Single-cell transcriptomic profiling of immune landscape in triple-negative breast cancer during neoadjuvant chemotherapy
Triple-negative breast cancer (TNBC) is the most aggressive subtype, typically requiring neoadjuvant chemotherapy (NAC) as an obligatory component of the treatment regimen. Achieving a pathological complete response to NAC is associated with improved long-term outcomes for patients with TNBC. The functional status of the immune system plays a critical role in NAC efficacy. Herein, we presented the first investigation of systemic and local immune landscape during the initial course of NAC treatment and identify factors that contribute to chemotherapy resistance of TNBC. Using single-cell RNA sequencing, we demonstrated that the transcriptional profile remained stable in a patient who responded to NAC, while a non-responder exhibited significant dysregulation in the expression of genes involved in stress response, apoptosis, immune cell proliferation, and differentiation within lymphocyte and monocyte populations. During the first course of NAC, circulating cytotoxic CD8 T cells in the non-responder patient overexpressed granzymes B and H, granulysin, and perforin. In contrast, expression of these factors decreased in CD8 T cells within the tumor. Finally, we identified for a first time a signature of myeloid-derived suppressor cells (MDSC) within the S100?highMHClow monocyte population and calculated an MDSC score for both the responder and the non-responder TNBC patients. An elevated MDSC score in the non-responder was validated using data from an independent cohort of patients with poor NAC response. Our data underscores the importance of immune system functionality in determining chemotherapy efficacy in TNBC. Keywords: triple-negative breast cancer, immune cell, cytotoxic CD8 T cell, chemotherapy, myeloid-derived suppressor cell, single-cell RNA sequencing. Overall design: The study enrolled two women who had been diagnosed with TNBC and underwent NAC. Both patients received 2-4 courses of adriamycin with cyclophosphamide followed by 4 NAC courses of taxanes before surgery. The first patient received a partial mastectomy and showed no evidence of tumors cells in the breast tissue or lymph nodes. The second patient demonstrated progression of TNBC during the fourth cycle of NAC. Therefore, the NAC was interrupted and a radical mastectomy was performed.  Two months after the surgery, metastases developed in her pelvic bones, leading to the patient's death two months later. The patient with a poor response to therapy and unfavorable outcome was referred to 'Non-responder,' while the patient with a good response and favorable outcome was referred to 'Responder.' Peripheral blood samples were collected from both TNBC patients at three time points: before NAC and on days 3 and 21 after the administration of the 1st NAC course. For non-responder patient were available biopsy samples of tumor tissue before NAC and on days 3 and 21 after the first course of NAC. The cellular viability of all PBMCs and tissue samples exceeded 75%, as evaluated by Calcein (BD Bioscience, USA) vs DRAQ7 (Thermo Fisher Scientific, USA) via flow cytometry. According to the manufacturer's protocol, 8 samples (6 PBMCs and 3 tumor cells suspensions) were labeled by Sample Tag and pooled for single-cell capture with the BD Rhapsody Single-Cell Analysis system (BD Bioscience, USA). Single-cell suspensions with PBS were loaded into microfluidic devices using the BD Rhapsody Single-Cell Analysis System (BD Bioscience, USA). Subsequently, the scRNA-seq libraries were constructed according to the protocol of the Immune Response Targeted Panel (Human) Single Cell RNA Library Kit (BD Bioscience, USA). Individual libraries were diluted and pooled according to the manufacturer's recommendations. Finally, Illumina NextSeq 2000 sequencing was performed using NextSeq 2000 P2 reagents (100 cycles). ***NOTE: FASTQ files contain multiple samples multiplexed by Sample Tag of BD Rhapsody. All required information to demultiplex was provided in metadata.tsv and sample_info.txt.