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The spatial organization of immune cells within the tumor microenvironment (TME) largely determines the anti-tumor immunity and also highly predicts tumor progression and therapeutic response. Tim-3 is a well-accepted immune checkpoint and plays multifaceted immunoregulatory roles via interaction with distinct Tim-3 ligands (Tim-3L), showing great potential as an immunotherapy target. However, the cell sociology mediated by Tim-3/Tim-3L and their contribution to tumor development remains elusive. Here, we analyzed the spatial distribution of Tim-3/Tim-3L in TME using multiplex fluorescence staining and revealed that despite the increased Tim-3 expression in various tumor-infiltrated lymphocytes, Tim-3(+)CD4(+) cells were more accumulated in parenchymal/tumor region compared with stromal region and exhibited more close association with patient survival. Strikingly, CD4 T cells surrounding Tim-3L(+) cells expressed higher Tim-3 than other cells in cancerous tissues. In vivo studies confirmed that depletion of CD4 T cells completely abrogated the inhibition of tumor growth and metastasis, as well as the functional improvement of CD8 T and NK, mediated by Tim-3 blockade, which was further validated in peripheral lymphocytes from patients with hepatocellular carcinoma. In conclusion, our findings unravel the importance of CD4 T cells in Tim-3/Tim-3L-mediated immunosuppression and provide new thoughts for Tim-3 targeted cancer immunotherapy.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. METHODS: ZHX2 overexpression and knockdown were introduced into HCC cell lines. BODIPY staining and D-Glucose- 13C6 labeled lipid mass spectrometry were involved to estimate lipogenesis in HCC cell lines. Colony formation and cell proliferation assays were used to determine HCC development. Hepatocytes specific Zhx2 knockout mice (Zhx2-KOhep) were included to induce spontaneous liver tumor. qRT-PCR, western blotting and dual luciferase assays were used to detect gene regulation. FINDINGS: Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC and SCD1. In accordance, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Further studies demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with SREBP1c inhibitor, fatostatin, dampened the spontaneous tumor formation in Zhx2-KOhep mice. Mechanically, ZHX2 transcriptionally increased expression of miR-24-3p, which targets on SREBP1c. INTERPRETATION: ZHX2 inhibits de novo lipogenesis in HCC cells by down-regulating the expression of SREBP1c via miR-24-3p, and further inhibits the development of HCC. These new findings may provide a new strategy for the treatment of HCC. FUNDING STATEMENT: This work was supported by grants from the National Science Foundation of China (Key project 81830017 and 81902443,81702647,81902051,81972819), Taishan Scholarship (No.tspd20181201), National Key Research and Development Program (2018YFE0126500), National Science Foundation for Distinguished Young Scholars of China (81425012), Shandong Provincial Key Innovation project (No.2018FYJH0503), Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong, Key Research and Development Program of Shandong (2019GSF108238) . DECLARATION OF INTERESTS: All authors declare no potential conflicts of interest. ETHICS APPROVAL STATEMENT: The Medical Ethics Committee of Shandong University approved the use of human specimens in accordance with the Helsinki Declaration. All mice were housed under conditions free of specific pathogens and euthanized according to regulations established by the Animal Care Committee of Shandong University.

Background: Liver cancer stem cells (CSCs) are critical determinants of cancer relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and cell lines by flow cytometry. Also, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: The expression of ZHX2 was reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of CSCs in supporting tumor initiation, self-renew and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significant poor survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Funding Statement: This work was supported by grants from the National Natural Science Fund for Outstanding Youth Fund (81425012), the National Key Research and Development Program (No.2016YFE0127000), the National Science Foundation of China (No. 81830017, 81370527, 81702647), Taishan Scholarship (No.tspd20181201), Shandong Provincial Key Innovation project (No.2018FYJH0503) and Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Informed consent was obtained from all patients before the study was initiated with approval of the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. All animal procedures were performed in according to protocols approved by the Shandong University Animal Care Committee and conducted with an animal ethical approval.

Background: Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Keywords: ZHX2, Tumor suppressor, Epigenetics, Cancer stem cells, Oncotherapy