Table_4_Single-Cell RNA Sequencing Reveals the Heterogeneity of Tumor-Associated Macrophage in Non-Small Cell Lung Cancer and Differences Between Sexes.xlsx

Non-Small Cell Lung Cancer (NSCLC) is a disease with high morbidity and mortality, which has sex-related differences in prognosis and immunotherapy efficacy. However, the difference in the mechanisms remains unclear. Macrophages, characterized by high plasticity and heterogeneity, act as one of the key cells that exert anti-tumor effects in the tumor microenvironment (TME) and play a complicated role in the process of tumor progression. To elucidate the subtype composition and functional heterogeneity of tumor-associated macrophages (TAMs) in NSCLC and further compare the sex-mediated differences, we conducted a single-cell level analysis in early-stage smoking NSCLC patients, combined with ssGSEA analysis, pseudotime ordering, and SCENIC analysis. We found two universally presented immune-suppressive TAMs with different functional and metabolic characteristics in the TME of NSCLC. Specifically, CCL18+ macrophages exerted immune-suppressive effects by inhibiting the production of inflammatory factors and manifested high levels of fatty acid oxidative phosphorylation metabolism. Conversely, the main metabolism pathway for SPP1+ macrophage was glycolysis which contributed to tumor metastasis by promoting angiogenesis and matrix remodeling. In terms of the differentially expressed genes, the complement gene C1QC and the matrix remodeling relevant genes FN1 and SPP1 were differentially expressed in the TAMs between sexes, of which the male upregulated SPP1 showed the potential as an ideal target for adjuvant immunotherapy and improving the efficacy of immunotherapy. According to the early-stage TCGA-NSCLC cohort, high expression of the above three genes in immune cells were associated with poor prognosis and acted as independent prognostic factors. Moreover, through verification at the transcription factor, transcriptome, and protein levels, we found that TAMs from women showed stronger immunogenicity with higher interferon-producing and antigen-presenting ability, while men-derived TAMs upregulated the PPARs and matrix remodeling related pathways, thus were more inclined to be immunosuppressive. Deconstruction of the TAMs at the single-cell level deepens our understanding of the mechanism for tumor occurrence and progress, which could be helpful to achieve the precise sex-specific tumor treatment sooner.

非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）是一种发病率与死亡率均居高不下的疾病，其预后及免疫治疗疗效存在性别相关差异，但相关机制尚未阐明。巨噬细胞具有高度可塑性与异质性，是肿瘤微环境（Tumor Microenvironment, TME）中发挥抗肿瘤作用的关键细胞之一，在肿瘤进展过程中扮演复杂角色。为阐明非小细胞肺癌中肿瘤相关巨噬细胞（Tumor-Associated Macrophages, TAMs）的亚型组成与功能异质性，并进一步比较性别介导的差异，本研究针对早期吸烟非小细胞肺癌患者开展了单细胞水平分析，结合单样本基因集富集分析（single-sample Gene Set Enrichment Analysis, ssGSEA）、拟时间排序分析及SCENIC分析。本研究在非小细胞肺癌肿瘤微环境中发现了两种普遍存在的免疫抑制性TAMs，二者具备不同的功能与代谢特征。具体而言，CCL18+巨噬细胞通过抑制炎症因子产生发挥免疫抑制作用，并呈现高水平的脂肪酸氧化磷酸化代谢模式；与之相反，SPP1+巨噬细胞的主要代谢通路为糖酵解，可通过促进血管生成与基质重塑助力肿瘤转移。在差异表达基因方面，补体基因C1QC以及基质重塑相关基因FN1与SPP1在不同性别的TAMs中存在表达差异，其中男性群体中高表达的SPP1有望成为辅助免疫治疗及提升免疫治疗疗效的理想靶点。基于早期癌症基因组图谱非小细胞肺癌（The Cancer Genome Atlas-Non-Small Cell Lung Cancer, TCGA-NSCLC）队列的分析显示，上述三个基因在免疫细胞中的高表达与不良预后相关，且为独立预后因子。此外，通过在转录因子、转录组及蛋白质水平进行验证，本研究发现女性来源的TAMs免疫原性更强，具备更高的干扰素产生能力与抗原呈递能力；而男性来源的TAMs则上调了过氧化物酶体增殖物激活受体（Peroxisome Proliferator-Activated Receptors, PPARs）及基质重塑相关通路，更易呈现免疫抑制表型。对TAMs开展单细胞水平解析，可深化我们对肿瘤发生与进展机制的理解，有助于早日实现精准的性别特异性肿瘤治疗。