Cancer-associated fibroblasts contributed to hepatocellular carcinoma recurrence and metastasis via CD36-mediated fatty-acid metabolic reprogramming.

Cancer-associated fibroblasts (CAFs) are the main components in the tumor microenvironment. Tumors activate fibroblasts from quiescent state into activated state by secreting cytokines, and activated CAFs may in turn promote tumor progression and metastasis. Therefore, studies targeting CAFs could enrich the therapeutic options for tumor treatment. In this study, we demonstrate that the content of lipid droplets and the expression of autophagosomes were higher in CAFs than in peri-tumor fibroblasts (PTFs), which was inhibited by 5-(tetradecyloxy)-2-furoic acid(TOFA). The expression of CD36 in CAFs was higher than that in PTFs at both mRNA and protein levels. Inhibition of CD36 activity using either the CD36 inhibitor SSO or siRNA had a significant negative impact on the proliferation and migration abilities of CAFs, which was associated with reduced levels of relevant activated genes (α-SMA, FAP, Vimentin) and cytokines (IL-6, TGF-ß and VEGF-α). SSO also inhibited HCC growth and tumorigenesis in nude mice orthotopically implanted with CAFs and HCC cells. Our data further show that CD36+CAFs affected the expression of PD-1 in CTLs leading to CTL exhaustion, and that patients with high CD36 expression in CAFs were correlated with shorter overall survival (OS). Together, our data demonstrate that CAFs were active in lipid metabolism with increased lipid content and lipophagy activity. CD36 may play a key role in the regulation of the biological behaviors of CAFs, which may influence the proliferation and migration of tumor cells by reprograming the lipid metabolism in tumor cells. Thus, CD36 could be an effective therapeutic target for the treatment of HCC.

Monocarboxylate transporter 4 inhibition potentiates hepatocellular carcinoma immunotherapy through enhancing T cell infiltration and immune attack.

BACKGROUND AND AIMS: Monocarboxylate transporter (MCT) 4 is a high-affinity lactate transporter that is primarily involved in the maintenance of intracellular pH homeostasis and highly expressed in different tumors. However, the role of MCT4 in modulating immune responses against HCC remains unknown. APPROACH AND RESULTS: In this study, we demonstrated that MCT4 was overexpressed in HCC, which was associated with poor prognosis in patients. Genetic or pharmacological inhibition of MCT4 using VB124 (a highly potent MCT4 inhibitor) suppressed HCC tumor growth in immunocompetent mice model by enhancing CD8 + T cell infiltration and cytotoxicity. Such improved immunotherapy response by MCT4 targeting was due to combined consequences characterized by the alleviated acidification of tumor microenvironment and elevated the chemokine (C-X-C motif) ligand (CXCL) 9/CXCL10 secretion induced by reactive oxygen species/NF-κB signaling pathway. Combining MCT4 inhibition improved the therapeutic benefit of anti-programmed cell death 1 immunotherapy in HCC and prolonged mice survival. Moreover, higher MCT4 expression was observed in tumor tissues from nonresponder patients with HCC receiving neoadjuvant therapy with toripalimab. CONCLUSIONS: Our results revealed that lactate exportation by MCT4 has a tumor-intrinsic function in generating an immunosuppressive HCC environment and demonstrated the proof of the concept of targeting MCT4 in tailoring HCC immunotherapeutic approaches.

Comprehensive analysis of complement-associated molecular features in hepatocellular carcinoma.

