Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma.

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+-CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Autophagy suppression facilitates macrophage M2 polarization via increased instability of NF-κB pathway in hepatocellular carcinoma.

The tumor microenvironment is a highly heterogeneous circumstance composed of multiple components, while tumor-associated macrophages (TAMs) are major innate immune cells with highly plastic and are always educated by tumor cells to structure an advantageous pro-tumor immune microenvironment. Despite emerging evidence focalizing the role of autophagy in other immune cells, the regulatory mechanism of autophagy in macrophage polarization remains poorly understood. Herein, we demonstrated that hepatocellular carcinoma (HCC) cells educated macrophages toward M2-like phenotype polarization under the condition of coculture. Moreover, we observed that inhibition of macrophage autophagy promoted M2-like macrophage polarization, while the tendency was impeded when autophagy was motivated. Mechanistically, macrophage autophagy inhibition inactivates the NF-κB pathway by increasing the instability of TAB3 via ubiquitination degradation, which leads to the M2-like phenotype polarization of macrophages. Both immunohistochemistry staining using human HCC tissues and experiment in vivo verified autophagy inhibition is correlated with M2 macrophage polarization. Altogether, we illustrated that macrophage autophagy was involved in the process of HCC cells domesticating M2 macrophage polarization via the NF-κB pathway. These results provide a new target to interfere with the polarization of macrophages to M2-like phenotype during HCC progression.

Synthetic miR-26a mimics delivered by tumor exosomes repress hepatocellular carcinoma through downregulating lymphoid enhancer factor 1.

BACKGROUND: The dysregulation of exosomal microRNAs plays an important role in the progression of hepatocarcinogenesis. In this study, we investigated the therapeutic potential of synthetic exosomal miR-26a against HCC cells and explored the feasibility of tumor-derived exosomes as drug delivery vehicles. METHODS: Proliferation and migration assays were performed to examine the effects of miR-26a on HCC in vitro. The direct target gene of miR-26a was identified through miRecords analysis and target validation. The transferring efficiency and anti-HCC effect of exosomes with different origin were studied and the optimal miR-26a delivery method was established and verified in vitro and in vivo. In addition, the relationships between prognosis of HCC patients and miR-26a expression in HCC serum and exosomes were retrospectively analyzed. RESULTS: Here, we found that tumor cell-derived exosomes were taken in preferentially by HCC cells and promoted HCC progression through Wnt pathway by low-density lipoprotein receptor-related protein 6 (LRP6). HCC cells with vacuolar protein sorting-associated protein 35 knocked down were adopted to generate engineered LRP6-exosomes. The engineered HCC-derived exosomes loading miR-26a inhibited HCC progression in vitro and in vivo effectively. Overexpression of miR-26a impaired the growth and migration of HCC by targeting lymphoid enhancer factor 1 (LEF1). Moreover, low expression of exosomal miR-26a was an independent prognostic factor for recurrence and survival in HCC patients. CONCLUSIONS: Our findings suggested the exosomal miR-26a could serve as a non-invasive prognostic marker for HCC patients. Genetically modified tumor-derived exosomes showed preferable transfection efficiency but reduced Wnt activity, which provides a novel therapeutic strategy for HCC.

Oncogenic miR-93-5p/Gal-9 axis drives CD8 (+) T-cell inactivation and is a therapeutic target for hepatocellular carcinoma immunotherapy.

