Tumor-initiating cells escape tumor immunity via CCL8 from tumor-associated macrophages in mice
Shuang Chen, Chensong Huang, Kang Li, Maosheng Cheng, Caihua Zhang, Jianqi Xiong, Guoli Tian, Ruoxing Zhou, Rongsong Ling, Xiaochen Wang, Gan Xiong, Zhihui Zhang, Jieyi Ma, Yan Zhu, Bin Zhou, Liang Peng, Zhenwei Peng, Heping Li, Demeng Chen
Shuang Chen, Chensong Huang, Kang Li, Maosheng Cheng, Caihua Zhang, Jianqi Xiong, Guoli Tian, Ruoxing Zhou, Rongsong Ling, Xiaochen Wang, Gan Xiong, Zhihui Zhang, Jieyi Ma, Yan Zhu, Bin Zhou, Liang Peng, Zhenwei Peng, Heping Li, Demeng Chen
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Research Article Oncology

Tumor-initiating cells escape tumor immunity via CCL8 from tumor-associated macrophages in mice

Abstract

Tumor-initiating cells (TICs) play a key role in cancer progression and immune escape. However, how TICs evade immune elimination remains poorly characterized. Combining single-cell RNA-Seq (scRNA-Seq), dual-recombinase–based lineage tracing, and other approaches, we identified a WNT-activated subpopulation of malignant cells that act as TICs in vivo. We found intensive reciprocal interactions between TICs and immune-regulatory tumor-associated macrophages (Reg-TAMs) via growth arrest–specific 6/AXL receptor tyrosine kinase/MER proto-oncogene, tyrosine kinase (GAS6/AXL/MERTK) signaling pathways, which facilitated the immune escape of TICs. In this study, we used chemical inhibitors and Axl/Mertk conditional double-KO (cDKO) mice to demonstrate that inhibiting the interaction between TIC-derived GAS6 and AXL/MERTK in Reg-TAMs reactivated antitumor immune responses. We identified CCL8 as a critical mediator of the GAS6/AXL/MERTK pathway, primarily by inhibiting Treg infiltration into the tumor. Furthermore, the AXL/MERTK signaling blockade sensitized tumor cells to anti–programmed cell death 1 (anti–PD-1) treatment. Thus, we elucidated a detailed mechanism by which TICs evade tumor immunity, providing insights into strategies to eradicate TICs that escape conventional immunotherapy.

Authors

Shuang Chen, Chensong Huang, Kang Li, Maosheng Cheng, Caihua Zhang, Jianqi Xiong, Guoli Tian, Ruoxing Zhou, Rongsong Ling, Xiaochen Wang, Gan Xiong, Zhihui Zhang, Jieyi Ma, Yan Zhu, Bin Zhou, Liang Peng, Zhenwei Peng, Heping Li, Demeng Chen

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Figure 2

WNT-activated cells constitute a TIC population in mouse ICC.

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WNT-activated cells constitute a TIC population in mouse ICC. (A) UMAP p...
(A) UMAP plot showing 4 epithelial cell subclusters. (B) RNA velocity–inferred developmental trajectory of epithelial cells. (C) UMAP plots showing the distribution of CytoTRACE scores for epithelial cells. Higher scores indicate higher stemness. (D) Monocle pseudotime trajectory showing cell differentiation of 4 epithelial cell subclusters. (E) Heatmap of GSVA-enriched pathways in epithelial cell clusters. (F) Schematic of lineage tracing. (G) Experimental strategies for lineage tracing of AXIN2+KRT19+ epithelial cells in ICC mice. d0, day 0. (H) Violin plots showing Sox4 and Klf6 expression among epithelial cell subclusters. (I and L) Representative fluorescence images of SOX4 (green) and KLF6 (green) staining and Tom+ cells (red) in ICC tumors. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. (J, K, M, and N) Statistical analysis of SOX4+ and KLF6+ expression in Tom+/– mouse ICC cells. (O and Q) Representative fluorescence images showing EdU+ (green), SCP2+ (green), ATP6V1F+ (green), and Tom+ (red) cells in ICC after tamoxifen treatment. Nuclei are stained with DAPI (blue). Scale bars: 50 μm. (P and R) Comparison of the percentage of Tom+EdU+, Tom+SCP2+, and Tom+ATP6V1F+ cells in ICC induced for 3 days, with results shown at 7, 14, and 21 days. (S) Flow cytometry plots of EpCAM+Tom+ tumor cells that were isolated and from ICC tissues and sorted. (T and U) Tumor formation frequency of EpCAM+Tom+ and EpCAM+Tom– ICC cells in vivo (T), analyzed by the single-hit model likelihood ratio test (U). Data represent the mean ± SD J, K, P and R). P values were calculated by 2-tailed, unpaired Student’s t test (H, J, K, M, and N) and 1-way ANOVA with Tukey’s multiple-comparison test (P and R).