| 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 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. |
