Abstract
Abstract The aim of this work is to elucidate the role of the CXCR4-CXCL12 axis in ER+ breast cancer with spatial proteomics profiling. The CXCR4-CXCL12 signaling pathway has emerged as an axis of interest in cancer tumorigenesis 1,2,3. Current clinical trials aim to increase immune cell inflammation in solid tumors by blocking this pathway. Our analysis of bulk-RNAseq revealed ER+ breast cancer has high gene expression levels of CXCL12 transcripts, making it a potential tumor indication to investigate. We spatially characterized 20 ER+ breast cancer FFPE samples by developing a 13-plex antibody panel for the Akoya PhenoCycler®-Fusion system. This panel was designed to identify immune cells (CD45, CD3e, CD4, CD8, FoxP3, CD68), stromal cells (FAP, SMA, Podoplanin, S100A4), tumor and epithelial cells (PanCK) as well as the signaling duo (CXCR4, CXCL12). We performed cell segmentation on the multiplex immunofluorescence images, and quantified marker intensity in each cell. Phenotypes were manually identified after normalization, integration, and clustering of over 10+ million cells across 20 samples. Overall, the composition of tumor and immune cells varies significantly between patients showing the heterogeneity of their microenvironments. We observed CD8 T-cell exclusion in tumors associated with infiltration of fibroblasts (FAP+, SMA+, PDPN+) that do not express CXCL12. Instead, the CXCL12 signal is highly correlated with PanCK in all samples suggesting that either tumor cells are producing the chemokine or are being “coated” with the CXCL12 produced by fibroblasts. We also identify at least three known CXCR4+ immunosuppressive populations (fibroblasts, macrophages and Tregs) in the tumors which are known to be recruited via CXCR4-CXCL12 signaling4,5. This immunosuppressive tumor micro-environment (TME) results in the CD8 T-cell exclusion we observe in our samples as well as mentioned in multiple studies1,6. In summary, these results show the exclusion of cytotoxic T cells in the TME and the intricate relationship between tumor cells and immunosuppressive cells through the CXCR4-CXCL12 axis. This study shows the benefits of the Akoya platform compared to conventional IHC as well as the power of spatial phenotyping to study the TME immune biology for development of therapeutic strategies.
Published Version
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