Mapping Heterogeneity of Breast Tissues

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 × 10⁶ 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 × 10⁶ 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 IgG_2a-PE, and Rat IgG_2a-PE-Cy5 or Ms IgG_2b-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.

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