolonged latency of tumor development [
19]. In spite of increased penetrance when mice heterozygous for the T
p53 tumor suppressor are crossed with mice harboring mutant
Brca1, these mice develop mammary tumors in over one year, at which time development of lymphomas, which is characteristic of p53-deficient background, also compromises animal survival [
20].
Expansion of Brca1 deficient mammary tumors by transplantation is a useful alternative for generating sufficient material for studying Brca1-associated tumorigenesis. The purpose of our study was to harvest, expand in vivo, and characterize tumors and cell lines derived from Brca1 mammary tumors. The original and transplanted tumors as well as well-characterized cell lines derived from original tumors provide the necessary tools for studying the biology of Brca1-deficient tumors, identification of putative cancer stem cells, and development of novel therapies to improve clinical outcome.
Materials and methods
Tumors and serial transplantations of cell suspensions
All studies were conducted in an AAALAC accredited facility in compliance with the PHS
Guidelines for the Care and Use of Animals in Research. Naïve 6-to-8 week old female
scid/NCr (BALBc) mice from the NCI Animal Production Program (Frederick, MD) were used as transplant recipients. Autoclaved feed and hyper-chlorinated water were provided
ad libitum. The
Brca1Co/Co Δexon 11,
p53+/-,
MMTV-Cre mouse mammary tumors [
21] were dissected and cell suspensions prepared as described by Varticovski and coworkers [
22]. In summary, tumors were excised, dissected, mechanically dissociated and forced through a 40 uM mesh. Viable cells were either frozen in freeze down media, or plated at low density in 100 or 150 mm plates in RPMI supplemented with Pen/Strep, glutamine, and 2% FCS for selection of cell lines. Non-adherent cells were removed after 48 hours and surviving clones were isolated using cloning cylinders. For implantation of cell suspensions into naïve recipients, the mouse fat pad #4 of SCID mice was visualized through a small skin incision just anterior to the rear leg, and one million cells were injected in 50μ l of RPMI-1640. The incision was closed with a sterile wound clip, which was removed in 7 days.
Tumor size was determined biweekly using caliper measurements (millimeters) in two perpendicular dimensions (length and width). Tumor weights (milligrams) were calculated using the formula for a prolate ellipsoid and assuming a specific gravity of 1.0 g/cm
3 [
23]. Mice were killed when tumors reached an approximate 1 g of wet weight. Tumors were divided into the following fragments: a portion was used to prepare cell suspensions and further passages
in vivo, the remaining was frozen or fixed in 10% neutral-buffered formalin.
Generation and genotyping of Brca1 tumor cell lines
Cells derived from Brca1 mammary tumors were grown at 37°C in 5% CO2 in RPMI-1640 media supplemented with increasing concentrations of FBS up to 10%, pen/strep, and glutamine. Over 40 clones were isolated. Sixteen cell lines were developed from 5 original primary tumors and maintained in culture for up to 50 passages in RPMI1640 supplemented with 10%FCS.
Brca1 primary tumors and cell lines were genotyped as described by PCR amplification of sequences specific for the null
Brca1 allele [
20], conditional
Brca1 (Brca1
Co/Co) allele [
13,
21], wild-type p53 allele [
20], and CRE [
20,
24].
RNA isolation, microarray hybridization and data analysis
Total RNA was isolated from 5 spontaneous (original, 0)
Brca1 mammary tumors, 4 tumors from first-passage transplantation into naïve recipients (first-passage, 1), 3 tumors from second-passage transplantation into naïve recipients (second-passage, 2), and 7 representative original tumor-derived cell lines. Normal virgin mammary glands from three #4 glands of 6–8 wk old C57Bl6 or SCID mice were included in the microarray analysis. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, followed by DNAse treatment and RNA clean-up (RNeasy Mini Kit, QIAGEN, Valencia, CA). RNA integrity was determined using the RNA 6000 Nano LabChip Kit on Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). RNA was amplified and hybridized overnight to the Agilent Mouse Oligo Microarrays 22 K (G4121AorB) against a common reference total RNA [
17]. The co-hybridized samples and reference RNA were washed and scanned on an Axon GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA), analyzed using GenePix 4.1 software, and uploaded to the UNC microarray database where Lowess normalization was performed [
25]. Genes were filtered by requiring the Lowess-normalized intensity values in both channels to be > 30. The log
2 ratio of Cy5/Cy3 was then reported for each gene. Hierarchical clustering and Gene Set Expression Comparison were performed using BRB Array Tools software developed by the Biometric Research Branch of the NCI [
26]. Analysis was restricted to probes that reported values in 75% or more of the samples for comparative analysis or probes that in addition had a minimum 2.5 – fold expression change in either direction from the median value in at least 20% of samples, for hierarchical clustering (defined as most variable genes).