Evading immune destruction is an emerging hallmark of cancer and a potential key step in tumorigenesis. Immune checkpoint blocker (ICB)-based combination therapies revolutionize the landscape of systemic therapy for HCC. However, the molecular underpinnings governing immune evasion and responses remain unclear. Our study aims to find new regulatory molecules that drive HCC immune escape and tumorigenesis and find new promising immunotherapeutic approaches for HCC. In our study, laser capture microdissection (LCM) and miRNA sequencing combined with in vitro and in vivo experiments identified miR-93-5p as a crucial initiating oncogene during liver progenitor cell (LPC) malignant transformation and immune escape. Mechanistically, miR-93-5p could directly target canonical tumour suppressors such as APC to promote LPC malignant transformation and hepatocarcinogenesis. More importantly, miR-93-5p could induce deviant GAL-9 augmentation to inactivate infiltrated CD8(+) T cells and induce immune evasion by targeting several epigenetic regulators, such as AEBP2, and then regulating H3K4me3/H3K27me3 bivalency. Experiments in C57BL/6 mice demonstrated that blockade of Gal-9 abrogated miR-93-5p-induced HCC progression and improved their prognosis. Clinically, we identified a unique subtype of HCC closely associated with high GAL-9 expression and anti-PD1 treatment resistance. Our study highlights the pivotal role of the miR-93-5p/Gal-9 axis in driving HCC immune escape and tumorigenesis. Blocking GAL-9 is an effective and promising immunotherapeutic approach for HCC.

Toripalimab combined with lenvatinib and GEMOX is a promising regimen as first-line treatment for advanced intrahepatic cholangiocarcinoma: a single-center, single-arm, phase 2 study.

Advanced intrahepatic cholangiocarcinoma (ICC) has a dismal prognosis. Here, we report the efficacy and safety of combining toripalimab, lenvatinib, and gemcitabine plus oxaliplatin (GEMOX) as first-line therapy for advanced ICC. Thirty patients with pathologically confirmed advanced ICC received intravenous gemcitabine (1 g/m2) on Days 1 and 8 and oxaliplatin (85 mg/m2) Q3W for six cycles along with intravenous toripalimab (240 mg) Q3W and oral lenvatinib (8 mg) once daily for one year. The expression of programmed death-ligand 1 (PD-L1) and genetic status was investigated in paraffin-embedded tissues using immunohistochemistry and whole-exome sequencing (WES) analysis. The primary endpoint was the objective response rate (ORR). Secondary outcomes included safety, overall survival (OS), progression-free survival (PFS), disease control rate (DCR) and duration of response (DoR). As of July 1, 2022, the median follow-up time was 23.5 months, and the ORR was 80%. Twenty-three patients achieved partial response, and one achieved complete response. Patients (21/30) with DNA damage response (DDR)-related gene mutations showed a higher ORR, while patients (14/30) with tumor area positivity ≥1 (PD-L1 staining) showed a trend of high ORR, but without significant difference. The median OS, PFS, and DoR were 22.5, 10.2, and 11.0 months, respectively. The DCR was 93.3%. Further, 56.7% of patients experienced manageable grade ≥3 adverse events (AEs), commonly neutropenia (40.0%) and leukocytopenia (23.3%). In conclusion, toripalimab plus lenvatinib and GEMOX are promising first-line regimens for the treatment of advanced ICC. A phase-III, multicenter, double-blinded, randomized study to validate our findings was approved by the National Medical Products Administration (NMPA, No. 2021LP01825).Trial registration Clinical trials: NCT03951597.

Inhibition of autophagy in macrophage promotes IL-1ß-mediated hepatocellular carcinoma progression via inflammasome accumulation and self-recruitment.

