ABSTRACT Purpose The liver is the most frequent metastatic site for colorectal cancer ( CRC ). Its microenvironment is modified to provide a niche that allows CRC cell growth. This study focused on characterizing the cellular changes in the metastatic CRC ( mCRC ) liver tumor microenvironment ( TME ). Experimental Design We analyzed a series of microsatellite stable (MSS) mCRCs to the liver, paired normal liver tissue and peripheral blood mononuclear cells using single cell RNA-seq ( scRNA-seq ). We validated our findings using multiplexed spatial imaging and bulk gene expression with cell deconvolution. Results We identified TME-specific SPP1 -expressing macrophages with altered metabolism features, foam cell characteristics and increased activity for extracellular matrix ( ECM ) organization. SPP1+ macrophages and fibroblasts expressed complementary ligand receptor pairs with the potential to mutually influence their gene expression programs. TME lacked dysfunctional CD8 T cells and contained regulatory T cells, indicative of immunosuppression. Spatial imaging validated these cell states in the TME. Moreover, TME macrophages and fibroblasts had close spatial proximity, a requirement for intercellular communication and networking. In an independent cohort of mCRCs in the liver, we confirmed the presence of SPP1 + macrophages and fibroblasts using gene expression data. An increased proportion of TME fibroblasts was associated with worst prognosis in these patients. Conclusions We demonstrated that mCRC in the liver is characterized by transcriptional alterations of macrophages in the TME. Intercellular networking between macrophages and fibroblasts supports CRC growth in the immunosuppressed metastatic niche in the liver. These features can be used to target these immune checkpoint resistant MSS tumors. TRANSLATIONAL RELEVANCE The liver is the commonest site for metastatic colorectal cancer ( mCRC ). Alterations in the tumor microenvironment ( TME ) allow metastatic cells to seed the distant liver site and grow. Leveraging single-cell RNA sequencing, we discovered a distinct SPP1 + macrophage cell state with pro-fibrogenic gene expression and altered metabolism. These SPP1 + macrophages communicated with fibroblasts, mutually influencing each other’s gene expression program. Using spatial imaging, we confirmed proximal colocalization between macrophages and fibroblasts in the mCRC TME, which is required for intercellular communication. These states and intercellular communication promoted immunosuppression in the TME, with a lack of dysfunctional anti-tumor CD8 T cells and prevalence of regulatory T cells. Increased fibroblasts were associated with worst prognosis in an independent patient cohort. Our results identified novel TME features that result in reshaping of the metastatic niche that allows progression of mCRC. These features can be potential targets for mCRC treatment, which is microsatellite stable and resistant to immune checkpoint blockade.

Colorectal cancer metastases in the liver establish immunosuppressive spatial networking between tumor associated <i>SPP1</i><sup>+</sup> macrophages and fibroblasts

Colorectal cancer metastases in the liver establish immunosuppressive spatial networking between tumor associated SPP1+ macrophages and fibroblasts