Introduction
Human breast cell lines have long served as models for a wide array of applications including the study of molecular, cellular, and biochemical mechanisms that regulate breast epithelial biology. Breast cancer cell lines are also commonly used in xenograft models for drug discovery and in the assessment of pre-clinical experimental therapeutic efficacy. Despite their crucial role for rational drug discovery and development and in understanding molecular pathophysiology of cancer, their ability to accurately reflect phenotypes of tumors remains controversial. Several studies have suggested that cell lines exhibit a narrow range of genetic profiles, harbor genetic alterations due to adaptation of tissue culture environment, and are poor predictors of
in vivo sensitivity to drug efficacy [
1‐
3]. Cell line-derived xenograft models also fail to recapitulate the heterogeneous histopathology characteristic of the parent tumor histology. However, other studies have indicated that cell lines, as a system, actually mirror many of the biological and genomic properties found within primary human tumors [
4,
5]. Genomic approaches have revealed that like primary tumors, the gene expression signatures of breast cancer cell lines can distinguish luminal from basal subtypes of breast cancer [
6‐
9]. Moreover, cell line-derived gene signatures can correctly classify human tumor samples [
6,
7,
10], suggesting that despite their acquired ability to grow
in vitro, and acquired mutations following adaptation to culture conditions, cell lines continue to share many of the molecular and genetic features of the primary breast cancers from which they were derived.
The use of primary human breast tissues for experimental studies and breast cancer research has been fueled by the notion that cell lines are not accurate models of the heterogeneity found
in vivo. As such, reduction mammoplasty and cancer tissues have been used to identify and characterize epithelial differentiation states and lineages since it is presumed that not all cell types are maintained or mirrored
in vitro. Expression of epithelial cell adhesion molecule (EpCAM) and CD49f
+ (α6 integrin) have been used to identify luminal and basal/myoepithelial cells from breast tissues [
11‐
14]. Mature luminal cells are reported to express an EpCAM
+/CD49f
- phenotype while luminal progenitors express an EpCAM
+/CD49f
+ marker profile. Myoepithelial cells and basal progenitor cells are defined by an EpCAM
-/CD49f
+ phenotype [
11,
13,
15]. In addition to EpCAM and CD49f, surface expression of CD44 and CD24 have also been used to identify luminal epithelial cells that express genes involved in hormone responses (CD24
+) and cells resembling progenitor cells that express genes involved in motility (CD44
+) [
16].
Reflecting the normal cell types within the breast, tumors are broadly classified histopathologically by expression of either luminal cytokeratins (CK8/18) or stratified epithelial cytokeratins (CK5/6/14, basal-type) [
17,
18]. Similarly, tumor subclasses identified by microarray were named to reflect the gene expression patterns of the normal breast luminal and myoepithelial/basal cells [
19‐
23]. Luminal-type breast cancers (Luminal A and Luminal B) express estrogen receptor (ER). Her2-type breast cancers typically overexpress or amplify Her2, are generally negative for ER expression and tend to express the genes associated with the Her2-amplicon. Lastly, Basal-like breast cancers are also often referred to as triple-negative tumors since they do not express ER, progesterone receptor (PR), or Her2 [
19‐
22].
To determine if cell lines mirror or maintain the cellular differentiation states found in primary tissues, we examined the molecular and cellular profiles of normal and malignant human breast epithelial cell lines and compared them to normal and cancerous tissues. In doing so, we found four distinguishable cell states across a collection of cell lines that mirrored the four differentiation states present within normal and malignant breast tissues. However, we also found that the cellular heterogeneity within cell lines was remarkably restricted in culture and was enriched for cellular phenotypes that were normally present as a minor component in vivo.
