Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide, with most CRC-related deaths resulting from liver metastases (CRC-LM). Neoadjuvant cytotoxic chemotherapy (CT), alone or combined with targeted agents (anti-EGFR or anti-VEGF), has improved resectability and short-term outcomes of CRC-LM. However, up to 60% of patients relapse within two years. A major unmet clinical need is therefore the identification of mechanisms underlying recurrence. Comprehensive characterization of the tumor immune microenvironment (TIME), and its modulation by neoadjuvant therapies, may provide mechanistic insights and identify immune correlates of durable disease control.

We performed multidimensional immune profiling of CRC-LM samples obtained at surgery from untreated patients and stratified by treatment status. Tumor core (TC) and invasive margin (IM) were analyzed using bulk RNA-seq, high-dimensional flow cytometry, scRNA-seq, scTCR-seq and CITE-seq. Integrated analyses were used to define immune cell composition, functional states, spatial compartmentalization, and clonal relationships.

Untreated CRC-LM lesions displayed a marked spatial immune dichotomy, characterized by a functionally active TIME at the IM and a profoundly immunosuppressed TC. The TC was enriched in immunosuppressive myeloid cells, activated regulatory T cells, and exhausted T cells, including both conventional CD103⁺CD69⁺CD8⁺ cells and unconventional CD4⁺ MAIT cells expressing a CD39hi phenotype. Neoadjuvant therapies reshaped the TC immune landscape, alleviating local immunosuppression. This was characterized by: (i) reduction of exhausted CD103⁺CD69⁺CD39hi CD8⁺ cells and expansion of non-exhausted LAG-3⁺CD39low CD8⁺ effector memory cells; (ii) decreased frequencies of activated Tregs; (iii) loss of putative M2-like macrophages. Notably, these immune remodeling effects were transient. LAG-3⁺CD39low CD8⁺ cells and double-negative (DN) γδ T cells and DN MAIT cells were identified as positive predictors of disease-free survival, whereas CD4⁺CD39hi MAIT cells correlated with relapse. Integrated scRNA-seq, scTCR-seq, and CITE-seq profiling of IM and TC from untreated CRC-LM patients uncovered distinct T cell subsets and their clonal relationships. Shared CD8⁺ T cell clonotypes were detected in both IM and TC, but displayed divergent expansion patterns and functional states.

Our study reveals profound spatial and functional heterogeneity of the TIME in CRC-LM and demonstrates that neoadjuvant therapies can transiently relieve immune dysfunction within the TC. We identify previously unrecognized conventional and unconventional T cell populations associated with disease outcome. These findings provide a framework for timing- and immune-informed therapeutic strategies aimed at achieving durable immune control of metastatic CRC.

Valeria Rossella