Single-Cell Rna Sequencing Reveals Priming Professional Antigen-Presenting Macrophages And Chemokine Expressing T Cells In Tumor Microenvironment By Axl Inhibitor, Ski-G-801.

Abstract The mechanistic investigations have defined that AXL inhibition reprograms the immunological microenvironment leading to increased T cells and antigen-presenting cells. However, previous reports did not clearly demonstrate the linchpin mechanism of anti-tumor effects with AXL inhibitors. It may be possible to elucidate the immunological mechanism of AXL inhibitor based on a comprehensive analysis of cell type-specific transcriptomic changes using scRNA-seq. The human CD34-NSG mice were engrafted LUSC tumor. These mice were divided into four dose groups: Vehicle, SKI-G-801, pembrolizumab, and combination groups. The tumor-associated cells were collected for flow cytometry and scRNA-seq analysis, and FFPE sample of tumor tissue was analyzed by a multispectral imager. The combination group demonstrated the superior anti-cancer efficacy to vehicle, pembrolizumab, and SKI-G-801 groups (all p<0.05). The scRNA-seq was performed with Seurat 4.0. The cells were separated into total 9 clusters. Following SKI-G-801 treatment, the T cells showed higher expressions of CD2, CCL5, CCL4, GZMA, and GZMB compared to both pembrolizumab and vehicle groups (both p<0.05). The gene expressions of CD2 and GZMA were higher than pembrolizumab and vehicle groups (both p<0.05). The CDR3 length differences of TCRs and diversity of T cell clones were higher increased in SKI-G-801, pembrolizumab and combination groups than vehicle group (p<0.05). In the macrophage cluster, the HLA-A, HLA-DRA, HLA-DRB1, and IL-1B genes were commonly expressed in SKI-G-801 and combination group (p<0.05). Mostly, the professional antigen-presenting macrophages (MHC class 2 protein complex high, p<0.05) were increased in the combination group. The combination effects between pembrolizumab and SKI-G-801 can be addressed by enhanced antigen presenting machinery, T cell activation and unique T cell clones. These transcriptomic results were highly correlated with the results of multispectral imaging and flow cytometry. Tumor infiltrations of helper T cells and cytotoxic T cells significantly increased in combination and SKI-G-801 groups (p < 0.01, p < 0.001 respectively). In multiplex IHC analysis, the proportion of CD4+ and CD8+ T cells were significantly increased in the tumor nest (both p<0.05), but the Tregs were not changed in the tumor nest which observations were validated by further flow cytometry analysis. A novel AXL inhibitor, SKI-G-801 drives priming of professional antigen-presenting cells and tumor-infiltrating T cells, leading to immunological synapsis for tumor killing, which is significantly enhanced by the combination with pembrolizumab. These results suggest the inhibition of AXL signal pathway by SKI-G-801 could confer a solid rationale for clinical investigation of lung cancer cells. Citation Format: Kyung-Ho Pyo, Hee Kyu Lee, Ha Ni Jo, Wongeun Lee, Seong Gu Heo, Sang Bin Lim, Dong Kwon Kim, Chun-Bong Synn, Youngseon Byeon, Young Seob Kim, Beung-Chul Ahn, Min Hee Hong, Sun Min Lim, Hye Ryun Kim, Byoung Chul Cho. Single-cell RNA sequencing reveals priming professional antigen-presenting macrophages and chemokine expressing T cells in tumor microenvironment by AXL inhibitor, SKI-G-801 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2755.