Supplementary materials and methods, Supplementary Figures and figure legends, Supplementary Tables

BACKGROUND & AIMS:Cholangiocarcinoma (CCA) is a lethal malignancy with limited therapeutic options. We investigated the oncogenic role of poly(ADP-ribose) glycohydrolase (PARG) and evaluated potential therapeutic strategies. METHODS:A tissue microarray comprising 275 CCA patient samples was analyzed by immunohistochemistry. Liquid chromatography-tandem mass spectrometry was utilized to identify downstream targets of PARG. Transgenic mice (Pargf/f) were employed to establish a spontaneous CCA model via hydrodynamic tail vein injection and biliary instillation. The efficacy of PARG inhibition was assessed in multiple preclinical models, including patient-derived organoids, patient-derived xenografts, orthotopic xenografts, and an immunocompetent syngeneic murine model. Cytometry by time-of-flight analysis was used to profile changes in the tumor microenvironment following PARG inhibition and anti-PD-1 therapy. RESULTS:PARG was highly expressed in CCA and predicted a dismal prognosis based on analysis of a large patient cohort. Genetic depletion of Parg in spontaneous CCA models induced by two methods, including hydrodynamic tail vein injection and biliary instillation, significantly halted carcinogenesis. PARG inhibition alone showed impressive efficacy and potentiated that of gemcitabine/cisplatin in patient-derived organoid, patient-derived xenograft, and orthotopic models. Mechanistically, PARG dePARylates ITCH, suppressing its autoubiquitination and thereby inhibiting the Hippo pathway, which promotes CCA proliferation and chemoresistance. Moreover, cytometry by time-of-flight analysis revealed crosstalk between the tumor and stroma, which could be suppressed by PARG inhibitors via the TEADs/CXCR4/CXCL12 axis. Combining PD-1 blockade and gemcitabine/cisplatin with PARG inhibitors resulted in a significantly greater reduction in tumor burden, as well as a survival benefit. CONCLUSIONS:Targeting PARG limits CCA progression, alleviates desmoplasia, and enhances response to both anti-PD-1 therapy and chemotherapy. IMPACT AND IMPLICATIONS:Little is known about the role of PARG in cholangiocarcinoma (CCA) development and progression. Herein, we show that PARG expression is upregulated and hyperactivated in CCA, promoting tumor cell proliferation, cancer-associated fibroblast recruitment, and resistance to therapy. Pharmacological inhibition of PARG suppresses CCA development and could be an effective therapeutic strategy when combined with chemotherapy and immunotherapy.

Hepatocellular carcinoma (HCC), which accounts for approximately 75–85% of primary liver cancers, ranks 4th in newly diagnosed cases among various types of cancer in China, and is the 2nd leading cause of cancer-related mortality, thereby posing a significant threat to the life and health of the Chinese population. Since the publication of the “Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China” in June 2017, which were updated by the China’s National Health Commission in December 2019 and December 2021, additional high-quality evidence from researchers worldwide regarding the diagnosis, staging, and treatment of HCC has emerged, necessitating another update to the guidelines. The new edition (2024 Edition) was written by more than 120 multidisciplinary experts in the field of HCC in China, which not only reflects the real-world situation in China but also may reshape the nationwide diagnosis and treatment of HCC. The new guideline aims to encourage the implementation of evidence-based practice and improve the national average 5-year survival rate for patients with HCC, as proposed in the “Healthy China 2030: A Vision for Health Care.”

The safety of immunotherapy in patients undergoing transplant remains unclear due to rejection risks. This study assessed the safety and efficacy of programmed death-1 (PD-1) inhibitors in patients with recurrent tumors who had undergone liver transplantation, emphasizing the value of using graft programmed death-ligand 1 expression as a predictor of rejection to guide patient selection. This single-center, open-label, prospective, single-arm study was conducted from July 2019 to May 2024 at Zhongshan Hospital, Fudan University (Shanghai, China). Eligible participants included patients with recurrent or metastatic liver cancer who had undergone liver transplantation and were unresponsive to locoregional or systemic therapies. The primary endpoints were the incidence and clinical outcomes of acute rejection. Secondary endpoints were overall survival and objective response rate. Twenty consecutive patients received PD-1 inhibitor therapy. Of these, 18 had HCC, and 2 had intrahepatic cholangiocarcinoma. Liver graft biopsies confirmed negative programmed death-ligand 1 expression in all participants before PD-1 inhibitor therapy. Three patients (15%) experienced acute rejection, with a 95% CI of 3.2%-37.9%. The 1-year and 2-year survival probabilities after PD-1 inhibitor treatment were 0.55 (95% CI: 0.33-0.77) and 0.24 (95% CI: 0.05-0.43), respectively. The median survival time after tumor recurrence was 24.6 months, exceeding the historically reported median survival time of 16.3 months. These exploratory findings suggest that, in selected recipients of liver transplant, PD-1 inhibitors may be associated with reduced rejection risk and potential survival benefit, although further validation is needed.