Hepatocellular carcinoma (HCC) has a complex and changeable tumor microenvironment. Despite emerging evidence focusing on autophagy process within immune cells, the function and regulatory mechanism of macrophage autophagy in tumor progression remains unclear. Our results of multiplex-immunohistochemistry and RNA-sequencing identified the reduced levels of autophagy in tumor macrophages in the HCC microenvironment, associated with a poor prognosis and increased microvascular metastasis in HCC patients. Specifically, HCC suppressed the macrophage autophagy initiation through the up-regulation of mTOR and ULK1 phosphorylation at Ser757. Knockdown of autophagy-related proteins to further inhibit autophagy significantly boosted the metastatic potential of HCC. Mechanistically, the accumulation of NLRP3 inflammasome mediated by autophagy inhibition promoted the cleavage, maturation, and release of IL-1ß, which facilitated the HCC progression, eventually accelerating HCC metastasis via the epithelial-mesenchymal transition. Autophagy inhibition provoked macrophage self-recruitment through the CCL20-CCR6 signaling was also a crucial account of HCC progression. Recruited macrophages mediated the cascade amplification of IL-1ß and CCL20 to form a novel pro-metastatic positive feedback loop through promoting HCC metastasis and increased macrophage recruitment, respectively. Notably, targeting IL-1ß/IL-1 receptor signaling impaired lung metastasis induced by macrophage autophagy inhibition in a mice HCC lung metastasis model. In summary, this study highlighted that inhibition of tumor macrophage autophagy facilitated HCC progression by increasing IL-1ß secretion via NLRP3 inflammasome accumulation and by macrophage self-recruitment through the CCL20 signaling pathway. Interruption of this metastasis-promoting loop by IL-1ß blockade may provide a promising therapeutic strategy for HCC patients.

Loss of 5-hydroxymethylcytosine induces chemotherapy resistance in hepatocellular carcinoma via the 5-hmC/PCAF/AKT axis.

Multidrug resistance is a major challenge in treating advanced hepatocellular carcinoma (HCC). Although recent studies have reported that the multidrug resistance phenotype is associated with abnormal DNA methylation in cancer cells, the epigenetic mechanism underlying multidrug resistance remains unknown. Here, we reported that the level of 5-hydroxymethylcytosine (5-hmC) in human HCC tissues was significantly lower than that in adjacent liver tissues, and reduced 5-hmC significantly correlated with malignant phenotypes, including poor differentiation and microvascular invasion; additionally, loss of 5-hmC was related to chemotherapy resistance in post-transplantation HCC patients. Further, the 5-hmC level was regulated by ten-eleven translocation 2 (TET2), and the reduction of TET2 in HCC contributes to chemotherapy resistance through histone acetyltransferase P300/CBP-associated factor (PCAF) inhibition and AKT signaling hyperactivation. In conclusion, loss of 5-hmC induces chemotherapy resistance through PCAF/AKT axis and is a promising chemosensitivity prediction biomarker and therapeutic target for HCC patients.

Pemigatinib in previously treated Chinese patients with locally advanced or metastatic cholangiocarcinoma carrying FGFR2 fusions or rearrangements: A phase II study.

OBJECTIVE: This study evaluated the antitumor activity and safety of pemigatinib in previously treated Chinese patients with advanced cholangiocarcinoma and fibroblast growth factor receptor 2 (FGFR2) fusions or rearrangements. BACKGROUND: Pemigatinib provided clinical benefits for previously treated patients with cholangiocarcinoma carrying FGFR2 fusions or rearrangements and was approved for this indication in multiple countries. METHODS: In this ongoing, multicenter, single-arm, phase II study, adult patients with locally advanced or metastatic cholangiocarcinoma carrying centrally confirmed FGFR2 fusions or rearrangements who had progressed on ≥1 systemic therapy received 13.5 mg oral pemigatinib once daily (3-week cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, or consent withdrawal. The primary endpoint was objective response rate (ORR) assessed by an independent radiology review committee. RESULTS: As of January 29, 2021, 31 patients were enrolled. The median follow-up was 5.1 months (range, 1.5-9.3). Among 30 patients with FGFR2 fusions or rearrangements evaluated for efficacy, 15 patients achieved partial response (ORR, 50.0%; 95% confidence interval [CI], 31.3-68.7); 15 achieved stable disease, contributing to a disease control rate of 100% (95% CI, 88.4-100). The median time to response was 1.4 months (95% CI, 1.3-1.4), the median duration of response was not reached, and the median progression-free survival was 6.3 months (95% CI, 4.9-not estimable [NE]). Eight (25.8%) of 31 patients had ≥grade 3 treatment-emergent adverse events. Hyperphosphatemia, hypophosphatasemia, nail toxicities, and ocular disorders were mostly

The clinical implications and molecular features of intrahepatic cholangiocarcinoma with perineural invasion.

