Immune suppressive landscape in the human non-small cell lung cancer microenvironment. Immune suppressive landscape in the human non-small cell lung cancer microenvironment

Background: Lung cancer is the one of the malignant tumors with the highest morbidity and mortality in China and even the world. The overall five-year survival rate is about 20%, and most patients die of tumor metastasis. Non-small cell lung cancer (NSCLC) is the main pathologic type of lung cancer. The development and metastasis of tumor cells is influenced by the interaction with other cells in the environment. To explore the molecular expression spectrum of tumor and tumor microenvironment can provide clues for the mechanism of tumor progression, drug resistance and metastasis. The emergence of single cell ribonucleic acid sequencing (scRNA-seq) technology can explore the genomic and transcriptional characteristics of different tumor cells at the single cell resolution, which can reveal the molecular characteristics of individual cells and the relationship between individuals and the environment, thus making it possible to further understand the genesis and progression of NSCLC. Methods: ScRNA-seq (10X genomics) was used to detect single-cell transcriptomes in 2 patients with NSCLC, aiming to more accurately explore the effects of the transcriptome in a single cell on its function and the progression of NSCLC, and to find new cell subtypes or molecular targets for drug therapy. Results: The tumor tissue, distant normal tissue, and lymph node tissue of 2 patients with NSCLC were analyzed. A total of 43779 cells were analyzed, which were classified into epithelial cells, T lymphocytes, natural killer cells (NK cells), B lymphocytes, myeloid cells, endothelial cells, fibroblasts, and MAST cells. Tumor cells showed signature markers of epithelial (EPCAM, KRT7, KRT8, KRT18, CDH1, etc.) and tumor (KRT19, ENO2, SLC25A2, UBE2C, etc.) markers. Additionally, the acquired immunity is activated in the tumor microenvironment, while the innate immunity is exhausted. The proportion of B lymphocytes increases, and immunosuppressive tumor-infiltrating B cells (CD19+, MS4A1+, CD69+, CD27-, CR2-) are observed, which are capable of participating in the induction of exhausted regulatory T cells. The proportion of cytotoxic CD8+ T cells in T lymphocyte population decreases, and the number of exhausted regulatory T cells and exhausted follicular helper T cells increases. They all express LAG3+, TIGIT+, PDCD1+, CTLA4+, and other suppressive markers, showing a state of immunosuppression. High expression of WFDC2 in T cells has a significant effect on prognosis. NK cells show a state of depletion, not only in number, but also gradually transform into a less cytotoxic subtype (CD56+, CD16-). In addition, myeloid cells also play an important role in disrupting the anti-tumor immune response. Tumor associated macrophages (TAMs) and plasmacytoid dendritic cells (pDCs) are enriched in tumor tissue. TAMs highly express anti-inflammatory markers (APOE+, C1QA+, C1QB+) and proliferative markers (MKI67+, STMN1+, NUSAP1+), which are closely related to tumor progression and metastasis. TAMs show M2 type polarization of macrophages, which has a tumor promoting effect. At the same time, TAMs express monocyte markers, which may be transformed from monocyte derived macrophages. Macrophages highly express CXCL8, CCL2, and other genes, which participate in Toll-like receptor, NOD-like receptor signaling pathway, and have a significant impact on the prognosis. pDCs are immunosuppressive cells. The high expression of LILR family genes and GZMB in pDCs may lead to poor tumor antigen presentation of lung squamous cell carcinoma. Dendritic cells highly express NLRP3, IRF7, and other genes, which participate in C-type lectin receptor, Toll-like receptor, NOD-like receptor signaling pathway, and have a significant impact on the prognosis. In addition to the immune system, fibroblasts in the stroma also show characteristics of immunosuppressive cells. Enrichment of Cancer-associated fibroblasts (CAFs) in tumor can promote immunosuppression and tumor progression. Among them, a special subtype termed inflammatory CAFs is found, which participate in the process of extracellular matrix remodeling, angiogenesis, and cell migration, resulting in promoting tumor progression. Fibroblasts highly express FN1, TIMP1, and hub gene SPP1, which interact with macrophages, participate in PI3K-AKT pathway, and have a significant impact on the prognosis. Conclusions: NSCLC shows heterogeneous tumor microenvironment. A variety of immune cells and stromal cells together lead to immunosuppressive environment and promote the progression of tumor. This study enhances our understanding of molecular and cellular dynamics of NSCLC, and reveals potential diagnostic and therapeutic targets in tumor microenvironment. Overall design: Single-cell RNA sequencing (10X Genomics) of non-small cell lung cancer, including two tumor tissues, two paired adjacent normal lung tissues, and two lymph node tissues from two patinets.

