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 3

WNT-activated cells and WNT/β-catenin signaling are responsible for murine ICC progression.

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WNT-activated cells and WNT/β-catenin signaling are responsible for muri...
(A) Schematic of DTR-mediated ablation of KRT19+AXIN2+ cells. (B) Experimental strategy for lineage ablation of KRT19+AXIN2+ cells. Sac, sacrifice. (C–E) Fluorescence staining for EdU (green, C), SCP2+ (green, D), ATP6V1F+ (green, E), and Tom+ (red) cells in ICC mice after DT treatment. Scale bars: 50 μm. (F) Quantification of Tom+, Tom+EdU+, Tom+SCP2+, and Tom+ATP6V1F+ cells in ICC mice after DT treatment. (G) Representative images of liver morphology after DT treatment. (H) Statistical analysis of liver-to-body weight ratio after DT treatment. (I–N) Representative images of H&E (I) and KRT19 (L) staining of liver sections after tamoxifen and DT treatment. Scale bars: 200 μm. Statistical analyses: ICC number (J), ICC diameter (K), KRT19+ cell area (M), and KRT19+ cell number (N). (O) Experimental strategy for XAV-939 treatment in ICC mice. (P) Representative image of liver morphology after XAV-939 treatment. (Q) Statistical analysis of liver to body weight ratio after XAV-939 treatment. (R–W) Representative images of H&E (R) and KRT19 (U) staining of liver sections after XAV-939 treatment. Scale bars: 200 μm. Statistical analyses: ICC number (S), ICC diameter (T), KRT19+ cell area (V), and KRT19+ cell number. (X and Y) Fluorescence images of lineage tracing at days 3, 7, 14, and 21 in ICC tumors after XAV-939 treatment, with nuclei stained with DAPI (blue). Scale bar: 50 μm (X). Quantification of Tom+ cells at these time points (Y). (Z) Fluorescence staining of EdU+, SCP2+, and ATP6V1F+ (green) and Tom+ (red) cells in ICC mice after XAV-939 treatment. Scale bars: 50 μm. Quantification of Tom+, EdU+, SCP2+, and ATP6V1F+ cells in ICC tumors (bottom right). Data represent the mean ± SD (F, H, J, K, M, N, Q, S, T, V, W, Y, and Z). P values were calculated by 2-tailed, unpaired Student’s t test for F, H, J, K, M, N, Q, S, T, V, W, Y, and Z.