Single-cell and bulk RNA-sequencing reveals PRRX2-driven cancer-associated fibroblasts-mediated perineural invasion for predicting immunotherapy outcome in colorectal cancer

Mingxiao ChenYue CaiFeng HanBo LiZhou XuKaili CuiWenqi BaiLi Feng^*

• Shanxi Province Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, China

Background: Perineural invasion (PNI) is common in a variety of solid tumors and has been identified as an important pathway promoting tumor local invasion and distant metastasis. Its presence is usually associated with increased aggressiveness, malignant biology, and a worse patient prognosis. However, their specific role and regulatory mechanisms in colorectal cancer (CRC) remain unclear. Methods: We integrated 20 CRC single-cell transcriptome datasets, which contained 575,768 high-quality cells, and used the Scissor algorithm to map PNI phenotypes in TCGA bulk samples to the single-cell level. Nine cancer-associated fibroblasts (CAF) subpopulations were identified and functionally annotated. We evaluated the clinical relevance of CAF subsets in TCGA and three independent cohorts using BayesPrism-based deconvolution. We analyzed transcriptional regulatory networks using pySCENIC and validated PRRX2 function by in vitro experiments. Immune infiltration characteristics were quantified using the ssGSEA score and the association between PRRX2 score and immune checkpoint inhibitor efficacy was analyzed in conjunction with two immunotherapy cohorts. In addition, we performed drug sensitivity analysis based on the GDSC pharmacogenomics database to screen potential therapeutic agents. Results: In this study, we systematically revealed the characteristics of the perineural invasion-associated fibroblast subsets and their regulatory mechanisms. In PNI-positive tumors, the proportion of fibroblasts was significantly increased, with the enrichment of MMP2+ myofibroblastic cancer-associated fibroblasts (myCAF) and facilitated perineural infiltration through extracellular matrix remodeling. Further analysis revealed that PRRX2 was a core regulator of MMP2+myCAF promoting perineural invasion through activation of TGF-β signaling pathways. PRRX2 knockdown significantly inhibited fibroblast proliferation, clonogenic formation, and invasive migration capacity, and reduced TGFB1 and NGF expression. Clinical cohort validation demonstrated a significant correlation between the PRRX2-score and advanced tumor stage, as well as vascular and lympho-vascular invasion (LVI). Furthermore, the patients with high PRRX2 scores had a significantly worse prognosis. In addition, high PRRX2-score patients responded poorly to immune checkpoint inhibitors but may be sensitive to targeted agents or antibody-coupled drugs, which may serve as potential targets for combination therapy. Conclusion: This analysis established PRRX2-driven MMP2+myCAF as pivotal mediators of CRC perineural invasion through TGF-β/ECM remodeling. The PRRX2 score serves as a biomarker for prognosis prediction and immunotherapy outcome.