The complement cascade plays a "complementing" role in human immunity. However, the potential roles of complement system in impacting molecular and clinical features of hepatocellular carcinoma (HCC) remain unclear. In this study, eleven public datasets are analyzed to compare the complement status between normal and cancerous samples based on 18 classical complement-associated genes. The complement scores are constructed to quantify complement signatures of individual tumors. HCC patients in the The Cancer Genome Atlas (TCGA) cohort are focused to perform systematical analyses between complement status and immune infiltration, miRNA expression, DNA methylation, clinicopathological features, and drug response. The results show that the complement scores in normal tissues are dramatically higher than those of tumor tissues. Tumor samples in the TCGA cohort are classified into complement score-low and score-high groups. Pathway analysis reveals that tumor-promoting pathways are typically inhibited in complement score-high group. This study also shows that tumor-killing immune cells, such as CD8 + T cells and natural killer cells are abundant and tumor-suppressing miRNAs are upregulated in complement score-high samples. In addition, we identify that complement scores are negatively correlated with certain clinical features, including pathological grade, clinical-stage, and portal vein invasion. Moreover, various molecular features together with complement scores are found to be correlated with response to anti-cancer drugs. This study provides a comprehensive and multidimensional analysis conducive to understanding the role of complement in cancer.

Prediction of overall survival in resectable intrahepatic cholangiocarcinoma: ISICC -applied prediction model.

Intrahepatic cholangiocarcinoma (ICC) remains a highly heterogeneous disease with poor prognosis. Tumor-infiltrating lymphocytes were predictive in various cancers, but their prognostic value in ICC is less clear. A total of 168 ICC patients who had received liver resection were enrolled and assigned to the derivation cohort. Sixteen immune markers in tumor and peritumor regions were examined by immunohistochemistry. A least absolute shrinkage and selection operator model was used to identify prognostic markers and to establish an immune signature for ICC (ISICC ). An ISICC -applied prediction model was built and validated in another independent dataset. Five immune features, including CD3peritumor (P) , CD57P , CD45RAP , CD66bintratumoral (T) and PD-L1P , were identified and integrated into an individualized ISICC for each patient. Seven prognostic predictors, including total bilirubin, tumor numbers, CEA, CA19-9, GGT, HBsAg and ISICC , were integrated into the final model. The C-index of the ISICC -applied prediction model was 0.719 (95% CI, 0.660-0.777) in the derivation cohort and 0.667 (95% CI, 0.581-0.732) in the validation cohort. Compared with the conventional staging systems, the new model presented better homogeneity and a lower Akaike information criteria value in ICC. The ISICC -applied prediction model may provide a better prediction performance for the overall survival of patients with resectable ICC in clinical practice.

Nine-factor-based immunohistochemistry classifier predicts recurrence for early-stage hepatocellular carcinoma after curative resection.

BACKGROUND: Immunoscore have shown a promising prognostic value in many cancers. We aimed to establish and validate an immune classifier to predict survival after curative resection of hepatocellular carcinoma (HCC) patients who have undergone curative resection. METHODS: The immunohistochemistry (IHC) classifier assay was performed on 664 patients with Barcelona Clinic Liver Cancer (BCLC) stage 0 or A HCC. A nine-feature-based HCC-IHC classifier was then constructed by the least absolute shrinkage and selection operator method. The associations between the HCC-IHC classifier and patient outcomes were assessed. Herein, a nomogram was generated from the Cox regression coefficients and evaluated by decision curve analysis. RESULTS: We constructed an HCC-IHC classifier based on nine features; significant differences were found between the low-HCC-IHC classifier patients and high-HCC-IHC classifier patients in the training cohort in the 5-year relapse-free survival rates (46.7% vs. 26.7%, respectively; P < 0.001). The HCC-IHC classifier-based nomogram presented better accuracy than traditional staging systems. CONCLUSIONS: In conclusion, the HCC-IHC classifier could effectively predict recurrence in early-stage HCC patients and supplemented the prognostic value of the BCLC staging system. The HCC-IHC classifier may facilitate patient decision-making and individualise the management of postoperative patients with early-stage HCC.

Adjuvant Transarterial chemoembolization does not influence recurrence-free or overall survival in patients with combined hepatocellular carcinoma and Cholangiocarcinoma after curative resection: a propensity score matching analysis.

