High-Resolution Spatial Transcriptomics Reveals Inflammatory Niches Orchestrating Immune Checkpoint Inhibitor Response in MSI-H Colorectal Adenocarcinoma

Background
Immune checkpoint inhibition (ICI) has transformed the treatment of microsatellite instability-high (MSI-H) colorectal cancer (CRC), although a significant subset of patients exhibit primary resistance. We reported two transcriptomic subtypes of MSI-H colon adenocarcinoma (COAD) with divergent outcomes following ICI: C1 Inflamed (responsive) and C2 Goblet-like (resistant). However, the tumor microenvironments (TME) driving these divergent outcomes remain poorly understood. Here, we analyzed a spatial transcriptomic atlas with linked clinical, molecular, and treatment data, and characterized the cellular interactomes that define the C1 and C2 phenotypes.

Methods
We leveraged the Tempus CRC Spatial Atlas cohort (n = 51), which captures the full molecular spectrum of CRC across MSI status, subtypes and actionable targets via the 10x Genomics Visium HD platform. We focused on five primary MSI-H COAD specimens comprising ~700k cells. Samples were stratified into C1 (n = 3) and C2 (n = 2) subtypes based on paired bulk RNA-sequencing data (Tempus xR). Cells were integrated using scVI, annotated via RCTD, and spatial tissue niches were characterized with CellCharter.

Results
Spatial analysis of 13 TME niches uncovered subtype-specific patterns. The C1 subtype was enriched for an inflammatory myeloid niche localized to the tumor-stroma boundary (logFC: 1.3, p: 0.007), whereas the C2 subtype was enriched for peritumoral mucosa (logFC: -3.18, p: 0.03) and goblet-program epithelium (logFC: -2.4, p: 0.04). C1 also exhibited high expression of genes promoting T cell activation (B2M, CD74), and was surrounded by lymphocytes (median distance: 29 µm). T and NK cells within C1 tumors demonstrated significantly increased expression of IFNG response genes (CXCL9, CXCL10, FDR < 0.05) vs. C2, indicative of T/NK cell activation and IFNG secretion. Conversely, the C2 subtype exhibited strict immune exclusion. C2 tumor-adjacent regions were characterized by the segregation of goblet-program epithelium from stromal- and immune-rich niches (p < 0.001).

Conclusions
High-resolution spatial profiling revealed that C1 tumors harbor a highly active inflammatory myeloid niche at the tumor-stroma boundary, heavily populated by M1 macrophages, surrounded by T cells, and with localized expression of IFNG response genes as a key orchestrator of anti-tumor immunity and ICI response. These findings provide a framework for developing predictive biomarkers and overcoming immune exclusion in ICI-refractory MSI-H COAD.