Image 1_Patient-aware single-cell analysis of fibrovascular remodeling in a predominantly colorectal cancer immunotherapy cohort.tiff

Independent CRC single-cell and spatial datasets, a cross-cancer immunotherapy cohort, and TCGA were used for external testing.</p>Results<p>After DecontX correction, tumor pericytes had higher ECM6 scores (median paired change 0.309; false discovery rate [FDR] 0.018). Scores were also higher for the matrix stiffness transcriptional proxy (0.277; FDR 0.012) and the hypoxia/angiogenesis transcriptional program (0.255; FDR 0.028).

All three signatures remained significant at the five-pericyte threshold (n=13). COL1A1 and FN1 increased in the analysis, although COL1A1 was borderline in the five-cell sensitivity analysis. Response-blind stability analysis identified a 2,983-cell ECM-remodeling CAF population represented in all 22 patients.

After excluding P02, patient-blocked pseudobulk analysis in 15 patients showed higher matrix, inflammatory, and myCAF-related programs and a lower iCAF program. Neither SPP1 nor baseline enrichment in stable disease was supported. In GSE205506, a disjoint 10-gene fibroblast gate retained higher inflammatory and composite CAF scores in non-pCR samples.

The highest-scoring unsupervised cluster was donor-concentrated and showed no patient-level association with response. Spatial and cross-cancer analyses yielded mixed or null results.</p>Conclusions<p>These results support pretreatment fibrovascular transcriptional remodeling at the patient level. They do not establish an SPP1-high response biomarker, a mechanical stiffness phenotype, or causal CAF-to-pericyte signaling.

Donor-aware analysis places the rare-cluster observation within a broader, reproducible ECM-remodeling CAF program while defining its evidential limits.</p>