Patient-aware single-cell analysis of fibrovascular remodeling in a predominantly colorectal cancer immunotherapy cohort
- 1Department of Hepatobiliary and Pancreatic Surgery, Ward I, Jingmen Central Hospital, Jingmen Central Hospital affiliated to Jingchu University of Technology , , ,
- 2Operating Room, Jingmen Central Hospital, Jingmen Central Hospital affiliated to Jingchu University of Technology , , ,
Abstract
Background: Cancer-associated fibroblasts (CAFs) and vascular mural cells may shape extracellular matrix organization in colorectal cancer. However, single-cell comparisons are susceptible to unequal cell recovery, ambient RNA, doublets, treatment-stage mixing, and repeated sampling within patients. We investigated fibrovascular remodeling using patients as the inferential unit and distinguished reproducible signals from donor-concentrated and context-dependent findings.
Methods: We reanalyzed a published single-cell RNA sequencing cohort of 22 patients, including 20 with colorectal cancer and 2 with duodenal carcinoma. The primary pericyte analysis included 14 pretreatment matched normal-tumor pairs with at least three pericytes per tissue. We assessed ambient contamination and doublets using DecontX and scDblFinder. CAFs were reclustered at six response-blind resolutions, and the stable parent population was tested by patient-blocked pseudobulk analysis after excluding its dominant donor. Independent CRC single-cell and spatial datasets, a cross-cancer immunotherapy cohort, and TCGA were used for external testing.
Results: 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.
Conclusions: 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.
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BibTeX
@article{gu2026patientaware,
title = {{Patient-aware single-cell analysis of fibrovascular remodeling in a predominantly colorectal cancer immunotherapy cohort}},
author = {Yang Gu and Li Li and Yuxian Zhang and Jie Tang and Zhigang Wang and Xiaofeng Luo},
journal = {Frontiers in immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1961075},
url = {https://europepmc.org/article/PMC/PMC13668821},
note = {CC BY},
}
RIS
TY - JOUR
TI - Patient-aware single-cell analysis of fibrovascular remodeling in a predominantly colorectal cancer immunotherapy cohort
AU - Yang Gu
AU - Li Li
AU - Yuxian Zhang
AU - Jie Tang
AU - Zhigang Wang
AU - Xiaofeng Luo
PY - 2026
DA - 2026/10/01
JO - Frontiers in immunology
DO - 10.3389/fimmu.2026.1961075
UR - https://europepmc.org/article/PMC/PMC13668821
AB - Background: Cancer-associated fibroblasts (CAFs) and vascular mural cells may shape extracellular matrix organization in colorectal cancer. However, single-cell comparisons are susceptible to unequal cell recovery, ambient RNA, doublets, treatment-stage mixing, and repeated sampling within patients. We investigated fibrovascular remodeling using patients as the inferential unit and distinguished reproducible signals from donor-concentrated and context-dependent findings. Methods: We reanalyzed a published single-cell RNA sequencing cohort of 22 patients, including 20 with colorectal cancer and 2 with duodenal carcinoma. The primary pericyte analysis included 14 pretreatment matched normal-tumor pairs with at least three pericytes per tissue. We assessed ambient contamination and doublets using DecontX and scDblFinder. CAFs were reclustered at six response-blind resolutions, and the stable parent population was tested by patient-blocked pseudobulk analysis after excluding its dominant donor. Independent CRC single-cell and spatial datasets, a cross-cancer immunotherapy cohort, and TCGA were used for external testing. Results: 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. Conclusions: 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.
SN - 1664-3224
ER -