BACKGROUND:The optimal conversion regimen that allows more patients with unresectable biliary tract cancer to access surgery remains unclear; there is currently no standard conversion therapy for biliary tract cancer in China, with commonly used regimens including immunotherapy-based combinations and local therapy. The ZSAB-TransGOLP study aimed to assess the efficacy and safety of tislelizumab plus lenvatinib and GEMOX (gemcitabine plus oxaliplatin) chemotherapy (GOLP) in patients with this disease. METHODS:This single-arm, phase 2 study was conducted at two centres in China. Eligible patients aged 18-70 years with previously untreated locally advanced unresectable biliary tract cancer (intrahepatic cholangiocarcinoma, perihilar bile duct cancer, and gallbladder cancer), and an Eastern Cooperative Oncology Group performance status of 0 or 1, Child-Pugh score of A, and at least 3 months' life expectancy were enrolled. Patients received 200 mg intravenous tislelizumab on day 1 and intravenous GEMOX (0·5 h of 1000 mg/m2 gemcitabine on days 1 and 8; and 2 h of 85 mg/m2 oxaliplatin on day 1) in a 21-day cycle for three cycles, and 8 mg oral lenvatinib once daily. Tumour resectability was determined by the multidisciplinary team every 3 cycles of conversion therapy; patients who were ineligible for R0 resection and did not require surgery after six cycles received maintenance therapy with tislelizumab plus lenvatinib at the same dose as used in conversion therapy until completing 1 year of treatment, disease progression, intolerable toxicity, death, consent withdrawal, or investigators' decisions. The primary endpoint was the R0 resection rate. All treated patients were evaluable for safety and primary endpoint. The trial is registered with ClinicalTrials.gov (NCT05156788) and is ongoing but closed for recruitment. FINDINGS:Between Dec 27, 2021 and July 3, 2023, 52 patients were screened, 11 were excluded for ineligiblity, and 41 patients were enrolled and received the GOLP regimen. All patients were Chinese, with median age of 58 years (IQR 54-65); 21 patients (51%) were male and 20 patients (49%) were female. Median duration of GOLP treatment was 3 cycles (IQR 3-6). 28 (68%) of 41 patients underwent surgery. At a median follow-up of 19·5 months (IQR 14·6-25·0) by data cutoff on Jan 20, 2025, the R0 resection rate was 63% (26 of 41 [95% CI 47-78]). All patients had at least one any-grade treatment-related adverse event (TRAE); grade 3-4 TRAEs occurred in 20 (49%) of 41 patients, with neutropenia (14 [34%] of 41) being most common. Serious TRAEs occurred in 4 (10%) of patients and included neutropenia (three [7%]) and decreased platelet count (one [2%]). No TRAE-related deaths occurred. INTERPRETATION:With promising efficacy and manageable safety, GOLP represents a potentially feasible and high-efficiency conversion regimen for unresectable locally advanced biliary tract cancer. FUNDING:Program of Shanghai Academic Research Leader, the Key Disease Joint Research Program of Xuhui District, Shanghai Health Commission Clinical Research Special Project, Fellowship from the China Postdoctoral Science Foundation, National Science and Technology Major Project of China, the Outstanding Resident Clinical Postdoctoral Program of Zhongshan Hospital Affiliated to Fudan University, National Natural Science Foundation of China, and the Shanghai Sailing Program.