BACKGROUND: Perineural invasion (PNI) is associated with metastasis in malignancies, including intrahepatic cholangiocarcinoma (ICC), and is correlated with poor prognosis. METHODS: The study included three large cohorts: ZS-ICC and TMA cohorts from our team, MSK cohort from a public database, and a small cohort named cohort 4. Prognostic implications of PNI were investigated in MSK cohort and TMA cohort. PNI-related genomic and transcriptomic profiles were analyzed in MSK and ZS-ICC cohorts. GO, KEGG, and ssGSEA analyses were performed. Immunohistochemistry was used to investigate the relationship between PNI and markers of neurons, hydrolases, and immune cells. The efficacy of adjuvant therapy in ICC patients with PNI was also assessed. RESULTS: A total of 30.6% and 20.7% ICC patients had PNI in MSK and TMA cohorts respectively. Patients with PNI presented with malignant phenotypes such as high CA19-9, the large bile duct type, lymph node invasion, and shortened overall survival (OS) and relapse-free survival (RFS). Nerves involved in PNI positively express tyrosine hydroxylase (TH), a marker of sympathetic nerves. Patients with PNI showed high mutation frequency of KRAS and an immune suppressive metastasis prone niche of decreased NK cell, increased neutrophil, and elevated PD-L1, CD80, and CD86 expression. Patients with PNI had an extended OS after adjuvant therapy with TEGIO, GEMOX, or capecitabine. CONCLUSION: Our study deciphered the genomic features and the immune suppressive metastasis-prone niche in ICC with PNI. Patients with PNI showed a poor prognosis after surgery but a good response to adjuvant chemotherapy.

Perioperative and oncologic outcomes of laparoscopic versus open liver resection for combined hepatocellular-cholangiocarcinoma: a propensity score matching analysis.

BACKGROUND: Laparoscopic liver resection (LLR) has now been established as a safe and minimally invasive technique that is deemed feasible for treating hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). However, the role of LLR in treating combined hepatocellular-cholangiocarcinoma (cHCC-CC) patients has been rarely reported. This study aimed to assess the efficacy of LLR when compared with open liver resection (OLR) procedure for patients with cHCC-CC. METHODS: A total of 229 cHCC-CC patients who underwent hepatic resection (34 LLR and 195 OLR patients) from January 2014 to December 2018 in Zhongshan Hospital, Fudan University were enrolled and underwent a 1:2 propensity score matching (PSM) analysis between the LLR and OLR groups to compare perioperative and oncologic outcomes. Overall survival (OS) and recurrence-free survival (RFS) parameters were assessed by the log-rank test and the sensitivity analysis. RESULTS: A total of 34 LLR and 68 OLR patients were included after PSM analysis. The LLR group displayed a shorter postoperative hospital stay (6.61 vs. 8.26 days; p value < 0.001) when compared with the OLR group. No significant differences were observed in the postoperative complications' incidence or a negative surgical margin rate between the two groups (p value = 0.409 and p value = 1.000, respectively). The aspartate aminotransferase (AST), alanine aminotransferase (ALT), and inflammatory indicators in the LLR group were significantly lower than those in the OLR group on the first and third postoperative days. Additionally, OS and RFS were comparable in both the LLR and OLR groups (p value = 0.700 and p value = 0.780, respectively), and similar results were obtained by conducting a sensitivity analysis. CONCLUSION: LLR can impart less liver function damage, better inflammatory response attenuation contributing to a faster recovery, and parallel oncologic outcomes when compared with OLR. Therefore, LLR can be recommended as a safe and effective therapeutic modality for treating selected cHCC-CC patients, especially for those with small tumors in favorable location.

Expert Consensus on Pathological Diagnosis of Intrahepatic Cholangiocarcinoma (2022 version).

