Revealing a novel tumor immune escaping mechanism induced by macrophage-tumor fusion cells in HPV- cervical cancer through spatial SCRNA analysis.

person Yan Zhang Hong Kong University of Shenzhen Hospital, Shenzhen, China info_outline Yan Zhang, Zhiyuan Xu, Xinyu Ye, Bin Ye, Huahui Li, Danyang Zheng, Caining Zhao, Wanli Xu, Li Yang, Zhongshi Lin, Yao Liu, Lingyu MA, Zhibing Liang, Xiangyu Xiao, Yi Lu, Jian Zhang, Feng-Ming Spring Kong, Hao Yu

Authors person Yan Zhang Hong Kong University of Shenzhen Hospital, Shenzhen, China info_outline Yan Zhang, Zhiyuan Xu, Xinyu Ye, Bin Ye, Huahui Li, Danyang Zheng, Caining Zhao, Wanli Xu, Li Yang, Zhongshi Lin, Yao Liu, Lingyu MA, Zhibing Liang, Xiangyu Xiao, Yi Lu, Jian Zhang, Feng-Ming Spring Kong, Hao Yu Organizations Hong Kong University of Shenzhen Hospital, Shenzhen, China, Department of Clinical Oncology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China, School of Medicine, Southern University of Science and Technology, Shenzhen, China, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China, The University of Hong Kong, Hong Kong, China, University of Hong Kong, Hong Kong, China, Department of Clinical Oncology,The University of Hong Kong-Shenzhen Hospital, Shenzhen, China, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China, Shenzhen Institute for Drug Control (Shenzhen Testing Center of Medical Devices), Shenzhen, China, University of Hong Kong - Shenzhen Hospital, Shenzhen, China, Hong Kong University Shenzhen Hospital, Shenzhen, China, Southern University of Science and Technology, Shenzhen, China Abstract Disclosures Research Funding Institutional Funding Shenzhen Science and Technology Program, ZDSYS20210623091811035 Background: Human papillomavirus negative (HPV-) cervical cancer is a rare type of cervical cancer whose tumor immune microenvironment is still unclear. In this study, by single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) analysis of HPV-cervical cancer, we discovered and characterized a novel cell subtype with the ability to promote tumor immune escape, which we called macrophage-tumor fusion cells. Methods: scRNA-seq and ST were integrated to analyze the newly diagnosed HPV- cervical cancer tumor tissue. Seurat 4.0 and CellChat were used to cluster and annotate cell subtypes and cellular interaction. U14 (RFP expressed in the nucleus) and macrophage (GFP expressed in cytoplasm and nucleus) were co-cultured to obtain macrophage-tumor fusion cells. In the tumor growth or antibody treatment model, C57BL/6L mice and Nude mice were injected subcutaneously or tail vein with 1*10 6 macrophage-tumor fusion cells or U14 cells, respectively. Statistical analysis was done by a two-sided paired t-test. P<0.05 was considered significant. Results: The HPV- cervical cancer tumor tissue includes 9800 cells with 90% tumor cells and 3% tumor-infiltrating immune cells with immunosuppressive features, similar to a cold tumor microenvironment. Especially, we found a special population of macrophage-tumor fusion cells, which highly expressed the marker genes of macrophages (CD68 and CD163) and of tumor cells (EPCAM and TP63). We confirmed they are not doublets or the endocytosis status of macrophages. ST showed that the macrophage-tumor fusion cells co-localized with tumor cells, macrophages, and also co-localized with T cells, CAFs, and endothelial cells. The NECTIN2/TIGIT was enriched in macrophage-tumor fusion cells to interact with T cells, promoting Treg activation and inhibiting cell proliferation. The LGALS9/HAVCR2 was also enriched in macrophage-tumor fusion cells to interact with macrophage, promoting immunological tolerance. Macrophage-tumor fusion cell hybrids were obtained by in-vitro co-culture of HPV-cervical mouse cell line (U14) and macrophages isolated from GFP transgenic mice. Macrophage-tumor fusion cells obtained higher proliferation and migration abilities than U14 cells in both the immunodeficient and syngeneic mouse tumor models (Ps<0.0001); and higher lung metastasis rate and intensity in the treated tail vein metastasis model (Ps = 0.039). Then we evaluated potential therapeutic targets found on macrophage-tumor fusion cells, identifying blocking TIGIT significantly inhibited the growth of macrophage-tumor fusion tumors in C57/BL6 mouse model, similar to the blocking PD1. Conclusions: This study sheds light on a novel tumor immune escape mechanism of HPV-negative cervical cancer by discovering macrophage-tumor fusion cells, which could provide a potential therapeutic strategy for the clinic.

Pre-clinical