A total of 64 specimens were obtained from patients with invasive breast cancer who received neoadjuvant therapy at the Department of Breast and Thyroid Surgery, Southwest Hospital, Third Military Medical University (Army Medical University) between 2012 and 2016. All patients met the following criteria: (1) patients pathologically diagnosed with invasive ductal carcinoma with ER positivity, PR positivity, and human epidermal growth factor receptor 2 (HER2) negativity; (2) patients with IIB to IIIC tumor-node-metastasis staging on admission; and (3) patients receiving four to six cycles of neoadjuvant chemotherapy based on epirubicin (EPI) and/or docetaxel (DOC). The patients enrolled in this study were divided into the chemosensitive (29 patients) and chemoresistant (35 patients) groups based on their response to EPI and/or DOC regimens. The response to these drugs was evaluated according to the standard guidelines of the Response Evaluation Criteria in Solid Tumors  as follows: complete response (CR) was defined as disappearance of all lesions in both primary tumor and lymph nodes, partial response (PR) was defined as at least a 30% reduction in the sum of the longest diameter of target lesions, progressive disease (PD) was defined as at least a 20% increase in the sum of the longest diameter of target lesions, and stable disease (SD) was defined as neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD. CR and PR were classified as chemosensitive, whereas SD and PD were classified as chemoresistant . This study was approved by the Southwest Hospital Research Ethics Committee. Written informed consent was obtained from all the participants. All methods were performed in accordance with the approved guidelines.
Cells and culture
The luminal breast cancer cell lines MCF-7 and T47D were purchased from the American Type Culture Collection. MCF-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA). T47D cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% FBS. All the cells were cultured in a humidified 5% CO2 incubator at 37°C.
Immunofluorescence and hematoxylin and eosin staining
Immunofluorescence was performed as previously described [22-24]. Briefly, paraffin-embedded samples were sectioned at a thickness of 4 μm. After being warmed for 30 min at 60°C, the slides were subjected to dewaxing in xylene for 3–4 min and rehydrated by passing them through graded alcohol in decreasing concentration order as 100%, 90%, 80%, 70%, 50%, and 30% for 1–2 min in each and rinsed in distilled water. Antigen retrieval was performed using a pressure cooker for 15–20 min in 0.01 M citrate buffer (pH, 6.0) to remove aldehyde links formed during initial fixation of tissues. The slides were then blocked with 2% bovine serum albumin for 1 h at room temperature. Subsequently, the sections were incubated with mouse-anti-human CD24 (1:1000, Abcam, ab31622) and rabbit anti-human CD44 (1:1000, Abcam, ab157107) primary antibodies overnight at 4°C. Following incubation with secondary antibody goat anti-mouse IgG H&L conjugated with Alexa Fluor 488 (1:1000, Abcam, ab150113) or goat anti-rabbit IgG H&L conjugated with Alexa Fluor 568 (1:1000, Abcam, ab175471) for 1 h at room temperature and counterstaining with 4′,6-diamidino-2-phenylindole, at least three randomly selected microscopic fields were obtained using a laser scanning confocal microscope under a high-power lens (LSM780, Zeiss) and processed using a software (ZEN 3.1, Zeiss). Hematoxylin and eosin (H&E) staining was performed to identify tumor cell positions in histology to perform image registration. After immunofluorescence staining and confocal laser scanning microscopic analysis, the sections were immersed in 0.5% (v/v) hydrochloric acid for 1–2 s followed by the diluted ammonia water for several times and rinsed in tap water. After dehydration with graded alcohol, the sections were dipped in eosin solution for 30 s to 1 min, rinsed with absolute alcohol, and immersed in xylene solution for 30 s. After the excess xylene was drained and mounted with DPX, the sections were observed under a whole-slide histological scanner (Axio Scan.Z1, Zeiss) and processed using a software (Image-Pro Plus software 6.0) . Quantification of the proportion of CD44+CD24− and CD44+CD24+ tumor cells in clinical samples was determined by expression analysis of CD44 and/or CD24 in tumor cells identified by H&E staining.
Induction and characterization of chemoresistant cells
EPI- and DOC-resistant variants of MCF-7 and T47D cells were generated by pulse selection [26, 27]. To generate resistant variants, the IC90 values of EPI and DOC in MCF-7 and T47D cells were determined. MCF-7 and T47D cells were seeded in 24-well plates at 2 × 105/well overnight. Different concentrations of EPI (Selleck, s1223) and DOC (Selleck, s1148) (0, 5, 10, 20, 100, 200, 400 ng/mL) were then added. After 72 h, the cells were counted using an automatic cell counter (Bio-Rad TC10, Bio-Rad, USA). Based on the growth inhibition curves, IC90s were calculated. MCF-7 and T47D cells were exposed to IC90 concentrations of EPI or DOC for 4 h and once a week for 7 weeks, respectively, to obtain resistant variants MCF-7/EPI, MCF-7/DOC, T47D/EPI, and T47D/DOC. At the endpoint of induction, the resistance index (RI), which refers to the ratio of the IC50 of resistant variants to that of their parental cells, was determined for each resistant variant by drawing growth inhibition curves, as described above.
Quantitative reverse transcription polymerase chain reaction
The expression of multidrug resistance-associated protein I (MRPI) in MCF-7/EPI, MCF-7/DOC, T47D/EPI, and T47D/DOC was detected using quantitative reverse transcription polymerase chain reaction. The assay was performed using the SYBR Premix Ex Taq kit (TaKaRa, Japan) according to the manufacturer’s instructions. The forward primer for MRPI was (5’-3’): AACCTGGACCCATTCAGCC. The reverse primer for MRPI was (3’-5’): GACTGGATGAGGTCGTCCGT. Data were collected and analyzed using a Bio-Rad RT-PCR CFX96.
Flow cytometry analysis
During induction, a small number of cells from each of the four resistant variants were marked with CD44 and CD24 antibodies and investigated by flow cytometry. Moreover, 1 × 106 cells were resuspended in phosphate-buffered saline (PBS) containing 1% FBS and stained with APC anti-human CD44 (1:400, BioLegend, 338806) and PE anti-human CD24 (1:400, BioLegend, 311106) antibodies for 30 min at 4°C. The cells were washed with assay buffer for flow cytometry analysis. Specimens were subsequently analyzed using a NovoCyte flow cytometer (ACEA Biosciences, Inc.) [28, 29].
For multidrug resistance protein 1 (MDR1) expression, 1 × 106 cells of four resistant variants (MCF-7/EPI, MCF-7/DOC, T47D/EPI, T47D/DOC) and their parental cells (MCF-7 and T47D) were resuspended in PBS containing 1% FBS and stained with APC anti-human MDR1 (BioLegend, 348607) for 30 min at 4℃. The cells were washed with an assay buffer for flow cytometry analysis. Specimens were subsequently analyzed using a NovoCyte flow cytometer (ACEA Biosciences, Inc.).
Statistical analyses were performed using the Statistical Package for the Social Sciences version 17.0 and GraphPad Prism version 8. Results are expressed as mean±standard error of the mean. Moreover, P<0.05 was considered statistically significant. Student’s t-test (two-tailed) was used to determine the significance of the differences between the two groups of data.