背景：肺癌是我国乃至全球发病率与死亡率最高的恶性肿瘤之一，总体五年生存率约为20%，多数患者因肿瘤转移死亡。非小细胞肺癌（Non-small cell lung cancer, NSCLC）是肺癌的主要病理类型。肿瘤细胞的发生发展与转移受其所处微环境中其他细胞的相互作用影响。探索肿瘤及肿瘤微环境的分子表达谱，可为肿瘤进展、耐药及转移的机制研究提供线索。单细胞核糖核酸测序（single cell ribonucleic acid sequencing, scRNA-seq）技术的出现，能够在单细胞分辨率下解析不同肿瘤细胞的基因组与转录组特征，揭示单个细胞的分子特性及其与环境的相互关系，从而为深入理解非小细胞肺癌的发生与进展提供可能。
方法：本研究采用单细胞核糖核酸测序（10X Genomics）技术，对2例非小细胞肺癌患者的单细胞转录组进行检测，旨在更精准地探究单细胞转录组对细胞功能及非小细胞肺癌进展的影响，并挖掘新的细胞亚型或药物治疗分子靶点。
结果：本研究对2例非小细胞肺癌患者的肿瘤组织、远端正常组织及淋巴结组织进行了分析，共纳入43779个细胞，可分为上皮细胞、T淋巴细胞、自然杀伤细胞（NK细胞）、B淋巴细胞、髓系细胞、内皮细胞、成纤维细胞及肥大细胞（MAST cells）。肿瘤细胞表达上皮细胞特征标志物（EPCAM、KRT7、KRT8、KRT18、CDH1等）及肿瘤特征标志物（KRT19、ENO2、SLC25A2、UBE2C等）。此外，肿瘤微环境中获得性免疫被激活，但先天免疫处于耗竭状态。B淋巴细胞比例升高，且可观察到免疫抑制性肿瘤浸润B细胞（CD19+、MS4A1+、CD69+、CD27-、CR2-），这类细胞可参与诱导耗竭性调节性T细胞。在T淋巴细胞群体中，细胞毒性CD8+T细胞比例降低，而耗竭性调节性T细胞及耗竭性滤泡辅助性T细胞数量增加，二者均表达LAG3+、TIGIT+、PDCD1+、CTLA4+等抑制性标志物，呈现免疫抑制状态。T细胞中高表达的WFDC2对患者预后具有显著影响。NK细胞处于耗竭状态，不仅数量减少，还逐渐向细胞毒性更低的亚型（CD56+、CD16-）转化。此外，髓系细胞在破坏抗肿瘤免疫应答中发挥重要作用。肿瘤组织中富集了肿瘤相关巨噬细胞（tumor associated macrophages, TAMs）及浆细胞样树突状细胞（plasmacytoid dendritic cells, pDCs）。TAMs高表达抗炎标志物（APOE+、C1QA+、C1QB+）及增殖标志物（MKI67+、STMN1+、NUSAP1+），与肿瘤进展及转移密切相关；TAMs呈现巨噬细胞M2型极化，具有促肿瘤作用，同时表达单核细胞标志物，提示其可能来源于单核细胞分化而来的巨噬细胞。巨噬细胞高表达CXCL8、CCL2等基因，参与Toll样受体、NOD样受体信号通路，且对患者预后具有显著影响。pDCs属于免疫抑制性细胞，其高表达的LILR家族基因及GZMB可能导致肺鳞癌的肿瘤抗原呈递功能受损。树突状细胞高表达NLRP3、IRF7等基因，参与C型凝集素受体、Toll样受体、NOD样受体信号通路，对患者预后具有显著影响。除免疫系统外，基质中成纤维细胞也呈现免疫抑制细胞特性。肿瘤相关成纤维细胞（Cancer-associated fibroblasts, CAFs）在肿瘤组织中富集，可促进免疫抑制及肿瘤进展。本研究发现一类特殊的炎性肿瘤相关成纤维细胞亚型，其参与细胞外基质重塑、血管生成及细胞迁移过程，进而促进肿瘤进展。成纤维细胞高表达FN1、TIMP1及核心基因SPP1，可与巨噬细胞相互作用，参与PI3K-AKT通路，且对患者预后具有显著影响。
结论：非小细胞肺癌的肿瘤微环境具有异质性，多种免疫细胞与基质细胞共同构成免疫抑制性微环境，促进肿瘤进展。本研究加深了我们对非小细胞肺癌分子与细胞动态变化的理解，并揭示了肿瘤微环境中潜在的诊断与治疗靶点。
整体设计：对非小细胞肺癌开展单细胞核糖核酸测序（10X Genomics），样本来自2例患者的2份肿瘤组织、2份配对的癌旁正常肺组织及2份淋巴结组织。