Materials and methods
Cell lines and tissue culture
SUM cell lines were obtained from Dr. Stephen Ethier (Kramanos Institute, Detroit, MI, USA) and are commercially available (Asterand, Detroit, MI, USA). The MCF7, T47 D, BT20, MCF10A, MCF10F, MDA.MB.231, MDA.MB.361 and HCC cell lines were obtained directly from the American Type Culture Collection (ATCC; Manassas, VA, USA). The MCF10A and MCF10F cell lines are non-tumorigenic mammary epithelial cell lines that were produced by long-term culture in serum-free medium with low calcium; the MCF10A cells were derived from an the adherent population in these cultures, while the MCF10F line was derived from floating cells within the MCF10 cultures [
24]. All of the ATCC cell lines used in this study were low passage (< 10). SUM225CWR, SUM149PT, and SUM159PT cells were cultured in F12 with 5% calf serum (CS), insulin (5 μg/ml), and hydrocortisone (1 μg/ml), while SUM1315 MO2 cells were cultured in F12 with 5% CS, insulin (5 μg/ml), and hEGF (10 ng/ml). MCF7, MDA.MB.361, BT20, and all HCC cell lines were cultured in DMEM with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). MDA.MB.231 and T47 D cells were cultured in Roswell Park Memorial Institute-1640 (RPMI; Hyclone, Logan, UT, USA) with 10% FBS. The TUM177 breast cancer cell line was established from a primary invasive ER-positive adenocarcinoma. An ER-negative cancer cell line spontaneously emerged after two months of cultivations. TUM177 cells were cultured in DMEM with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA).
HME I and HME II cells were derived from reduction mammoplasty tissues from two different patients grown in Mammary Epithelial Growth Medium (MEGM) until the generation of variant cells [
25] and then immortalized through the ectopic expression of the catalytic subunit of human telomerase (hTERT) [
26].
MCF10F cells were cultured in Dulbecco's modified Eagle's medium-Ham's F12 (DMEM/F12; 1:1) with 5% horse serum, insulin (5 μg/ml), hydrocortisone (1 μg/ml), and human epidermal growth factor (hEGF; 10 ng/ml), and cholera toxin (100 ng/ml) (all, Sigma, St. Louis, MO, USA). MCF10A and immortalized human mammary epithelial (HME) cell lines were cultured in MEGM supplemented with bovine pituitary extract (52 μg/ml), hydrocortisone (0.5 μg/ml), hEGF (10 ng/ml) and insulin (5 μg/ml) (MEGM Bullet Kit, Lonza Corporation, Walkersville, MD, USA). MCF10A cells were further supplemented with cholera toxin (100 ng/ml). For serum differentiation experiments, HME or MCF10A cells were switched to growth in the MCF10F medium with substitution of 5% CS for the horse serum and omission of the cholera toxin, or 5% CS was added to MEGM and cells were allowed to differentiate for six days before use in experiments. For mammosphere culture, cells were plated at 20,000 cells/ml and grown on ultra-low adherence six-well plates for one week (Corning Life Sciences, Lowell, MA, USA). Quantification of mammospheres was accomplished using a Multisizer 3 COULTER COUNTER (Beckman-Coulter, Brea, CA USA) that provides number, and size distributions with an overall sizing range of 14 μm to 336 μm.