Intrahepatic cholangiocarcinoma (iCCA) can originate from the large bile duct group (segment bile ducts and area bile ducts), small bile duct group (septal bile ducts and interlobular bile ducts), and terminal bile duct group (bile ductules and canals of Hering) of the intrahepatic biliary tree, which can be histopathological corresponding to large duct type iCCA, small duct type iCCA and iCCA with ductal plate malformation pattern, and cholangiolocarcinoma, respectively. The challenge in pathological diagnosis of above subtypes of iCCA falls in the distinction of cellular morphologies, tissue structures, growth patterns, invasive behaviors, immunophenotypes, molecular mutations, and surgical prognoses. For these reasons, this expert consensus provides nine recommendations as a reference for standardizing and refining the diagnosis of pathological subtypes of iCCA, mainly based on the 5th edition of the World Health Organization Classification of Tumours of the Digestive System.

Effectiveness and Safety of Avatrombopag in Liver Cancer Patients with Severe Thrombocytopenia: Real-World Data and Challenges.

Background: Avatrombopag has been approved in patients who have severe thrombocytopenia (<50 × 109/L) and chronic liver disease (CLD) while receiving invasive procedures. The real-world application and effectiveness of avatrombopag in the subgroup patients with liver cancer remain unknown. Methods: Liver cancer patients (including primary liver cancer and colorectal cancer liver metastasis) who had severe thrombocytopenia and received avatrombopag were retrospectively enrolled. Avatrombopag dose, peak and absolute platelet count increase, combination treatment with other thrombopoietic agents, responder (peak count ≥50 × 109/L with absolute increase ≥20 × 109/L) rate, and anticancer treatment effect were analyzed. Thrombosis and bleeding events were assessed. Results: In total, 93 patients were enrolled, with 72 and 21 in the CLD and non-CLD groups, respectively. Patients with CLD had hepatitis B or C, larger spleen volume, and a higher cirrhosis degree. Baseline platelet counts were similar between two groups (median, 37.0 × 109/L vs. 39.0 × 109/L; P=0.594), while patients without CLD had higher peak platelet (median, 134.0 × 109/L vs. 74.0 × 109/L; P=0.015) and absolute increase (median, 101.0 × 109/L vs. 41.0 × 109/L; P=0.020) after avatrombopag treatment. The responder rate was higher in patients without CLD (100% vs. 76.4%; P=0.010). Combined avatrombopag with other thrombopoietic agents significantly increased platelet count; repeated use of avatrombopag produced similar effects with that of initial treatment. Concerning anticancer treatment effect, patients who responded to avatrombopag had a higher disease control rate. No thrombosis or hemorrhagic events were observed, even in patients with portal vein tumor thrombosis. Conclusion: Avatrombopag was safe and effective and ensured successful implementation of anticancer treatment in liver cancer patients with severe thrombocytopenia, accompanied with or without CLD.

KSR2-14-3-3ζ complex serves as a biomarker and potential therapeutic target in sorafenib-resistant hepatocellular carcinoma.

BACKGROUND: Kinase suppressor of Ras 2 (KSR2) is a regulator of MAPK signaling that is overactivated in most hepatocellular carcinoma (HCC). We sought to determine the role of KSR2 in HCC pathogenesis. METHODS: We tested the level of KSR2 in HCC tissues and cell lines by tissue microarray, qPCR, and western blotting. Functionally, we determined the effects of KSR2 on the proliferation, migration, and invasion of HCC cells through colony formation assays, scratch assays, transwell migration assays, and xenograft tumor models. Co-immunoprecipitation (co-IP) experiments were used to assess the interaction of phospho-serine binding protein 14-3-3ζ and KSR2, and the effects of this interaction on growth and proliferation of human HCC cells were tested by co-overexpression and knockdown experiments. Additionally, we used flow cytometry to examine whether the KSR2 and 14-3-3ζ interaction conveys HCC resistance to sorafenib. RESULTS: KSR2 was significantly upregulated in HCC tissues and cell lines, and high KSR2 expression associated with poor prognosis in HCC patients. KSR2 knockdown significantly suppressed HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, co-IP experiments identified that 14-3-3ζ complexed with KSR2, and elevated 14-3-3ζ increased KSR2 protein levels in HCC cells. Importantly, Kaplan-Meier survival analysis showed that patients with both high KSR2 and high 14-3-3ζ expression levels had the shortest survival times and poorest prognoses. Interestingly, HCC cells overexpressing both KSR2 and 14-3-3ζ, rather than either protein alone, showed hyperactivated MAPK signaling and resistance to sorafenib. CONCLUSIONS: Our results provide new insights into the pro-tumorigenic role of KSR2 and its regulation of the MAPK pathway in HCC. The KSR2-14-3-3ζ interaction may be a therapeutic target to enhance the sorafenib sensitivity of HCC.