A comparative analysis of previously published microarray expression data of Brca1-deficient tumors on the same array platform [
17] was performed using Gene Cluster 3.0. To this end, we compared gene expression from 22 arrays encompassing the 5 original
Brca1 tumors from our study, with 7 tumors from
Brca1+/-,
p53+/- irradiated (IR, Kohler) mice, and 10 tumors from
Brca1Co/Co,
p53+/-,
MMTV-Cre (Furth) from a previous study [
17]. Genes [20,988] were filtered for 80% present (to remove genes that have missing values in greater than 20% of the arrays) and Standard Deviation (SD Gene Vector) >= 1.5 (to remove genes that have standard deviations of observed values less than 1.5, thus selecting for more variable genes across experiments). This filter yielded 339 genes that were used for Hierarchical Clustering. The dendrograms were visualized with Java TreeView.
Analysis of cell surface markers
Cells from each cell line were grown to 70% confluence, scraped or trypsinized, stained with PE Anti-mouse CD44 (BD Pharmingen, San Jose, CA), APC Anti-mouse CD24 (Biolegend, San Diego, CA), FITC Anti-mouse CD44 (Southern Biotech, Birmingham, AL), or APC Anti-mouse CD117 (Biolegend). Rat IgG (CHEMICON, Billerica, MA) was used as the isotype control according to manufacturer's instructions. For analysis of Cytokeratins, cells were washed in ice-cold PBS, fixed in 70% ethanol, and stained with either Cytokeratin 5 (Covance, Berkeley, CA) or Cytokeratin 18 (Abcam, Cambridge, MA), and counter-stained with Alexa 488-conjugated anti-rabbit or anti-mouse IgG, respectively. Cells were analyzed by flow cytometry using FACScalibur or LSR II (BD Biosciences, San Jose, CA). Data were collected with Cell Quest Pro software (BD) from no fewer than 10,000 cells and FACSDiVa software (BD) from no fewer than 30,000 cells, respectively.
Immunohistochemistry
Paraffin sections of original and transplanted tumors were stained for H&E for histological analysis. Histology of original tumors was compared to transplanted tumors derived from cell suspensions. Immunofluorescent staining was performed for smooth muscle actin (SMA) using mouse A2537 antibody (Sigma, St Louis, MO) at 1:1000 dilution and vimentin, using guinea pig anti-Vimentin antibody (RDI, Concord, MA) at 1:400 dilution. Slides were de-paraffinized and hydrated through a series of xylenes and graded ethanol steps. Heat-mediated epitope retrieval was performed in boiling citrate buffer (pH 6.0) for 15 min. Samples were then cooled to room temperature for 30 min. Secondary antibodies for immunofluorescence were conjugated with Alexa Fluor-488 or -594 fluorophores (1:200, Molecular Probes, Invitrogen, Carlsbad, CA).
Cell lines were plated in chamber glass slides, allowed to attach overnight, stained with APC Anti-mouse CD117 (c-kit) from Biolegend, Cytokeratins 5 and 18, and counter stained with Alexa 488-conjugated anti-rabbit or anti-mouse IgG, respectively.
Immunoblot analysis
The analysis of protein levels was performed essentially as described [
27]. Membranes were blocked at room temperature for 1 h I nTBS/0.05% Tween-20 (TBS-T) containing 5% non-fat dry milk (monoclonal antibodies) or 5% BSA (polyclonal antibodies) and exposed to primary antibodies overnight at 4°C in blocking solution. The following commercial antibodies were used: Keratin 5 (Covance); Keratin 18 (Abcam); CD117 (Abgent; San Diego, CA), p53(S15), PDGF Receptor (Anti-CD140a) (Chemicon), Vimentin (BD Pharmingen), and Actin (Ab-1) (Oncogene Research Products, Cambridge, MA). Membranes were washed twice in TBS-T and incubated with species-specific HRP-labeled secondary antibodies in TBS-T for 1 h. Finally, the membranes were washed in TBS-T three times and the proteins were visualized using ECL Western blotting detection reagents (Amersham, Piscataway, NJ).
Discussion
In spite of previously established genomic instability associated with Brca1 deficiency [
16,
27,
32‐
34], transplantation of original Brca1 mouse mammary tumors into naïve recipients and generation of cell lines from these tumors provides a reliable source of material with relatively stable expression profile. However, due to significant heterogeneity among the original tumors, a panel of individual original tumors will be required for selection of a subset of tumors with appropriate characteristics and correlation with human disease.