Reduction mammoplasty and tumor tissue specimens
All human breast tissue procurement for these experiments was obtained in compliance with the laws and institutional guidelines, as approved by the Institutional Review Board committee from Beth Israel Deaconess Hospital and Tufts Medical Center. Fresh disease-free reduction mammoplasty tissues (n = 12) and tumor tissues (n = 15; 8 fresh, 15 formalin-fixed paraffin embedded) were obtained from discarded material from patients undergoing elective reduction mammoplasty surgeries or from patients undergoing partial or complete mastectomy for excision of tumor tissue from the Pathology departments at BIDMC or Tufts Medical Center. All samples were obtained from de-identified discarded material and therefore, informed consent was not required for these studies. All samples were evaluated histologcially and confirmed to be invasive ductal carcinomas. The following histopathologic variables, determined for all tumor tissue specimens, were done on full sections, and cases with 10% or more positive for ER, p53 or EGFR staining were grouped as positive. The scoring of Her2 was performed using the ASCO/CAP guidelines, as follows: Cases with 30% or more strongly positive cells with strong complete membrane staining were defined as Her2+ tumors. Cases with 10% or more positive cells with weak to moderate complete membrane staining were considered Her2+ but were not defined as Her2+ tumors solely on this basis. IHC analysis for estrogen receptor (ER), progesterone receptor (PR), Her2, p53 and EGFR were independently reviewed by expert breast pathologists (HG and SN). Breast tumor subtypes were defined as follows: Luminal A (ER+ and/or PR+, Her2-), Luminal B (ER+ and/or PR+, Her2+), Her2+ (ER-, PR-, Her2+), and Basal-like (ER-, PR-, Her2-, and epidermal growth factor receptor (EGFR)+/-) and p53+.
Uncultured cells from reduction mammoplasty or human breast tumor organoid preps [
27] were dissociated to a single-cell suspension by trypsinization and filtered through a 20 μm nylon mesh (Millipore, Danvers, MA, USA). Human breast tumors were plated in DMEM supplemented with 10% CS for one to two hours to deplete stromal cells.
Immunohistochemical analysis and scoring
Immunohistochemistry was performed by the Histology Special Procedures Laboratory at Tufts Medical Center on paraffin-embedded tissue sections on a Ventana (Tucson, Arizona, USA) automated slide stainer with the iVIEW DAB detection kit for visualization. Antibodies used were CK14 (1:500, clone LL002, Vector (Burlingame, CA, USA)), CK8/18 1:500, clone DC-10, Vector), Vimentin (1:500, clone V9, Vector), S100A4 (1:200, clone 1F12-1G7, Sigma), S100A6 (1:200, clone CACY-100, Sigma), p53 (Ventana Medical Systems), ER (Ventana Medical Systems), Her2 (Ventana Medical Systems), EGFR (1:20, clone 31G7, Zymed), and PR (Ventana Medical Systems). All Ventana antibodies are prediluted.
IHC and IF results were semi-quantitatively analyzed in a blinded fashion across multiple patient samples using a scoring metric in 10% increments. Negative staining represents 0 to 10% of the cell staining and was given a score of 1; mixed staining represents moderate to strong intensity staining of cells with > 10% but < 50% positive cells and was given a score of 2; and positive staining represents strong intense staining with > 50% cells staining positive and was given a score of 3. The staining intensity and percent staining scores were added to obtain a total stain score for each field. An average total stain score was calculated for the staining for a particular sample. Statistical analysis was performed using the student's t-test across the different patient samples.
Flow cytometry and FACS
Uncultured cells from reduction mammoplasty tissues (n = 12) or primary breast tumor tissues (n = 8) from organoid preparations were dissociated to single-cell suspensions, as described above. For reduction mammoplasty tissues, endothelial, lymphocytic, monocytic, and fibroblastic lineages were depleted with antibodies to CD31, CD34 and CD45 (all Thermo/LabVision, Fremont, CA, USA) and Fibroblast Specific Protein/IB10 (Sigma) using a cocktail of Pan-mouse IgG and IgM Dynabeads (Dynal, Invitrogen) according to the manufacturers instructions and as described previously [
28]. Depleted single cells suspensions were resuspended at 1 × 10
6 cells/ml in phosphate-buffered saline containing 1% calf serum (FACS buffer, FB) and bound with fluorescently-conjugated antibodies to human EpCAM (APC), CD49f (PE), and CD24 (FITC) (all, BD Biosciences, San Jose, CA, USA) for 20 minutes at 4°C. Antibody-bound cells were washed and resuspended at 1 × 10
6 cells/ml in FB and run on a FACSCalibur flow cytometer. Flow cytometry data was analyzed with the Flowjo software package (TreeStar, Ashland, OR, USA).
