Spatial profiling of the immune microenvironment by multiplex imaging identifies barriers to immunotherapy in colorectal liver metastases

Colorectal liver metastasis (CRLM) is the most frequent site of distant metastasis in colorectal cancer (CRC), affecting around 50% of CRC patients. Only 10–30% of CRLM patients were eligible for curative resection, with a low 5-year survival rate (35–55%) and a high recurrence rate (70%). Notably, most CRLMs are resistant to immune checkpoint blockade, prompting a need to map how immune failure is organised in space. In this study, we profiled the CRLM tumour microenvironment across tumour core, peritumour region, and healthy background liver using two highly multiplexed, single-cell datasets: Cell DIVETM spatial proteomics (13 patients; 1,617 500μm × 500μm regions of interest (ROIs); 2.9 M cells) and CosMxTM 6k spatial transcriptomics (4 patients; 1,233 ROIs; 1.34 M cells). Our multiscale pipelines quantified cell abundances, defined neighbourhood clusters from ten-nearest neighbours, tested short-range interactions with permutation-controlled proximity statistics, resolved distance-dependent associations with cross-pair correlation functions (cross-PCF), and captured higher-order geometry with persistent homology-based topological data analysis. Inference was performed at the slide level with multiplicity control. Across cohorts, the peritumour rim concentrated adaptive and antigen-presenting programmes. Short-range coupling of CD8+ T-cells with conventional dendritic cells, together with CD4–dendritic, CD4–B, and CD4–CD8 proximities, demonstrated the formation of tertiary lymphoid structure (TLS)-like hubs at the tumour border. CosMx identified a CCL19/CCL21-expressing fibroblast subset, consistent with fibroblastic reticular cell-like stromal support, while Cell DIVE confirmed higher frequencies of both activated and exhausted T cells at the rim, indicating active yet regulated immunity. The tumour core exhibited layered suppression. Cell DIVE revealed adjacency between T-cells and aSMA⁺/FAP⁺ cancer-associated fibroblasts (CAF). CosMx resolved extracellular matrix remodelling fibroblasts, enriched regulatory T-cells, and abundant myeloid programmes, which together tracked with exclusion of cytotoxic effectors. Cross-PCFs showed reductions in TLS couplings within the tumour core and reinforcements of CAF to immune cell proximities. Topological data analysis separated regions using component and loop statistics, highlighting potential fragmented T-cell geometry interleaved with stromal interfaces at the peritumour region and interface-dominated topology with granulocytic admixture in the tumour core. The healthy liver displayed simple parenchymal vascular segregation consistent with baseline tolerance. These spatial maps nominate promising therapeutic strategies to mitigate immunotherapy resistance. Peritumour-focused strategies include expanding and licensing cross-priming dendritic cells with CD40 agonism and increasing dendritic cell supply at the border. While tumour core-focused approaches include reducing immunosuppressive activities/secretions from CAF and T-regs, and targeting immunosuppressive myeloid cells.