Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted therapies in colon cancer

Single cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application towards dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a conventional dendritic cell population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing. For Patient P0104, P0305, P0309, P0411, P0720, P0728, P1212, P1228, P0413, and P0825, single-cell RNA-seq were performed by the SMART-seq2 platform. Single cell suspensions collected from CRC samples were stained with antibodies against CD45 for FACS sorting, performed on a BD Aria III instrument. To enrich myeloid cells, single cells were further enriched either by gating CD3-, CD3-CD19-/CD79A- or CD3-CD19-CD56- cells to exclude lymphoid cells. For B cells and NK cells, single cells were enriched by gating 7AAD-CD45+CD3-CD19+CD56- and 7AAD-CD45+CD3-CD19-CD56+. For non-immune cells, single cells were enriched by gating 7AAD-CD45-. For Patient P0104, P0123, P0202, P0305, P0323, P0408, P0410, P0613, P1025, P1026, single-cell RNA-seq were performed by the 10x Genomic single cell 3’ library platform. Single cell suspensions collected from CRC samples were stained with antibodies against CD45 for FACS sorting, performed on a BD Aria III instrument. To enrich myeloid cells, single cells were further enriched either by gating CD3- or CD3-CD19-/CD79A-CD56-, cells to exclude lymphoid cells. For T and monocyte-lineage cells, single cells were enriched by gating 7AAD-CD45+CD3+ and 7AAD-CD45+CD3-CD14+ cells, respectively. *** Raw sequence data access provided at: China Genomic Sequence Archive (GSA) (under accession HRA000056 https://bigd.big.ac.cn/gsa-human/browse/HRA000056).