For fluorescence-activated cell sorting (FACS), cells from reduction mammoplasty tissue were prepared as above for flow cytometry and resuspended at 5 x106 cells/ml in FB and sorted on a BD Influx Cell sorter (BD Biosciences) into culture medium (MEGM) containing 50% CS.
For cell lines, non-confluent cultures of cells were trypsinized into single cell suspension, counted, washed with PBS, and stained with antibodies specific for human cell CD24 (PE) and CD44 (APC) (BD Biosciences). The cells were stained with antibodies specific for human cell surface markers: EpCAM-fluorescein isothiocyanate (FITC), CD24-phycoerythrin (PE), and CD49f-PE-Cy5 or CD44-allophycocyanin (APC) (BD Biosciences). Additional cells were stained with isotype controls for each antibody: Ms IgG1-FITC, Ms IgG2a-PE, and Rat IgG2a-PE-Cy5 or Ms IgG2b-APC (BD Biosciences). A total of 200,000 to 800,000 cells were incubated with antibodies or isotype controls for 20 minutes on ice. The cells were washed with PBS to remove any unbound antibody and analyzed no later than one hour post-staining on a FACSCalibur flow cytometer (BD Biosciences). Antibody-bound cells were resuspended at 1 × 106 cells/ml in FB and run on a FACSCalibur flow cytometer (BD Biosciences) or sorted on an BD Influx FACS sorter (BD Biosciences). Flow cytometry data was analyzed with the Flowjo software package (TreeStar). Each cell line was analyzed in three to five different biological replicates.
Immunofluorescence
Collected cell fractions from FACS were counted and cytospun onto glass slides at 10,000 cells per spot with a Cytospin 4 cytospinner (Thermo Scientific, Waltham, MA, USA). Cultured cell lines were plated at 10 to 20,000 cells per well in eight-well chamber slides (BD Biosciences) and grown two to three days. Cytospins and cells in chamber slides were fixed in 100% methanol and stained overnight at 4°C with primary antibodies directed to EpCAM (VU-ID9, 1:100, Stem Cell Technologies, Vancouver, BC, Canada), CK8/18 (5D3, 1:500, Vector Labs, Burlingame, CA, USA), ERα (1D5, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) CK14 (ASM-1, 1:500, Thermo Scientific/LabVision), α-smooth muscle actin (SMA; 1:250, Vector Labs) and vimentin (V9, 1:500, Vector Labs) followed by secondary antibodies (1:500 Alexa488 or Alexa555 conjugated anti-mouse and anti-rabbit H+L IgG, Invitrogen) for one hour at room temperature. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI) and images were captured with the Spot imaging software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA); staining was analyzed by counting the total number of cells positive stain compared to the total number of cells in multiple fields with at least 50 cells analyzed per condition. Negative staining represents no cells staining positive, Mixed staining is > 1% but < 50% of the cells staining positive, while positive staning is > 80% of the cells staining positive.
An average total stain score of a cell line was calculated using three to five different regions of the plate. Statistical analysis was performed using the student's T-test across the different patient samples.
Animals and surgery
All animal procedures were performed in accordance with an approved protocol submitted to the Tufts University Institutional Animal Care and Use Committee. A colony of NOD/SCID mice was maintained under sterile conditions and received food and water ad libum. Nulliparous female mice aged 8 to 10 weeks were utilized in all experiments. For tumor latency studies, 1 × 106 human breast cancer cells were resuspended in media and Matrigel (1:1; BD Biosciences) and injected orthotopically in a total of 4 to 10 different glands. Tumor formation was assessed by palpitation at least once a week, and tumor growth curves were calculated from weekly caliper measurements as previously described. Tumor latency is described as the time it takes for a tumor to reach a diameter of 1 cm.
