Cxcr3 promotes protection from colorectal cancer liver metastasis by driving NK infiltration and plasticity

The anti-metastatic activity of NK cells is well established in several cancer types, but the mechanisms underlying NK cell metastasis infiltration and acquisition of anti-tumor characteristics remain unclear. Herein, we investigate the cellular and molecular factors required to facilitate the generation of an ILC1-like CD49a+NK cell population within the liver metastasis environment in orthotopic mouse models of colorectal cancer (CRC). We show that CD49a+NK cells have the highest cytotoxic capacity among metastasis-infiltrating NK cells in the MC38 mouse model. Furthermore, the chemokine receptor CXCR3 promotes CD49a+NK cell accumulation and persistence in metastasis where NK cells co-localize with macrophages in CXCL9 and CXCL10 rich areas. We confirmed the accumulation of CXCR3+ NK cells in metastatic samples mined from a published sc-RNAseq dataset of a cohort of treatment-naïve CRC patients. Conditional deletion of Cxcr3 in NKp46+ cells and antibody-mediated depletion of metastasis-associated macrophages markedly impair CD49a+NK cell development, indicating that CXCR3 and macrophages contribute to efficient NK cell localization and polarization in liver metastasis. Conversely, CXCR3neg NK cells maintain a CD49a- phenotype in metastasis with reduced parenchymal infiltration and tumor killing capacity. Furthermore, CD49a+NK cell accumulation is impaired in an independent SL4-induced CRC metastasis model, which fails to accumulate CXCL9+ macrophages. Together, our results highlight a role for CXCR3/ligand axis in promoting macrophage-dependent NK cell accumulation and functional sustenance in liver metastasis from colorectal cancer. Overall design: We performed MC38 cell intrasplenic injection in C57BL6/J mice to induce liver metastasis. For bulk RNA-seq cells from 6 to 8 pooled samples of metastasis-free liver or metastasis were sorted with FACS Aria III (BD Bioscience) as following: NK cells (live lin- CD45+ NKp46+ CD49a- CD49b+), ILC1 (live lin- CD45+ NKp46+ CD49a+ CD49b-) and CD49+ NK cells cells (live lin- CD45+ NKp46+ CD49a+ CD49b+).