PURPOSE: Digital proteomic profiling was performed to identify spatial context in relationship to patient response and survival after anti-PD-1 therapy in metastatic colorectal cancer. EXPERIMENTAL DESIGN: Primary colorectal cancers with deficient mismatch repair from patients treated with anti-PD-1 antibodies were analyzed (N = 30) using digital spatial profiling (GeoMx nCounter). At the invasive margin, 71 proteins were profiled in 10 regions of interest/slide that were segmented into 3 compartments labeled with pan-cytokeratin (epithelia), CD45 (stromal cells), and SYTO13 (nuclei). In an independent cohort (n = 13), digital spatial profiling data and single-cell transcriptomic data were analyzed. Differential protein abundance, after Benjamini-Hochberg correction, was examined by clinical response and progression-free survival (PFS) using multivariable Cox regression. RESULTS: Protein abundance varied significantly between epithelial and stromal compartments. Nonresponders to anti-PD-1 showed higher fibronectin and smooth muscle actin abundance in the epithelial compartment that was associated with significantly shorter PFS (adjusted HR: 6.49 and 4.52, respectively; P < 0.05). In CD45+ stroma, increased expression of proteins related to T cells (CD3 and CD4), NK cells (CD56), antigen presentation (CD40), immune activation (CD27, ICOS), and apoptosis (GZMA) were found in responders (vs nonresponders) to anti-PD-1; each marker was significantly associated with longer patient PFS (0.02 < adjusted HR < 0.17; P < 0.05). In a separate cohort, consistent results by compartment were found for fibronectin and CD56. Gene expression data revealed that fibronectin and smooth muscle actin were primarily derived from cancer-associated fibroblasts. CONCLUSIONS: Spatially resolved protein profiles within microenvironments of deficient mismatch repair colorectal cancers can influence patient response and survival after anti-PD-1, highlighting their potential clinical significance.