Cell lines, cultures, drug treatments, and plasmid transfections
This study used breast cancer cell lines obtained from ATCC, including ERα+ (MCF-7 and T47D) and ERα- (MDA-MB-231). The MCF-7 cells were maintained in Dulbecco's Modified Eagle Medium (Cat No. 11995, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Cat No. 16000, Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin (Cat No. 15140, Invitrogen). The T47D and MDA-MB-231 cells were grown in Roswell Park Memorial Institute 1640 (Gibco, El Paso, TX) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cells were cultured at 37°C in the presence of 5% CO2. The cells were starved for 24 hours and treated with ICI (fulvestrant, Sigma-Aldrich, St. Louis, MO, 1 to 10 µM), IFN-γ (R&D Systems, Minneapolis, MN, 100 units/ml), or β-estradiol (Sigma-Aldrich, 1 nM) in 2 ml of medium for an appropriate time. The cells were then used in the protein expression assays.
For the ESR1 plasmid transfection, ERα-cells were plated and cultured in a 6-well plate at 90% confluency and transfected with 2.5 µg of hESR-GFP (Cat No. #28230, Addgene, Cambridge, MA) using 3.75 µl of Lipofectamine® 3000 reagent (Life Technologies) and 5 µl of P3000™ reagent (Life Technologies) per well according to the manufacturer’s protocol.
Protein isolation and Western blotting
The cells were lysed with RIPA buffer, and the Pierce BCA Protein Assay Reagent Kit (Cat No. 23225, Thermo Fisher, Waltham, MA) was used to measure the protein concentration. Approximately 10 µg of protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA). Table 1 summarizes the antibodies used for protein detection. Equal loading of the protein samples was verified with an antibody to β-actin. Immunoreactive signals were detected with the Promega Western Blot Detection System (Cat No. W1008).
GEO data analysis
We analyzed one dataset from the GEO database that included Affymetrix RNA microarray analysis data from MCF-7 breast cancer cells that were transfected with ESR1 shRNA (5’- GCTTCAGGCTACCATTATGttcaagagacataATGGTAGCCTGAAGCttttttacgcgt -3’) (accession number: GDS4061). The fold changes of ESR1, HLA-A, and MxA mRNA expressions were calculated.
Patients and tissue specimens
A total of 126 patients who were diagnosed with ER+/HER2- invasive ductal carcinoma were randomized to receive either estrogen modulator treatment or chemotherapy for 24 weeks as their neoadjuvant systemic therapy. We analyzed the clinicopathologic data of the patients and the HLA-ABC and ER protein expressions and TIL levels in the pre-neoadjuvant biopsy tissues and the post-neoadjuvant resected tissues.
The histologic type was defined based on the 2012 WHO classification criteria, and the histologic grade was assessed using the modified Bloom–Richardson classification. The hematoxylin and eosin (H&E)-stained slides were histopathologically analyzed for TILs (defined as the percentage of the invasive carcinoma’s stroma that was infiltrated by lymphocytes in 10% increments, if less than 10% of the stroma was infiltrated by TILs, 1% or 5% criteria were used, all available full sections were evaluated), histological subtype and grade, tumor size, pT stage, pN stage, and lymphovascular invasion.[1, 20] The tumor response to neoadjuvant systemic therapy was evaluated based on the Miller-Payne grade (1, no change, 2, up to 30% reduction, 3, 30–90% reduction, 4, more than 90% reduction, 5, no residual malignant cells). A pathologic complete response (pCR) was defined as the absence of residual invasive cancer cells in the breast and lymph nodes.
Tissue microarray construction and immunohistochemical evaluation
Among the 126 patients, fifty-six were available for formalin-fixed, paraffin-embedded (FFPE) tissue blocks of both biopsies and resected specimens. Each resected tissue sample was arrayed in three 1-mm diameter cores to minimize tissue loss and overcome tumor heterogeneity. Full sections of biopsy tissues and tissue microarray sections of the resected tissues were stained with an automatic immunohistochemical staining device (Benchmark XT, Ventana Medical Systems, Tucson, AZ). Table 2 summarizes the antibodies used for the immunohistochemical staining.
ER and progesterone receptor (PR) levels were regarded as positive if there was at least 1% positive tumor nuclei staining. Additionally, the Allred score, which is the sum of the intensity score (0–3) and the proportion score (0–5), was calculated for the ER and PR. HR+ tumors were defined as those determined to be ER-positive and/or PR-positive. HER2-overexpressing tumors were defined as those with scores of 3+ according to the immunohistochemistry or gene amplification by silver in situ hybridization. The HLA-ABC staining was semi-quantitatively evaluated as the H-score, which is the product of the actual percentage of positive-stained cells and the intensity score (0–3), the H-score can range from 0–300. We then categorized HLA-ABC expression in tumor cells as one of two levels (negative, H-score < 50, positive, H-score ≥ 50).
All statistical analyses were performed using R version 3.2.3. The Wilcoxon rank-sum test, chi-square test, Fisher’s exact test, and log-rank test were used as appropriate. All tests were two-sided, and statistical significance was set at 5%.