The literature suggests that hepatocellular Yes-associated protein 1 (YAP1) signaling is activated following hepatectomy and that such activation can suppress the growth of metastatic liver tumors. The prognosis of a real-world cohort of 240 patients with colorectal cancer liver metastasis (CRLM) undergoing major and minor hepatectomy was compared after adjusting for confounding factors. To model CRLM, we induced liver metastasis in mice by transsplenically injecting MC38 cells. We found that patients with CRLM and mice undergoing major hepatectomy had better survival compared to those undergoing minor hepatectomy. Mechanistically, extensive hepatectomy activates hepatocellular YAP1 by regulating the epidermal growth factor receptor, altering glutamine metabolism-related gene expression and increasing liver glutamine consumption. This metabolic shift leads to glutamine scarcity in tumor cells, causing increased reactive oxygen species production, which promotes loss of YAP1 activity in tumor cells. Consequently, the production of the chemokine CXCL5 is suppressed, which inhibits myeloid-derived suppressor cell infiltration and enhancing the immunological function of CD8+ T cells.

BACKGROUND:The combination of atezolizumab and bevacizumab (ATZ/BVZ) therapy has significantly advanced therapeutic approaches for hepatocellular carcinoma (HCC). However, less than 30% of patients achieve durable responses, highlighting the urgent need to understand mechanisms underlying resistance. OBJECTIVE:This study aimed to elucidate the mechanisms of resistance to ATZ/BVZ therapy in HCC and identify druggable targets associated with resistance, thus improving the treatment efficacy of ATZ/BVZ-resistant HCC. DESIGN:We employed single-cell RNA sequencing and a prospective clinical cohort (NCT04649489) to identify and characterise potential genes that contribute to ATZ/BVZ therapy resistance. Multiple preclinical HCC models and a coculture system were constructed, and cytometry by time-of-flight technology was used to further explore the relevant molecular mechanism. RESULTS:Elevated baseline serum galectin-4 levels correlated with resistance to ATZ/BVZ therapy and unfavourable prognosis in HCC. Galectin-4 overexpression nullified ATZ/BVZ therapy efficacy through promoting metabolic adaptation and fostering an immunosuppressive tumour microenvironment characterised by reduced infiltration and impaired cytotoxicity of CD8+ T cells and accumulation of PD-L1+ tumour-associated neutrophils. Mechanistically, galectin-4 inhibited proteasomal degradation of lactate dehydrogenase A (LDHA) by competitively decreasing tripartite motif containing 28 binding, thereby enhancing glycolysis and amplifying HIF-1α-mediated C-X-C motif chemokine ligand 6 (CXCL6) expression. Genetic knockdown or pharmacological inhibition of galectin-4 reversed metabolic adaptation and immune exclusion, and restored sensitivity to anti-PD-L1/BVZ therapy in preclinical models. CONCLUSION:Activation of the galectin-4/LDHA/HIF-1α and CXCL6 axis plays a pivotal role in ATZ/BVZ therapy resistance. Galectin-4 serves as a promising therapeutic target to improve immunotherapy efficacy and an effective predictive biomarker for immunotherapy response in HCC.

Anti-PD-L1-based combination immunotherapy has become the first-line treatment for unresectable hepatocellular carcinoma (HCC). However, the objective response rate is lower than 40%, highlighting the need to identify mechanisms of tolerance to immune checkpoint inhibitors and accurate biomarkers of response. In this study, we used next-generation sequencing to analyze HCC samples from 10 patients receiving anti-PD-L1 therapy. Activation of the renin-angiotensin system was elevated in nonresponders compared with responders, and angiotensin-converting enzyme 2 (ACE2) expression was significantly downregulated in nonresponders. ACE2 deficiency promoted HCC development and anti-PD-L1 resistance, whereas ACE2 overexpression inhibited HCC progression in immune-competent mice. Mass cytometry by time of flight revealed that ACE2-deficient murine orthotopic tumor tissues featured elevated M2-like tumor-associated macrophages, displayed a CCR5+PD-L1+ immunosuppressive phenotype, and exhibited high VEGFα expression. ACE2 downregulated tumor-intrinsic chemokine (C-C motif) ligand 5 expression by suppressing NF-κB signaling through the ACE2/angiotensin-(1-7)/Mas receptor axis. The lower chemokine (C-C motif) ligand 5 levels led to reduced activation of the JAK-STAT3 pathway and suppressed PD-L1 and VEGFα expression in macrophages, blocking macrophage infiltration and M2-like polarization. Pharmacologic targeting of CCR5 using maraviroc enhanced the tumor-suppressive effect of anti-PD-L1 therapy. Together, these findings suggest that activation of the ACE2 axis overcomes the immunosuppressive microenvironment of HCC and may serve as an immunotherapeutic target and predictive biomarker of response to PD-L1 blockade. Significance: ACE2 regulates the immune landscape of hepatocellular carcinoma by abrogating M2-like macrophage polarization and sensitizes tumors to anti-PD-L1, suggesting that harnessing the ACE2 axis could be a promising strategy to improve immunotherapy efficacy.

