Desmoplasia is associated with carcinoma associated fibroblasts’ heterogeneity in non-small cell lung cancer patients

Cancer associated fibroblasts (CAFs) are well known to strongly influence tumor development, progression, and metastasis. Their characteristics and prognostic gene signature in non-small cell lung cancer (NSCLC) patients have been recognized. Heterogeneity of CAFs has long been observed, however, the functional heterogeneity of CAFs between patients and their genetic basis are far less known. Here, we report the distinction of two CAF subtypes obtained from primary cultures of CAFs and matched normal fibroblasts (NFs) from 28 resected NSCLC. These 28 pathological NSCLC (82 % adenocarcinoma) were categorized into two types according to the histological properties of the peritumoral stroma: High desmoplasia (HD; n=14), and low desmoplasia (LD; n=14), and the prognostic significance evaluated. This classification seems to align with functional CAFs, as those with HD have higher rate of gel contraction, higher rate of tumor growth and poor prognosis. To demonstrate a gene expression profile specific to CAF activity we used Illumina DASAL Microarray gene expression analysis on extracted RNA from 24 CAF cell lines (12 CAFs-HD and 12 CAFs-LD) that were embedded in collagen gel for 24 hours. From the CAF cohort, the most significant genes that are correlated with the desmoplasia are identified to be enriched in the clinical cohort of 181 NSCLC patients. Overall, our studies showed that in NSCLC, desmoplasia is significantly associated with poor prognosis and is able to significantly subgroup CAF between NSCLC patients. Early passage (passage 2) cultures of 4 LD-CAF (CAF extracted from low desmoplasia (LD) primary non-small cell lung cancer) and 3 HD-CAF (CAF extracted from high desmoplasia (HD) primary non-small cell lung cancer) were cultured in collagen gel for 24 hours.

癌相关成纤维细胞（Cancer associated fibroblasts, CAFs）是一类被广泛证实可显著影响肿瘤发生、进展与转移的细胞群。学界此前已明确非小细胞肺癌（non-small cell lung cancer, NSCLC）患者体内CAFs的特征及其预后基因特征。尽管CAFs的异质性早已被观察到，但不同患者间CAFs的功能异质性及其遗传基础仍鲜为人知。
本研究从28例手术切除的NSCLC患者的癌相关成纤维细胞及配对正常成纤维细胞（normal fibroblasts, NFs）原代培养体系中，分离得到两种CAF亚型并对其进行区分。该28例病理确诊的NSCLC患者（其中82%为腺癌）根据肿瘤周围间质的组织学特征分为两类：高促结缔组织增生型（High desmoplasia, HD；n=14）与低促结缔组织增生型（Low desmoplasia, LD；n=14），并对其预后意义进行了评估。
该分类与CAFs的功能特征高度吻合：HD组CAFs的凝胶收缩活性更强、促肿瘤生长能力更高，且对应患者预后更差。为揭示CAFs活性相关的特异性基因表达谱，本研究对在胶原凝胶中培养24小时的24株CAF细胞系（12株HD-CAFs与12株LD-CAFs）提取的RNA进行了Illumina DASAL微阵列基因表达分析。
从该CAF队列中筛选出的与促结缔组织增生显著相关的核心基因，在181例NSCLC患者的临床队列中得到了富集验证。综上，本研究证实：在NSCLC中，促结缔组织增生与不良预后显著相关，且可有效区分不同患者体内的CAFs亚群。本研究同时将4株LD-CAFs（源自低促结缔组织增生型原发性NSCLC的CAFs）与3株HD-CAFs（源自高促结缔组织增生型原发性NSCLC的CAFs）的早期传代（第2代）细胞在胶原凝胶中培养24小时。