Spatial predictors of response to chemo-immunotherapy in microsatellite stable metastatic colorectal cancer

Microsatellite-stable (MSS) colorectal cancers (CRC) are largely unresponsive to immune checkpoint inhibition (ICI). The MAYA trial used temozolomide (TMZ) in MGMT-silenced MSS mCRC, hypothesizing that TMZ-induced hypermutation could sensitize tumors to ICI; the primary endpoint was met, showing durable responses with TMZ plus ipilimumab and nivolumab. We perform integrated spatial, transcriptomic, and immune profiling of longitudinal tumor and blood samples from patients treated on the MAYA trial. Post-TMZ increases in tumor mutational burden associate with improved progression-free survival. Spatial profiling demonstrates that clinical benefit is greatest in permissive tumor microenvironments. Responders exhibit enrichment of cytotoxic T cells across tumor and stromal compartments, whereas non-responders display heterogeneous cellular neighborhoods, with fibroblasts in close spatial proximity to T cells, consistent with barriers to immune-mediated clearance. Longitudinal peripheral immune profiling shows that early upregulation of TIGIT and PD-1 following TMZ exposure predicts resistance. Together, these findings indicate that both mutational evolution and spatial immune architecture contribute to immune sensitization in MGMT-silenced MSS CRC. Clinical Trial Identification: NCT03832621. The MAYA trial utilized temozolomide in microsatellite stable (MSS) colorectal cancers (CRC) sensitizing them to immune checkpoint inhibition. Here, the integration of spatial, transcriptomic, and immune profiling of longitudinal tumor and blood samples identify molecular determinants of therapeutic response in MSS-CRC.