Peritumoral immune suppressive mechanisms impede intratumoral lymphocyte infiltration into CRC liver versus lung metastases.

Microsatellite stable (MSS) colorectal cancer (CRC) patients with liver metastases are resistant to immune checkpoint inhibitor (ICI) therapy, while about one-third of CRC patients without liver metastases, particularly those with lung-only metastases, respond to ICI. We analyzed primary CRC tumors and major metastatic sites (liver, lung, peritoneal) using multiplex immunofluorescence (mIF) and whole slide spatial analyses to identify variations in immune contexture and regional localization within the tumor microenvironment (TME). While levels of T and B cells within peritumoral regions were similar, their levels were significantly lower within the tumor core of liver and peritoneal metastases compared to lung metastases. In contrast, antigen presenting cells (APCs) and APC-T cell interactions were more abundant in all regions of lung metastases. We also identified an abundance of lymphoid aggregates throughout lung metastases, but these were present only within peritumoral regions of liver and peritoneal metastases. Larger lymphoid aggregates consistent with features of tertiary lymphoid structures (TLSs) were observed within or adjacent to primary tumors, but not metastatic lesions. Our findings were validated using NanoString GeoMx DSP, which further showed that liver metastases had higher expression of immune-suppressive markers, while lung metastases showed higher proinflammatory activity and T cell activation markers. Peritoneal metastases demonstrated higher expression of cancer-associated fibroblast-related proteins and upregulated PD-1/PD-L1 signaling molecules. Our results demonstrate that functional status and spatial distribution of immune cells vary significantly across different metastatic sites. These findings suggest that metastatic site dependent immune contexture may underlie discordant responses to ICI therapy in MSS CRC patients.