Identification of tumor microenvironment for high infiltration of lymphocytes in triple negative breast cancer

Elucidating the roles of intra-tumoral macrophage subtypes that promote a high-TIL microenvironment in triple-negative breast cancer. High levels of tumor-infiltrating lymphocytes (TILs) within the stromal compartment are associated with favorable prognosis and improved response to immune checkpoint blockade in triple-negative breast cancer (TNBC). To elucidate the mechanisms underlying the divergence between TIL-high and TIL-low tumor microenvironments (TMEs), we analyzed treatment-naïve, early-stage TNBC samples. We profiled the TME of 21 patients using single-cell-resolution spatial transcriptomics on FFPE tissue microarray blocks. Patients were classified into high TIL group with over 60% ratio of TIL in stroma. sTILs (0-100%) were evaluated on H&E-stained slides, and tumors with sTIL ≥ 60% and ≤ 10% were classified as high and low TIL, respectively. These thresholds are stricter than the commonly used sTIL ≥ 50% and < 30% cutoffs associated with distant RFS. Using this classification, 11 patients were assigned to the TIL-high group and 10 to the TIL-low group. We clustered 40 cell types for TNBC samples using Xenium 5K platform and identified 12 spatial domains by uncovering recurrent cellular neighborhoods (RCNs) that share similar spatial patterns using SCIMAP(v2.2.11). Among those 12 spatial domains, domain 9 and domain 0 represented tumor center and tumor edge, respectively. We identified 10 myeloid cell subtypes and discovered macrophage subtype MC2 expressing LDHA and PLAUR are enriched specifically in tumor domains consisting of domain 9 and 0. Interestingly, the MC2(LDHA_PLAUR) subtype present low PLAUR expression in TIL-high group compared to TIL-low group in tumor domains. Although the proportion of infiltrating MC2 (LDHA_PLAUR) macrophages within tumor domains was higher in the TIL-high group compared with the TIL-low group, the expression level of PLAUR within MC2 cells was paradoxically lower in the TIL-high group. Additionally, the MC2(LDHA_PLAUR) expressed more IFN-stimulated genes such as CXCL9, GBP5, and IRF1 in TIL-high group compared to TIL-low group. PLAUR has been known for inducing HIF1A highly in tumor microenvironment. As HIF1A hinders IFNG-signaling important to establish TIL-high environments, high expression of PLAUR promotes inhibition of IFNG-signaling in macrophages. So, low expression of PLAUR for MC2 in TIL-high group helps normal IFNG-signaling pathways in macrophages. Taken together, these findings indicate MC2 macrophages located in both the cancer center and edge regions showed reduced PLAUR expression, rendering them less susceptible to HIF1A-driven suppression and allowing sustained IFNG-signaling in high-TIL tumors. This suggests that these MC2 cells help establish an intra-tumoral environment conducive to TIL infiltration by continuously supplying CXCL9, CXCL10, and CXCL11.