Vessels encapsulating tumor clusters promote noninvasive metastasis of hepatocellular carcinoma by shaping an immunosuppressive microenvironment
Bi-Yu Huang, Zheng-Qi Mi, Xiao-Yu Zhang, Yu-Chen Ji, Meng-Zhi Wu, Zi-Feng Cheng, Chen Xie, Shuai He, Jing Zhu, Jian-Hong Fang, Chong Wu, Bin-Kui Li, Yun-Fei Yuan, Limin Zheng, Shi-Mei Zhuang
Bi-Yu Huang, Zheng-Qi Mi, Xiao-Yu Zhang, Yu-Chen Ji, Meng-Zhi Wu, Zi-Feng Cheng, Chen Xie, Shuai He, Jing Zhu, Jian-Hong Fang, Chong Wu, Bin-Kui Li, Yun-Fei Yuan, Limin Zheng, Shi-Mei Zhuang
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Research Article Hepatology Immunology Vascular biology

Vessels encapsulating tumor clusters promote noninvasive metastasis of hepatocellular carcinoma by shaping an immunosuppressive microenvironment

Abstract

Vessels encapsulating tumor clusters (VETC), a distinct vascular pattern in hepatocellular carcinoma (HCC), facilitates noninvasive metastasis in whole clusters. The interaction between VETC and the tumor microenvironment requires exploration. Here, we found that, compared with human non-VETC-HCCs, VETC-tumors exhibited more PD1+CD8+ T cells and Tregs, especially TNFRSF4+ Tregs and Ki67+ Tregs, which showed increased immunosuppressive and proliferative activity. Such immunosuppressive status was also detected in tumor emboli of VETC-HCCs, and Treg density in emboli was positively associated with metastatic cell proliferation. VETC-HCCs revealed abundance correlation, closer spatial proximity, and stronger immunosuppressive ligand-receptor interactions between TNFRSF4+ Tregs/Ki67+ Tregs and PD1+CD8+ T cells. Depleting Tregs in mice reduced PD1+CD8+ T cells in primary lesions, tumor emboli, and metastatic foci of VETC-allografts, and attenuated allograft metastasis. TGF-β1 levels were upregulated in endothelial cells of VETC-HCCs and associated with TNFRSF4+ Tregs/Ki67+ Tregs enrichment. Disrupting VETC formation decreased endothelial TGF-β1 expression and reduced TNFRSF4+ Tregs, Ki67+ Tregs, PD1+CD8+ T cells, and Treg/CD8+ T cell ratios. Collectively, VETC may enhance Treg activity via TGF-β1, while Tregs promote and sustain CD8+ T cell exhaustion through immune inhibitory ligand-receptor interaction, thereby shaping the immunosuppressive microenvironment and enabling tumor clusters to retain such niche to disseminate. These findings disclose mechanisms of tumor immune microenvironment formation and provide rationales for precision medicine.

Authors

Bi-Yu Huang, Zheng-Qi Mi, Xiao-Yu Zhang, Yu-Chen Ji, Meng-Zhi Wu, Zi-Feng Cheng, Chen Xie, Shuai He, Jing Zhu, Jian-Hong Fang, Chong Wu, Bin-Kui Li, Yun-Fei Yuan, Limin Zheng, Shi-Mei Zhuang

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Figure 3

CD8+ T cells exhibit a more exhausted phenotype in human VETC tumors compared to non-VETC HCCs.

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CD8+ T cells exhibit a more exhausted phenotype in human VETC tumors com...
(A) Pseudotime trajectory analysis on CD8+ T cell differentiation. (B) The score of exhausted genes in CD8_PDCD1 cells. Genes are listed in Supplemental Table 9. The central line of violin plot denotes median value (50th percentile), the upper and lower lines denote the 75th and 25th percentile, respectively. (C) The proportion of PD1+CD8+ T cells in CD8+T cells. (D) The proportion of GZMB+ or GZMK+CD8+ T cells in CD8+T cells. (E) Positive correlation between the number of Tregs and the proportion of PD1+CD8+ T cells in HCC tissues (n = 28). R represents the Pearson correlation coefficient. (F) Spatial proximity between Tregs and PD1+CD8+ T cells. Yellow arrows indicate close proximity of these cells. (G) CellPhoneDB analysis illustrated ligand-receptor interactions between Treg_TNFRSF4/Treg_MKI67 and CD8_PDCD1 cells. Dot size indicates average expression levels and color intensity denotes P value. (H) The proportion of Galectin9+ Tregs in Tregs. (I) The proportion of TIM3+PD1+CD8+ T cells in CD8+ T cells. (J and K) Spatial proximity between Galectin9+ Tregs and TIM3+PD1+CD8+ T cells. non-VETC HCCs, n = 6 (A, B, and G), 14 (C and E), 10 (D), 9 (F, H, and I), 8 (K); VETC HCCs, n = 6 (A, B, and G), 16 (C), 10 (D, H, and I), 14 (E),11 (F), 9 (K). scRNA-seq data were analyzed in A, B, and G, multiplex immunofluorescent staining in C–F and H–K. Scale bar: 25 μm. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by Mann-Whitney U test (B–D), 2-tailed Student’s t test (F, H, I, and K), Pearson’s correlation test (E).