Liver transplantation represents a complex surgical procedure and serves as a curative treatment for patients presenting an acute or chronic end-stage liver disease, or carefully selected liver malignancy. A significant gap still exists between the number of available donor organs and potential recipients. The use of an otherwise-wasted resected liver lobe from patients with benign liver tumors is a new, albeit small, option to alleviate the allograft shortage. This review provides evidence that resected liver lobes may be used successfully in liver transplantation.

Lenvatinib is a multiple receptor tyrosine kinases inhibitor (TKI) authorized for first-line treatment of hepatocellular carcinoma (HCC). However, Lenvatinib resistance is common in HCC clinical treatment, highlighting the urgent need to understand mechanisms of resistance. Here, we identified Golgi membrane protein 1 (GOLM1), a type II transmembrane protein originally located in the Golgi apparatus, as a novel regulator of Lenvatinib resistance. We found GOLM1 was overexpressed in Lenvatinib resistant human HCC cell lines, blood and HCC samples. Additionally, GOLM1 overexpression contributes to Lenvatinib resistance and HCC progression in vitro and in vivo. Mechanistically, GOLM1 upregulates CSN5 expression through EGFR-STAT3 pathway. Reversely, CSN5 deubiquitinates and stabilizes GOLM1 protein by inhibiting ubiquitin-proteasome pathway of GOLM1. Furthermore, clinical specimens of HCC showed a positive correlation between the activation of the GOLM1-EGFR-STAT3-CSN5 axis. Finally, GOLM1 knockdown was found to act in synergy with Lenvatinib in subcutaneous and orthotopic mouse model. Overall, these findings identify a mechanism of resistance to Lenvatinib treatment for HCC, highlight an effective predictive biomarker of Lenvatinib response in HCC and show that targeting GOLM1 may improve the clinical benefit of Lenvatinib.

Background & Aims: The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in resistance to anti-PD-1 therapy in HCC. Methods: Gene expression in HCC specimens from 10 patients receiving anti-PD-1 therapy was identified by RNA-sequencing. A total of 404 HCC samples from tissue microarrays were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) Trp53 fl/fl ) received hydrodynamic tail vein injections of a CRKL-overexpressing vector. Mass cytometry by time of flight was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. Results: CRKL was identified as a candidate anti-PD-1-resistance gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 treatment efficacy by mobilizing tumor-associated neutrophils (TANs), which block the infiltration and function of CD8+ + T cells. PD-L1+ + TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits APC (adenomatous polyposis coli)-mediated proteasomal degradation of (3-catenin by competitively decreasing Axin1 binding, and thus promotes VEGFa and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/(3-catenin/VEGFa and CXCL1. Targeting CRKL using CRISPRCas9 gene editing ( CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid model. Conclusions: Activation of the CRKL/(3-catenin/VEGFa and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 could be useful for the treatment of HCC. (c) 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Functional roles of SIGLEC15 in hepatocellular carcinoma (HCC) were not clear, which was recently found to be an immune inhibitor with similar structure of inhibitory B7 family members. SIGLEC15 expression in HCC was explored in public databases and further examined by PCR analysis. SIGLEC15 and PD-L1 expression patterns were examined in HCC samples through immunohistochemistry. SIGLEC15 expression was knocked-down or over-expressed in HCC cell lines, and CCK8 tests were used to examine cell proliferative ability in vitro. Influences of SIGLEC15 expression on tumor growth were examined in immune deficient and immunocompetent mice respectively. Co-culture system of HCC cell lines and Jurkat cells, flow cytometry analysis of tumor infiltrated immune cells and further sequencing analyses were performed to investigate how SIGLEC15 could affect T cells in vitro and in vivo. We found SIGLEC15 was increased in HCC tumor tissues and was negatively correlated with PD-L1 in HCC samples. In vitro and in vivo models demonstrated inhibition of SIGLEC15 did not directly influence tumor proliferation. However, SIGLEC15 could promoted HCC immune evasion in immune competent mouse models. Knock-out of Siglec15 could inhibit tumor growth and reinvigorate CD8+ T cell cytotoxicity. Anti-SIGLEC15 treatment could effectively inhibit tumor growth in mouse models with or without mononuclear phagocyte deletion. Bulk and single-cell RNA sequencing data of treated mouse tumors demonstrated SIGLEC15 could interfere CD8+ T cell viability and induce cell apoptosis. In all, SIGLEC15 was negatively correlated with PD-L1 in HCC and mainly promote HCC immune evasion through inhibition of CD8+ T cell viability and cytotoxicity.

