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 6

CCL8 is the downstream mediator of AXL/MERTK signaling in Reg-TAMs.

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CCL8 is the downstream mediator of AXL/MERTK signaling in Reg-TAMs. (A) ...
(A) Experimental design for induction of ICC in Lyz2-CreER (Control, CTL) and Lyz2-CreER Ccl8fl/fl (Ccl8cko) mice. (B) The KO efficiency of CCL8 in Lyz2+cells was validated by Western blotting. (C) Representative liver morphology images of control and Ccl8cko mice for survival outcome analysis (left). Kaplan-Meier OS curve for control and Ccl8cko mice is shown (right). (D) Representative liver morphology images of control and Ccl8cko mice after 6 weeks of plasmid injection (left) and statistical analysis of liver-to-body weight ratio (right). (E–G) Flow plots show immune cell frequencies and counts in control and Ccl8cko mice: CD4+ and CD8+ T cells (E), NK and NKT cells (F), and Tregs (G). Each plot displays cell frequencies (left) and quantitative analysis (right). (H) Experimental strategy for ICC mice using R428 alone or R428 with CCL8 (R428+CCL8). (I) Representative liver morphology image of different treatment groups for survival outcome analysis (left). Kaplan-Meier OS curve for mice with ICC in different treatment groups (right). (J) Liver morphology (left) in different treatment groups after 6 weeks of treatment and statistical analysis of liver-to-body weight ratios (right). (K) Fluorescence images (left) of lineage tracing in different treatment groups, statistical analysis of Tom+ cell number and areas (right); Scale bar: 50 μm. (L–N) Flow plots (left) and graphs (right) showing frequencies of tumor-infiltrating CD4+ and CD8+ T cells (L), NK and NKT cells (M), and Tregs (N) in different treatment groups. Data represent the mean ± SD (D–G and J–N). P values were calculated by 2-tailed, unpaired Student’s t test (D–G), 1-way ANOVA with Tukey’s multiple-comparison test (J–N), and log-rank test (C and I).