Table 1_Single-cell and bulk RNA-sequencing reveal PRRX2-driven cancer-associated fibroblast-mediated perineural invasion for predicting the immunotherapy outcome in colorectal cancer.xlsx

BackgroundPerineural 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, its specific role and regulatory mechanisms in colorectal cancer (CRC) remain unclear.
MethodsIn this study, 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 fibroblast (CAF) subpopulations were identified and functionally annotated. We evaluated the clinical relevance of CAF subsets in TCGA and three independent cohorts (silu_2022, GSE39582, and GSE17536) 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 the PRRX2 score and immune checkpoint inhibitor efficacy was analyzed in conjunction with two immunotherapy cohorts. In addition, we performed a drug sensitivity analysis based on the GDSC pharmacogenomics database to screen potential therapeutic agents.
ResultsIn 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 (myCAFs), and facilitated perineural infiltration through extracellular matrix remodeling. Further analysis revealed that PRRX2 was a core regulator of MMP2+myCAFs, promoting perineural invasion through the activation of TGF-β signaling pathways. PRRX2 knockdown significantly inhibited fibroblast proliferation, clonogenic formation, and invasive migration capacity, and it reduced TGFB1 and NGF expressions. The clinical cohort validation demonstrated a significant correlation between the PRRX2-score and advanced tumor stage, along with vascular and lympho-vascular invasion (LVI). Furthermore, patients with high PRRX2 scores had a significantly worse prognosis. In addition, patients with high PRRX2 scores 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.
ConclusionThis analysis established PRRX2-driven MMP2+myCAFs 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.

背景：神经周围浸润（Perineural invasion, PNI）广泛存在于多种实体瘤中，是促进肿瘤局部侵袭与远处转移的关键通路。该现象通常与肿瘤侵袭性增强、恶性生物学行为恶化及患者不良预后密切相关。然而，其在结直肠癌（Colorectal cancer, CRC）中的具体作用及调控机制仍未明确。
方法：本研究整合了20套CRC单细胞转录组数据集，共包含575768个高质量细胞；通过Scissor算法将TCGA bulk转录组样本中的PNI表型映射至单细胞水平。研究共鉴定出9种癌症相关成纤维细胞（Cancer-associated fibroblast, CAF）亚群并完成功能注释。采用基于BayesPrism的细胞反卷积分析，评估了TCGA队列及3个独立队列（silu_2022、GSE39582、GSE17536）中CAF亚群的临床相关性。利用pySCENIC分析转录调控网络，并通过体外实验验证PRRX2的功能。通过ssGSEA评分量化免疫浸润特征，结合两个免疫治疗队列分析PRRX2评分与免疫检查点抑制剂疗效的关联。此外，本研究基于GDSC药物基因组学数据库开展药物敏感性分析，以筛选潜在治疗药物。
结果：本研究系统揭示了神经周围浸润相关成纤维细胞亚群的特征及其调控机制。在PNI阳性肿瘤中，成纤维细胞占比显著升高，且伴随MMP2+肌成纤维样癌症相关成纤维细胞（myofibroblastic cancer-associated fibroblasts, myCAFs）的富集，并通过细胞外基质重塑促进神经周围浸润。进一步分析显示，PRRX2是MMP2+myCAFs的核心调控因子，可通过激活TGF-β信号通路促进神经周围浸润。PRRX2基因敲低可显著抑制成纤维细胞增殖、集落形成及侵袭迁移能力，并降低TGFB1与NGF的表达水平。临床队列验证结果表明，PRRX2评分与肿瘤晚期分期、血管及淋巴管浸润（LVI）显著相关。此外，PRRX2高评分患者的预后显著更差。同时，PRRX2高评分患者对免疫检查点抑制剂响应不佳，但可能对靶向药物或抗体偶联药物敏感，上述制剂或可作为联合治疗的潜在靶点。
结论：本分析确立了PRRX2介导的MMP2+myCAFs通过TGF-β/细胞外基质重塑参与结直肠癌神经周围浸润的关键作用。PRRX2评分可作为预后预测及免疫治疗疗效评估的生物标志物。