Fu Zhao ¹ Junjie Hong ² Guangyao Zhou ^3* Tian Jiao Huang ⁴ Zhiheng Lin ¹ Yining Zhang ^5* Lei L. Liang ^5* Huarong Tang ^5*

• ¹ Shandong University of Traditional Chinese Medicine, Jinan, China
• ² Department of Gynecologic Oncology, Zhejiang Cancer Hospital, Hangzhou, Jiangsu Province, China
• ³ Tianjin Medical University Cancer Institute and Hospital, Tianjin, Tianjin, China
• ⁴ Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang Province, China
• ⁵ Zhejiang Cancer Hospital, Hangzhou, China

Background Cervical cancer (CC) is the fourth most common malignancy among women globally and serves as the main cause of cancer-related deaths among women in developing countries. The early symptoms of CC are often not apparent, with diagnoses typically made at advanced stages, which lead to poor clinical prognoses. In recent years, numerous studies have shown that there is a close relationship between Mast cells (MCs) and tumor development. Method The present study acquired single-cell RNA sequencing data from ten CC tumor samples in the ArrayExpress database. Slingshot and AUCcell were utilized to infer and assess the differentiation trajectory and cell plasticity of MCs subpopulations. Differential expression analysis of MCs subpopulations in CC was performed, employing Gene Ontology, gene set enrichment analysis, and gene set variation analysis. Result The obtained 93,524 high-quality cells were classified into ten cell types, including T_NK cells, endothelial cells, fibroblasts, smooth muscle cells, epithelial cells, B cells, plasma cells, MCs, neutrophils, and myeloid cells. Furthermore, a total of 1,392 MCs were subdivided into seven subpopulations: C0 CTSG+ MCs, C1 CALR+ MCs, C2 ALOX5+ MCs, C3 ANXA2+ MCs, C4 MGP+ MCs, C5 IL32+ MCs, and C6 ADGRL4+ MCs. Notably, the C2 subpopulation showed close associations with tumor-related MCs, with Slingshot results indicating that C2 subpopulation resided at the intermediate-to-late stage of differentiation, potentially representing a crucial transition point in the benign-to-malignant transformation of CC. CNVscore and bulk analysis results further confirmed the transforming state of the C2 subpopulation. CellChat analysis revealed TNFRSF12A as a key receptor involved in the actions of C2 ALOX5+ MCs. Moreover, in vitro experiments confirmed that TNFRSF12A promotes the proliferation, migration, and invasion of CC cells. Additionally, a prognosis model and immune infiltration analysis based on the marker genes of the C2 subpopulation provided valuable guidance for patient prognosis and clinical intervention strategies. Conclusions We first identified the transformative tumor-associated MCs subgroup C2 ALOX5+ MCs within CC, which was at a critical stage of tumor differentiation and impacted the progression of CC.