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
View: Text | PDF
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

×

Figure 2

Tregs are more abundant and exhibit greater immunosuppressive and proliferative activity in human VETC HCCs compared with non-VETC tumors.

Options: View larger image (or click on image) Download as PowerPoint
Tregs are more abundant and exhibit greater immunosuppressive and prolif...
(A) Score of tumor-specific Treg signature genes. The central line of violin plot denotes median value (50th percentile), the upper and lower lines denote the 75th and 25th percentiles, respectively. (B) UMAP plot visualization and quantification of four Treg clusters. non-VETC, VETC HCCs: n = 6, 6. (C) Kaplan-Meier overall survival analysis. TCGA HCC patients (n = 368) were stratified into high and low Treg_TNFRSF4 or Treg_MKI67 infiltration groups based on their signature genes in Supplemental Table 6. Infiltration levels were estimated by CIBERSORTx deconvolution on the RNA-seq data from TCGA. (D and E) Proportions of TNFRSF4+ Tregs and Ki67+ Tregs. Yellow arrows indicate TNFRSF4+ (D) or Ki67+ Tregs (E). (F) Pseudotime trajectory of Treg differentiation based on the signature genes of Treg_MKI67 and Treg_TNFRSF4. Genes are listed in Supplemental Table 6. Cells are assigned pseudotime scores, visualized as a gradient from dark to light blue, indicating progression from early to terminal states (left panel). (G) Score of tumor-specific Treg signature genes along the differentiation trajectory. For A and G, genes are listed in Supplemental Table 5. (H) The ratio of TNFRSF4+ Tregs and TNFRSF4–Tregs coexpressing TIM3 or CD39. n = 19. (I and J) The proportion of TNFRSF4+Ki67+ Tregs in Ki67+ Tregs (I) or TNFRSF4+ Tregs (J). non-VETC HCCs, n = 6 (A and B), 49 (D and E), 51 (I and J); VETC HCCs, n = 6 (A and B), 66 (D), 67 (E), 70 (I and J). scRNA-seq data were analyzed in A, B, F, and G, multiplex immunofluorescent staining in D, E, and H–J. Scale bars: 25μm. Data are shown as mean ± SEM.; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, by Mann-Whitney U test (A, E, I and J), 2-tailed Student’s t test (B, D, and H), log-rank test (C).