KRAS acting through ERK signaling stabilizes PD-L1 via inhibiting autophagy pathway in intrahepatic cholangiocarcinoma.

BACKGROUND: While the correlation between PD-L1 expression and KRAS mutation has been previously reported in other solid tumors such as non-small cell lung cancer (NSCLC), whether PD-L1 can be modulated by ERK signaling downstream of KRAS in intrahepatic cholangiocarcinoma (iCCA) and the underlying molecular regulatory mechanism remain unclear. METHODS: The expression of ERK, p-ERK, PD-L1 and autophagy markers following KRAS knockdown or Ras/Raf/MEK/ERK signaling inhibitors treatment was examined in two human iCCA cell lines (HuCCT1 and RBE) using western blotting and immunofluorescence. Both pharmacological autophagy inhibitors and short-interfering RNA against ATG7 were applied to inhibit autophagy. The apoptosis rates of iCCA cell lines were detected by flow cytometry and CCK-8 after co-culturing with CD3/CD28-activated human CD8+ T lymphocytes. Immunohistochemistry was applied to detect the correlation of ERK, p-ERK and PD-L1 in 92 iCCA tissues. RESULTS: The present study demonstrated that the PD-L1 expression level was distinctly reduced in KRAS-mutated iCCA cell lines when ERK signaling was inhibited and ERK phosphorylation levels were lowered. The positive association between p-ERK and PD-L1 was also verified in 92 iCCA tissue samples. Moreover, ERK inhibition induced autophagy activation. Both inhibiting autophagy via autophagy inhibitors and genetically silencing the ATG7 expression partially reversed the reduced PD-L1 expression caused by ERK inhibition. In addition, ERK-mediated down-regulation of PD-L1 via autophagy pathways induced the apoptosis of iCCA cells when co-cultured with CD3/CD28-activated human CD8+ T lymphocytes in vitro. CONCLUSIONS: Our results suggest that ERK signaling inhibition contributes to the reduction of PD-L1 expression through the autophagy pathway in iCCA. As a supplement to anti-PD-1/PD-L1 immunotherapy, ERK-targeted therapy may serve as a potentially novel treatment strategy for human KRAS-mutated iCCA.

Future liver volume combined with platelet count predicts liver failure after major hepatectomy.

BACKGROUND: Major hepatectomy is associated with high incidence of post-hepatectomy liver failure (PHLF). This study aimed to evaluate the effect of future remnant liver volume combined with liver function tests on predicting PHLF. METHODS: Patients who underwent major hepatectomy from April 2009 to May 2017 were enrolled in the training cohort. Univariate and multivariate logistic regression analyses were performed to identify independent risk factors of PHLF and generate a logistic regression model for the prediction of PHLF. A conditional inference tree was generated based on the optimal cutoff value of independent predictive factors of PHLF. The precedent results were validated in an independent cohort from June 2017 to March 2018. RESULTS: One hundred and eighteen patients were included in the training cohort, while another 34 in the validation cohort. Future remnant liver volume/estimated standard total liver volume (FLV/eTV) and preoperative platelet count were independent predictive factors of PHLF (P = 0.0021 and P = 0.012, respectively). The conditional inference tree showed that patients with FLV/eTV ≤0.56 and PLT count ≤145 × 109/L were at high risk of developing PHLF. CONCLUSION: FLV/eTV combined with preoperative PLT count is effective in predicting PHLF after major hepatectomy.