PDF file - 656K, CXCL16 expression in HCC and its relationship with clinical outcome

PDF file - 856K, Effects of CXCL16-knockdown on HCC cell invasion and cytokine secretion in vitro, and tumor growth in vivo

Background:Liver metastasis (LM) accounts for most colorectal cancer (CRC)-related deaths. However, how metastatic CRC cells gain the ability to survive and grow in liver remains largely unknown.Methods:First, we screened differentially expressed genes (DEGs) between LM and paired primary tumors (PTs) in Gene Expression Omnibus (GEO) database, and identified cytochrome P450 1B1 (CYP1B1) as the only common differential gene. Then, we verified messenger RNA (mRNA) and protein expression level in clinical specimens. After constructing stable up-regulated CYP1B1 versions of HCT116 and RKO CRC cells and stable down-regulated CYP1B1 versions of SW480 and HT29 CRC cells, cell proliferation assays, subcutaneous tumor formation, and mouse LM models were used to comprehend its function. Next, we used RNA-seq to uncover specific mechanisms of growth; cell cycle, polymerase chain reaction (PCR), western blot (WB) and GEO series (GSE) datasets were used to verify its mechanism. Last, gas chromatography tandem mass spectrometry (GC-MS/MS) was adopted to examine which fatty acids were changed.Results:A significantly higher level of CYP1B1 was found in LM than in PT in paired clinical CRC LM samples (P<0.05). After CYP1B1 overexpression in HCT116 and RKO cells, cell proliferation abilities in vitro and in vivo were enhanced; LM of NOD.Cg-PrkdcscidIl2rgem1Smoc (NSG) mice were enhanced. And knockdown of CYP1B1 in SW480 and HT29 cells, cell proliferation abilities in vitro and in vivo were reduced; LM of NSG mice were declined (P<0.05). RNA-seq showed 59 common genes from upregulated genes of RKO overexpression group and downregulated genes of SW480 knockdown group were enriched in cell cycle and DNA replication. Further investigation revealed CYP1B1 regulated alternation of MCM5, PCNA, and FEN1 genes, and G1/S transition in CRC cells. GC-MS/MS revealed long chain fatty acids (LCFAs) made a difference in SW480 knockdown group (P<0.05). Through adding LCFAs into SW480 and HT29 knockdown groups, cell proliferation abilities in vitro and in vivo were enhanced, and expressions of MCM5, PCNA, FEN1 were upregulated (P<0.05).Conclusions:CYP1B1 exerts a significant influence on LM of CRC by modulating tumor cell proliferation via "CYP1B1-LCFAs-G1/S transition". This finding suggests CYP1B1 could be a promising target for CRC LM.

PDF file - 1.6MB, The effects CXCR6-konckdown on expression of epithelial-to-mesenchymal transition markers and signaling molecules of hepatoma cells

Background: Primary liver cancer, of which around 75–85% is hepatocellular carcinoma in China, is the fourth most common malignancy and the second leading cause of tumor-related death, thereby posing a significant threat to the life and health of the Chinese people. Summary: Since the publication of Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China in June 2017, which were updated by the National Health Commission in December 2019, additional high-quality evidence has emerged from researchers worldwide regarding the diagnosis, staging, and treatment of liver cancer, that requires the guidelines to be updated again. The new edition (2022 Edition) was written by more than 100 experts in the field of liver cancer in China, which not only reflects the real-world situation in China but also may reshape the nationwide diagnosis and treatment of liver cancer. Key Messages: The new guideline aims to encourage the implementation of evidence-based practice and improve the national average 5-year survival rate for patients with liver cancer, as proposed in the “Health China 2030 Blueprint.”