Single-cell RNA sequencing identifies dysregulated NOTCH3 signaling in tumor stroma of lung adenocarcinoma

Cancer immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD); however, a significant fraction of patients fails to respond. Recent studies to understand transcriptomic determinants of immunotherapy response have pinpointed stroma-mediated resistance mechanisms. To gain a better understanding of the stromal biology at the cellular and molecular levels in LUAD, we obtained the transcriptomes of 256,379 single cells with 13,857 mesenchymal cells from 9 treatment-naïve patients. Among the mesenchymal cells, we identify subsets of FAP+PDPN+ cancer-associated fibroblasts (CAFs) and ACTA2+MCAM+ pericytes, which enrich in tumors, differentiate from lung resident fibroblasts and possess a myofibroblast phenotype. By utilizing imaging-mass cytometry, we found that both subsets are topographically adjacent to the perivascular niche and have close spatial interaction with endothelial cells (ECs). Modelling of ligand and receptor interactomes between mesenchymal and ECs identifies that NOTCH3/NOTCH signaling drives the cell-cell interaction in tumors, with pericytes and CAFs as the signal receivers and arterial and PLVAPhigh immature neovascular ECs as the signal senders. Either pharmacologically blocking NOTCH signaling or tuning down NOTCH3 level in mesenchymal cells reduced collagen production and suppressed cell invasion. By evaluating the clinical relevance of NOTCH signaling using bulk-RNA sequencing data, we demonstrate that NOTCH3 correlates with poor survival in stroma-rich patients and that a T-cell inflammation gene signature predicts survival only in low-NOTCH3 expressing patients. Our study underscores the importance of NOTCH3 in regulating tumor stroma biology and may serve as a potential target for immunotherapy combination strategies. All tissue samples from 9 patients for the scRNA sequencing (cohort A) were obtained from Brigham and Women’s Hospital, with patient consent approved by the Institutional Review Board protocol DFCI #98-063. LADC samples from a cohort B of 7 patients for IMC were purchased from commercial suppliers with patient informed consents. Tumors (T) or Adjacent Non-Tumor (ANT) tissues from cohort A were minced with scissors and digested with a human tumor dissociation kit (Miltenyi Biotec, 130-095-929) as per the manufacturer’s protocol with some modifications. For digesting lung samples for MRC001-005, the digestion cocktail from the kit was used, whereas the digestion cocktail with the addition of 0.8 mg/mL dispase II (Sigma, 4942078001) was used to digest the rest of samples.

癌症免疫治疗已彻底革新了肺腺癌（LUAD, lung adenocarcinoma）的临床治疗，但仍有相当比例的患者无法对治疗产生应答。当前为解析免疫治疗应答的转录组学决定因素，已有研究明确了肿瘤间质介导的耐药机制。为在细胞与分子层面更深入解析肺腺癌的肿瘤间质生物学特性，本研究从9名初治患者体内获取了256379个单细胞的转录组数据，其中包含13857个间质细胞。在这些间质细胞中，我们鉴定出两类细胞亚群：FAP+PDPN+的癌症相关成纤维细胞（cancer-associated fibroblasts, CAFs）以及ACTA2+MCAM+的周细胞（pericytes）。这两类亚群在肿瘤组织中富集，与肺部常驻成纤维细胞存在分化差异，并具有肌成纤维细胞表型。借助成像质谱流式技术（imaging-mass cytometry），我们发现这两类亚群均在空间上紧邻血管周围微环境，并与内皮细胞（endothelial cells, ECs）存在紧密的空间相互作用。通过构建间质细胞与内皮细胞间的配体-受体相互作用组模型，我们发现NOTCH3/NOTCH信号通路可介导肿瘤内的细胞间相互作用：周细胞与癌症相关成纤维细胞作为信号接收方，动脉内皮细胞及PLVAP高表达的未成熟新生血管内皮细胞则作为信号发送方。无论是通过药理学手段阻断NOTCH信号通路，还是下调间质细胞中的NOTCH3表达水平，均可减少胶原蛋白生成并抑制细胞侵袭。通过基于批量RNA测序数据评估NOTCH信号通路的临床相关性，我们证实NOTCH3在间质富集患者群体中与不良预后相关，而T细胞炎症基因特征仅在NOTCH3低表达患者中可预测生存结局。本研究强调了NOTCH3在调控肿瘤间质生物学特性中的重要性，或可成为免疫治疗联合策略的潜在靶点。本研究中用于单细胞RNA测序（single-cell RNA sequencing, scRNA测序）的9名患者组织样本均采集自布莱根妇女医院，相关患者知情同意流程已通过达纳-法伯癌症研究所（DFCI）伦理审查委员会方案#98-063的审批。用于成像质谱流式检测（IMC）的队列B共7名患者的肺腺癌（LADC, lung adenocarcinoma）样本均购自商业供应商，且均已获得患者知情同意。队列A的肿瘤组织（T）及癌旁非肿瘤组织（Adjacent Non-Tumor, ANT）均经剪刀剪碎后，使用人肿瘤组织解离试剂盒（Miltenyi Biotec，货号130-095-929）按照制造商说明书进行消化，并进行了部分优化调整。针对编号为MRC001-005的肺部样本消化，直接使用试剂盒自带的消化液；其余样本则使用添加了0.8 mg/mL分散酶II（Sigma，货号4942078001）的消化液进行消化。