Amplification of spatially isolated adenosine pathway by tumor-macrophage interaction induces anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND: Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. METHOD: The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP-ADO pathway amplified by HCC-macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. RESULTS: Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP-adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. CONCLUSION: In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP-adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

Simulation of portal/hepatic vein associated remnant liver ischemia/congestion by three-dimensional visualization technology based on preoperative CT scan.

BACKGROUND: Remnant liver hypoperfusion is frequently observed after hepatectomy, and associated with a higher risk of postoperative complications and poorer survival. However, the development of remnant liver hypoperfusion was not fully understood. METHODS: We retrospectively analyzed patients who received hepatectomy and took contrast-enhanced computed tomography (CT) scans before, 1-week (POW1) and 4-week (POW4) after resection in our department from June 2017 to July 2019. We simulated and estimated the occurrence of portal-vein-related remnant liver ischemia (RLI) and hepatic-vein-related remnant liver congestion (RLC) after hepatectomy via three-dimensional visualization technology (3DVT) according to blood vessels ligated in the resection; then we analyzed association between the estimated RLI, RLC, and postoperative clinical outcomes. RESULTS: A total of 102 eligible patients were analyzed. Remnant liver hypoperfusion was observed in 47 (46%) patients in the POW1 CT scans and shrunk in the POW4 CT scans. RLC had better diagnostic significance than RLI in predicting remnant liver hypoperfusion [area under receiver operating characteristic (ROC) curve: 0.745 vs. 0.569, P=0.026]. Multivariate analysis showed that larger RLI [odds ratio (OR), 1.154; 95% confidence interval (CI), 1.075-1.240; P<0.001] was independent risk factor for post-hepatectomy liver failure (PHLF). Besides, larger RLC (OR, 1.114; 95% CI, 1.032-1.204; P=0.006) was independent risk factor for major postoperative complications. CONCLUSIONS: Remnant liver hypoperfusion can be predicted during the preoperative surgical plan by 3DVT. Portal vein related RLI was associated with PHLF, and hepatic vein related RLC was associated with major postoperative complications. Preservation of the hepatic vein and complete removal of the perfusion territory of ligated vessels are essential procedures to reduce RLI/RLC and the risk of PHLF or other surgical complications.

CircMEMO1 modulates the promoter methylation and expression of TCF21 to regulate hepatocellular carcinoma progression and sorafenib treatment sensitivity.

BACKGROUND: Cirrhosis is a recognized risk factor for developing hepatocellular carcinoma (HCC). Few studies have reported the expression profile of circRNAs in HCC samples compared to paratumour dysplastic nodule (DN) samples. METHODS: The Arraystar Human circRNA Array combined with laser capture microdissection (LCM) was used to analyse the expression profile of circRNAs in HCC samples compared to paratumour DN samples. Then, both in vitro and in vivo HCC models were used to determine the role and mechanism of key circRNA in HCC progression and treatment sensitivity. RESULTS: We found that circMEMO1 was significantly downregulated in HCC samples and that the level of circMEMO1 was closely related to the OS and disease-free survival (DFS) of HCC patients. Mechanistic analysis revealed that circMEMO1 can modulate the promoter methylation and gene expression of TCF21 to regulate HCC progression by acting as a sponge for miR-106b-5p, which targets the TET family of genes and increases the 5hmC level. More importantly, circMEMO1 can increase the sensitivity of HCC cells to sorafenib treatment. CONCLUSION: Our study determined that circMEMO1 can promote the demethylation and expression of TCF21 and can be considered a crucial epigenetic modifier in HCC progression.