Transplantation of multiple mouse mammary tumor models into naïve recipients revealed remarkable stability of the cancer genome in MMTV-PyMT and -Wnt1 mammary models [
22,
35]. Similarly, transplantation of a Brca1 tumor for 2 subsequent passages preserved the molecular features associated with the tumor of origin. However, we found significant differences among the original Brca1 tumors, not previously detected in other mouse mammary models. Thus, in contrast to other mouse mammary tumor models in which the genomic features allow pooling multiple original tumors for expansion
in vivo to provide virtually unlimited starting material, Brca1 mammary tumors should be analyzed individually and not pooled. Previous studies found significant discrepancies between human disease and some mouse Brca1 deficient tumor models. These differences are likely to reflect the innate heterogeneity of Brca1 mouse mammary tumors and analysis of multiple individual tumors would be required for selection of appropriate tumors that correlate with human disease [
28]. In addition, human BRCA1-associated breast and ovarian cancers are multifocal and frequently arise in the contralateral breast. Future studies need to be performed to determine whether differences in gene expression at multiple sites correlate with the heterogeneity found in the mouse models and whether these differences correspond to mixed responses to therapy.
We have previously described that MMTV-PyMT mouse mammary tumors arise earlier in anterior mammary gland, have accelerated tumor growth and can be separated from posterior tumors by gene expression [
22]. Our current results pose the intriguing possibility that anterior mammary tumors, such as the original 0_A1 tumor, may be enriched in cancer stem cells. Previous studies indicated that normal CD24
low mammary cells are myoepithelial and have high mammary fat pad reconstitution capacity, whereas CD24
+ population have low capacity for reconstitution and thus are devoid of normal mammary stem cells [
30]. In addition, a recent report showed that freshly isolated normal mammary CD24+ cells have the capacity to form mammospheres. However, when these cells are briefly cultured, the CD24
- population becomes enriched in mammosphere-forming and mammary-gland repopulating cells, indicative of a switch in stem cell population [
36]. In our studies, the cell lines derived from the original 0_A1 mammary tumor, but not cell lines from posterior tumors contained a population of CD44
+/CD24
-/low cells. We recently confirmed that cells expressing these markers define a cancer stem cell in
Brca1 deficient cell lines [
31]. Analysis of additional markers that distinguish the anterior and posterior
Brca1 tumors, and their capacity for tumor reconstitution need to be performed to test this hypothesis.
Although transplantation preserves gene expression profile of the original Brca1 deficient tumor, it offers only a limited potential for tumor expansion for further studies since the starting material is a single mouse tumor. Analysis of multiple cell lines derived from each one of the original
Brca1 tumors revealed additional characteristics of these tumors. In spite of changes consistent with exposure to tissue culture conditions and adaptation to growth as monolayer, such as further loss of basal marker, Cytokeratin 5 [
37], and gain in expression of Pdgfr α and β, each group of cell lines segregated with their own tumor of origin. Cell lines derived from a tumor with EMT features, 0_A1, lost expression of keratins, and contained a distinct subpopulation of breast cancer stem cells that express CD44+/CD24- markers [
31]. Loss of keratin expression has been correlated with poor prognosis in human breast cancer [
38]. To our knowledge, the correlation between enrichment in breast cancer stem cells and EMT features has not previously been reported. It would be important to determine whether other BRCA1-deficient human and mouse tumors with EMT features are enriched in putative breast cancer stem cells.
Acknowledgements
The authors thank Crystal Salcido for assistance in data analysis, Barbara J. Taylor for assistance with flow cytometry, Karen MacPherson and Dorothea Dudek for bibliographical assistance. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute. Additional support was provided by Federal funds from the National Cancer Institute, under Contract No. N01-C0-12400, and by funds for CMP from the NCI Breast SPORE program to UNC-CH (P50-CA58223-09A1) and RO1-CA-101227-01.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MHW generated cell lines from tumors, characterized them by flow cytometry and western blot, genotyped and isolated RNA from tumors and cell lines, and drafted the manuscript. AIR contributed to the analysis and interpretation of microarray data and helped to draft the manuscript. JIH performed mRNA microarray hybridization, immunohistochemistry and contributed to the analysis and interpretation of microarray data. AIR and JIH contributed equally to the manuscript. MGH performed serial transplantation of tumor suspensions. MRA performed pathological assessment of tumor specimens. CMP supervised microarray analysis, data interpretation and critical review of the manuscript. LV designed the study, participated in data interpretation and manuscript preparation. All authors read and approved the final manuscript.