DOI: 10.25442/hku.29528033.v1
title: Supporting data for “HDAC9 Regulates Macrophage Polarization and Modulates the Progression of Intrahepatic Cholangiocarcinoma”

description: How tumor-related epigenetic abnormalities affect the interactions between cancer cells and the immune system is still not fully understood. Tumor genome studies have revealed that chromatin modifiers can reshape tumorphenotype through the induction of immune response. Macrophages, as one kind of immune cells which are common seen in tumor microenvironment have a controversial role in pro- and antitumoral effects.<br>Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer after hepatocellular carcinoma (HCC). iCCA originates from bile duct epithelial cells, and it’s well-known for its desmoplastic tumor microenvironment. iCCA is difficult to detect by routine examination due to its cryptic anatomical location, it progresses asymptomatically in the early stage but is highly biologically invasive. More than 90% of patients survived less than four years. Only early-stage cholangiocarcinoma can undergo surgery, radiotherapy and chemotherapy for mid- and late-stage cholangiocarcinoma have little effect. There are no targeted drugs for the treatment of cholangiocarcinoma. The first-line treatment drugs are gemcitabine and cisplatin, but the effect is not ideal. Therefore, it is crucial to understand the molecular mechanisms behind the invasive behavior and progression of iCCA.<br>In our study, we found that there was a higher HDAC9 expression level in iCCA tissue compared with corresponding para-cancerous tissues, high tumoral expression of HDAC9 was associated with longer disease-free survival and overall survival. TCGA (N=36) and local (N=57) cohorts were included in this study. The expression of HDAC9 in iCCA tissues and corresponding para-cancerous tissues were assessed by immunohistochemistry. We demonstrated that inactivation of histone deacetylase 9 (HDAC9) promotes the proliferation and invasion of intrahepatic cholangiocarcinoma (iCCA), and that patients with iCCA with low HDAC9 expression exhibit shorter survival. HDAC9 remodels the tumor microenvironment (TME) through the control of H4K16 deacetylation in iCCA. The effects of HDAC9 knockdown were investigated in orthotopic xenograft iCCA mouse models, and the tumor and para-cancerous liver of mouse models were used to analysis the polarization of macrophages. In orthotopic mouse models of iCCA, HDAC9-knocked down tumor microenvironments had more exhausted T cells and M2 macrophages, with M2 macrophages resulting from high levels of CCL2 secretion from iCCA. Cell co-culture assay confirmed the same result that HDAC9 knockdown in iCCA cells promoted more M0 macrophages polarized to M2 macrophages.<br>Mechanistically, the deletion of HDAC9 leads to an increase in acetylation of lysine 16 on histone H4 (H4K16ac), and at the same time affects the binding between lysine on histone H4 position 20 (H4K20me2) and p53-binding protein 1 (53BP1), thereby inhibiting the transcriptional activation of the p53 pathway, and the expression of cell cycle arrest gene GADD45A in the p53 pathway is blocked, resulting in uncontrolled cell cycle and faster progression of iCCA.<br>In summary, this study clarified the role of histone deacetylase HDAC9 in regulating tumor cells and microenvironment through the TGF-β signaling pathway from the perspective of genetics and immunology, filling the gap in epigenetic research on cholangiocarcinoma tumor cells regulating the immune microenvironment, and providing inspiration for the targeted treatment and clinical combination drug use of cholangiocarcinoma.