Statistical analysis
Fisher exact tests were used when comparing the binary categories of expression of proteins between groups. All P-values reported are two-sided.
Discussion
We have used flow cytometry and immunostaining for lineage markers to identify four epithelial cell states present within normal human breast epithelial tissues and have shown that these cell states can be used to stratify a panel of human breast cancer cell lines. Through use of a three-marker strategy, we have subdivided human breast tissue into Luminal 1 cells, characterized by the majority of cells having an EpCAM
hiCD24
+CD49f
- profile; Luminal 2 cells, characterized by a majority of EpCAM
hiCD24
+CD49f
+ cells; Basal cells, characterized by EpCAM
+/loCD24
-CD49f
+ cells, and Mesenchymal cells, characterized by EpCAM
-CD24
-CD49f
+ cells. Our description of four major cell types within breast tissue is similar to previously published reports describing epithelial populations through the use of EpCAM and CD49f staining [
11‐
15]. Notably, Villadsen
et al. described two luminal populations representing lobular and ductal-oriented luminal cells characterized as EpCAM
hiCD49f
- and EpCAM
hiCD49f
+, respectively, and lobular and ductal myoepithelial/basal populations with EpCAM
lo/-CD49f
+ phenotypes [
11].
Recently, several groups have identified breast bi-potent progenitor/stem-like activity in EpCAM
+/hiCD49f
+ populations but also in EpCAM
-/loCD49f
+ populations [
11‐
15]. These conflicting differences may arise from use of different fluorescently conjugated antibodies for flow cytometry and gating strategies. Alternatively, it could be that human breast tissue may contain two distinct populations of bi-potent stem/progenitor cells. Consistent with this notion, ductal (CD24
loCD49f
hi) and lobular/alveolar (CD24
hiCD49f
lo) progenitors that both give rise to luminal and myoepithelial cells have been described in the mouse mammary gland [
36,
37]. By using CD24 to further define luminal populations in human breast tissues, it may be that EpCAM
hi/+/CD24
-/CD49f
+ and EpCAM
lo/+/CD24
-/CD49f
+ represent the lobule and ductal progenitors in the human breast. CD24
+ cells have been previously described to be associated with the EpCAM
+CD49f
+ luminal progenitors [
14]. However, we have observed that CD24
+ cells are found in both the EpCAM
hiCD49f
- and EpCAM
hiCD49f
+ populations. It is worth speculating that the use of CD24 as an additional marker might reveal different bi-potent potentials of progenitor cells. Indeed, we found that HMEC lines with bi-potent and differentiation potential contained EpCAM
+/CD24
-/CD49f
+ cells, while those that were nearly all EpCAM
-/CD49f
+ cells were only able to differentiate into an EpCAM
-/CD24
+ phenotype which does not exists in human breast tissue. Therefore, future studies that further define the normal breast epithelial cell hierarchy using additional markers will be necessary to fully understand the complex cell types and differentiation states in human tissues.
In this small study, we surprisingly found that the majority of human breast cancer tissues exhibited a EpCAM
+/CD49f
+ luminal epithelial differentiation phenotype regardless of their molecular subtype. This is consistent with immunohistochemistry studies that have reported that breast cancers largely express luminal makers despite being of the basal molecular subtype [
38]. We found that in tissues and cell lines, the EpCAM
+/CD49f
+ phenotype contains both CD24
+ and CD24
- cells. In reduction mammoplasty tissues, EpCAM
+/CD24
-/CD49f
+ cells exhibited a basal cytokeratin phenotype while breast cancer cell lines with a basal-like phenotype also contained a unique population of EpCAM
+/CD24
-/CD49f
+ cells. Gene expression profiling of cell lines that exhibit a large EpCAM
+/CD49f
+ population most closely corresponded with the expression profile of Basal-like breast tumors [
14] suggesting that EpCAM
+/CD49f
+ cells may be the cellular precursors to both luminal and basal-like tumors. Future studies will need to be performed to determine if this is indeed the case.
