研究背景：皮肤鳞状细胞癌（cutaneous squamous cell carcinoma ,CSCC）是一 类起源于皮肤角质形成细胞的恶性肿瘤，是非黑素瘤皮肤癌中发病率第二高的肿 瘤，也是全球范围内发病率和死亡率较高的恶性肿瘤之一。目前关于 CSCC 的确切 病因和发病机制目前尚未完全清楚，因此，亟需进一步阐明皮肤鳞癌发生发展的病 因机制及肿瘤异质性特点，为临床诊疗提供新的参考依据。前期基于组织样本的混 合转录组学测序（bulk RNA-seq）方法初步揭示了皮肤鳞癌的转录表达失调特征， 然而，CSCC 是一类异质性较高的肿瘤，是由包括肿瘤、免疫、成纤维细胞等亚群在 内构成的复杂微环境系统，肿瘤微环境是 CSCC 发生发展的必要土壤，然而当前对 CSCC 进展过程中微环境细胞亚群组成及其功能了解较少。近年来发展的单细胞转录 组测序技术（scRNA-seq）可以在单细胞水平上揭示细胞亚群比例及其相互作用， 它可提供肿瘤异质性及肿瘤微环境的精细分辨率图谱。 研究方法：（1）纳入 3 例病理诊断明确的并行手术治疗的 CSCC 患者，对手术中获 取的癌及配对癌旁组织样本制备单细胞悬液后应用 10x Genomics 技术进行 scRNA-seq。（2）对获得的数据在质控和细胞周期效应评估后进行 PCA 降维，利用 UMAP 使细胞聚类和可视化，对各细胞亚群的标记基因进行鉴定，使用 Single R 包 注释各细胞类型，独立地推断每个单细胞的起源细胞并确定主要细胞簇。（3）对 CSCC 组织与癌旁组织之间的主要细胞亚群进行再聚类，并进行通路富集分析。（4）使用 Monocle 2 进行拟时序分析，绘制中性粒细胞亚群演化的动态轨迹图谱。 研究结果： （1）质控后对 58209 个细胞进行了 scRNA-seq，降维聚类后共获得 11 类细胞亚群，11 类主要细胞亚群包括：T 和 NK 细胞，上皮细胞，内皮细胞，成纤 维细胞，巨噬细胞和树突状细胞，中性粒细胞，B 细胞和浆细胞，肥大细胞，黑素 细胞，周细胞，汗腺上皮细胞。（2）与细胞增殖、癌症进展和转移密切相关的通路 在肿瘤上皮细胞中高度富集，而与免疫活化相关的通路显著下调，提示肿瘤上皮细 胞在癌症发生发展中起重要作用。（3）肿瘤中浸润的中性粒细胞可能通过激活 Th2 型细胞因子，发挥抑制肿瘤的作用。（4）T&NK 细胞可通过干扰素刺激通路，白介素 通路，TCR 通路等参与肿瘤微环境重编程，而巨噬细胞和树突状细胞则可能通过三 羧酸循环促进巨噬细胞向 M2 型极化，发挥促肿瘤作用。 研究结论：（1）本研究通过 scRNA-seq 描绘了 CSCC 肿瘤微环境的单细胞图谱，证 IV 明了 CSCC 细胞亚群组成的广泛异质性。（2）CSCC 肿瘤微环境中免疫细胞可能通过 调节免疫通路，细胞增殖，生物代谢等过程重塑肿瘤微环境，中性粒细胞可能通过 激活 Th2 型细胞因子，发挥抑制肿瘤的作用。（3）研究发现的多种细胞亚群特异差 异表达基因和关键通路有助于进一步理解 CSCC 的发生机制，为开发新的免疫治疗 新靶点提供基础。 

Background: Cutaneous squamous cell carcinoma (CSCC) is a kind of malignant tumor originated from skin keratinocytes, which has the second highest incidence among non-melanoma skin cancers, and is also one of the malignant tumors with high morbidity and mortality worldwide. At present, the exact etiology and pathogenesis of CSCC are still not completely clear. Therefore, it is urgent to further clarify the etiology and pathogenesis of skin squamous cell carcinoma and the characteristics of tumor heterogeneity, so as to provide new reference for clinical diagnosis and treatment. Previous hybrid transcriptomics sequencing based on tissue samples (bulk RNA-seq) method preliminarily revealed the transcriptional expression disorder characteristics of skin squamous cell carcinoma. However, CSCC is a kind of tumor with high heterogeneity, which is a complex microenvironment system composed of tumor, immune cells, fibroblast and other subsets. Tumor microenvironment (TME) is the necessary soil for the occurrence and development of CSCC. However, the dynamic changes of cell subsets in microenvironment during the progression of CSCC and their functions are less well understood. Recently, single-cell transcriptome sequencing (scRNA-seq) can reveal the proportion of cell subsets and their interactions at the single-cell level, which can provide a fine resolution map of tumor heterogeneity and tumor microenvironment. Methods: (1) Three patients with pathologically confirmed CSCC who underwent surgery were included. Single cell suspensions were prepared from cancer and matched adjacent tissue samples obtained during surgery, and then scRNA-seq was performed using 10x Genomics technology. (2) PCA dimensionality reduction was performed after quality control and cell cycle effect evaluation of the obtained data. UMAP were used for cell clustering and visualization. Marker genes of each cell subpopulation were identified. Cell types were annotated using Single R packages, and the origin cells of each single cell were independently inferred and the main cell clusters were identified. (3) The main cell subsets between CSCC tissues and adjacent cancer tissues were reclustered, and pathway enrichment analysis was performed. (4) Monocle 2 was used for pseudotime analysis to map VI the dynamic trajectory of neutrophil subpopulation evolution. Results: (1) After quality control, scRNA-seq was performed on 58209 cells, and a total of 11 cell subsets were obtained after dimensionality reduction clustering, and the main cell subsets of 11 types included: T and NK cells, epithelial cells, endothelial cells, fibroblasts, macrophages and dendritic cells, neutrophils, B cells and plasma cells, mast cells, melanocytes, pericytes, sweat gland epithelial cells. (2) Pathways closely related to cell proliferation, cancer progression and metastasis were highly enriched in tumor epithelial cells, while pathways related to immune activation were significantly downregulated, suggesting that tumor epithelial cells play an important role in cancer development. (3) Infiltrated neutrophils in tumors may inhibit tumor immunity by activating Th2-type cytokines. (4) T&NK cells can participate in reprogramming the tumor immune microenvironment through interferon stimulation pathway, interleukin pathway, TCR pathway and so on, while macrophages and dendritic cells may promote the polarization of macrophages to M2 type through tricarboxylic acid circulation. Conclusions: (1) The present study demonstrated the wide heterogeneity and diversity of cell subsets in CSCC by single-cell mapping of the tumor microenvironment using scRNA-seq. (2) Immune cells in the CSCC can reprogram the TME by regulating immune pathways, cell proliferation, biological metabolism and other processes, infiltrated neutrophils in tumors may inhibit tumor immunity by activating Th2-type cytokines. (3) The differentially expressed genes and key pathways specifically expressed in multiple cell subsets found in the study are helpful to further understand the pathogenesis of CSCC and provide a basis for developing new targets for immunotherapy.