We found that adherent cultures of normal human breast epithelial cells and to a lesser extent, cancer cell lines lead to enrichment of cells that exhibited basal and mesenchymal differentiation states with limited capacity to differentiate into fully-committed luminal cells. This suggests that standard adherent culture may select preferentially for cells of basal-orientation, or may result in epigentic loss of luminal differentiation programs.
Data from studies in mouse mammary glands and human tissues suggest that bi-potent progenitor/stem-like activity is correlated with the formation of colonies that contain cells of both luminal and basal lineages, defined by keratin CK8/18/19 or CK14/5 expression, respectively. However, since luminal cells are lost following in vitro cultivation, this suggests that bi-potent progenitor/stem-like activity from luminal cells has not been well studied. This does not discount the evidence that mammary stem-like cells have basal characteristics but it does suggest that in vitro methods need to be improved to allow for maintenance or cultivation of cells of the luminal lineage to better model cells that are likely of great importance for human breast tumor development.
In this study, we found that the morphology and molecular classification of several cell lines differed from those previously reported by others [
7,
40,
41]. In this study, all the commercially available cell lines were obtained directly from ATCC or from Dr. Ethier, were characterized at low passage (less than 10 passages) and were grown in specified medium. Under these conditions, we found a strong association between epithelial or spindle-cell morphology, marker expression (CK14, CK18, vimentin, and EpCAM), and the proportion of CD44
+/CD24
- cells. It is well established that cancer cell lines evolve over time in culture and may be influenced by a variety of factors including confluency, media compositions as well as passage number. Thus, it is highly likely that as certain cell lines have evolved in culture when grown under differing conditions and in turn have acquired different morphological features. However, it is likely the case that such cell lines could still be classified on the basis of cell surface phenotypes and be grouped into one of the four breast epithelial differentiation states. Future studies will be needed to determine whether the plasticity of the cell state dynamics within cancer cell lines is due to
de novo acquired mutations or due to epigenetic changes associated with extracellular environment.
Conclusions
Our data indicate that, while cell lines as a group indeed represent the heterogeneity of human breast tumors, individually, they exhibit a notable increase in lineage-restricted profiles that falls short of truly representing the intratumoral heterogeneity of individual breast tumors, regardless of their molecular classification. This is in large part due to the loss of Luminal 1 cells in culture, which represents a major cell phenotype of normal and malignant breast tissues. Additionally, we found that normal human breast epithelial cell lines, like cancer cell lines, have a Basal/Mesenchymal-restricted lineage phenotype under normal serum-free culture conditions but that they can be induced to partially differentiate under serum-containing conditions. However, the four normal breast cell lines tested, representing some of the most commonly used cell lines for studying the behavior of mammary epithelial cells in culture, have a phenotype that does not represent the major cell types within breast tissue, namely, differentiated luminal epithelial cells and luminally-oriented progenitors. These results serve as a resource for further understanding the behavior and origins of breast cell lines, which are crucial and widely used research models. However, they also demonstrate that additional models and cell lines are needed to more accurately depict and study human breast epithelial cell types and tumors in a manner that is more efficient for developing effective therapies. These findings also indicate that further studies are needed to identify culture conditions that can allow for the growth and expansion of Luminal 1 cells, which seem to be unable to survive or expand in vitro.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PJK, AL, LMA and ADJ took part in the conception and design of the study, the collection of data, data analysis and interpretation, and manuscript writing. IK collected and/or assembled data. CF and CMP took part in the collection of data, and data analysis and interpretation. JAR and TAD collected data. HG, SS, RAG, DJ and SN dealt with the provision of study materials, including the procurement of resources and samples (cell lines or reduction mammoplasty and tumor tissues). CK took part in the conception and design of the study, the collection and/or assembly of data, data analysis and interpretation, manuscript writing and financial support. All authors approved of the final manuscript.