BACKGROUND: The prognosis of patients with combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma (CHC) is usually poor, and effective adjuvant therapy is missing making it important to investigate whether these patients may benefit from adjuvant transarterial chemoembolization (TACE). We aimed to evaluate the efficiency of adjuvant TACE for long-term recurrence and survival after curative resection before and after propensity score matching (PSM) analysis. METHODS: In this retrospective study, of 230 patients who underwent resection for CHC between January 1994 and December 2014, 46 (18.0%) patients received adjuvant TACE. Univariate and multivariate regression analyses were used to identify the independent predictive factors of survival. Cox regression analyses and log-rank tests were used to compare overall survival (OS) and disease-free survival (DFS) between patients who did or did not receive adjuvant TACE. RESULTS: A total of 230 patients (mean age 52.2 ± 11.9 years; 172 men) were enrolled, and 46 (mean age 52.7 ± 11.1 years; 38 men) patients received TACE. Before PSM, in multivariate regression analysis, γ-glutamyl transpeptidase (γ-GT), tumour nodularity, macrovascular invasion (MVI), lymphoid metastasis, and extrahepatic metastasis were associated with OS. Alanine aminotransferase (ALT), MVI, lymphoid metastasis, and preventive TACE (HR: 2.763, 95% CI: 1.769-4.314, p < 0.001) were independent prognostic factors for DFS. PSM created 46 pairs of patients. Before PSM, adjuvant preventive TACE was not associated with an increased risk of OS (HR: 0.911, 95% CI: 0.545-1.520, p = 0.720) or DFS (HR: 3.345, 95% CI: 1.686-6.638, p = 0.001). After PSM, the 5-year OS and DFS rates were comparable in the TACE group and the non-TACE group (OS: 22.7% vs 14.9%, respectively, p = 0.75; DFS: 11.2% vs 14.4%, respectively, p = 0.06). CONCLUSIONS: The present study identified that adjuvant preventive TACE did not influence DFS or OS after curative resection of CHC.

Histopathology-based immunoscore predicts recurrence for intrahepatic cholangiocarcinoma after hepatectomy.

Intrahepatic cholangiocarcinoma (ICC) is a rare malignancy with poor prognosis. The evaluation of recurrence risk after liver resection is of great importance for ICCs. We aimed to assess the prognostic value of intra- and peritumoral immune infiltrations and to establish a novel histopathology-related immunoscore (HRI) associated with ICC recurrence. A total of 280 ICC patients who received curative resection between February 2005 and July 2011 were enrolled in our study. Patients were randomly assigned to the derivation cohort (n = 176) or the validation cohort (n = 104). Sixteen immune biomarkers in both intra- and peritumoral tissues were examined by immunohistochemistry. The least absolute shrinkage and selection operator (LASSO) Cox model was used to establish the HRI score. Cox regression analysis was used for multivariate analysis. Nine recurrence-related immune features were identified and integrated into the HRI score. The HRI score was used to categorize patients into low-risk and high-risk groups using the X-tile software. Kaplan-Meier analysis presented that the HRI score showed good stratification between low-risk and high-risk groups in both the derivation cohort (P < 0.001) and the validation cohort (P = 0.014), respectively. Multivariate analysis demonstrated that serum γ-glutamyl transpeptidase, carbohydrate antigen 19-9, lymphoid metastasis, tumor numbers, and the HRI score were independent risk factors associated with recurrence-free survival (RFS). The combination of Shen's model and HRI score provided better performance in recurrence prediction compared with traditional staging systems. The HRI score might serve as a promising RFS predictor for ICC with prognostic values.

Tissue-infiltrating lymphocytes signature predicts survival in patients with early/intermediate stage hepatocellular carcinoma.

BACKGROUND: Intratumoral immune infiltrates have manifested a robust prognostic signature in patients with hepatocellular carcinoma (HCC). We hypothesized that a novel tissue-related immune signature (TRIS) could improve the prediction of postoperative survival for patients diagnosed with early/intermediate HCC. METHODS: Twenty-eight immune features were immunohistochemically examined on 352 HCC specimens. The LASSO Cox regression model was used to construct a five-feature-based TRIS. The univariate and multivariate Cox analyses were performed. Based on independent predictors, the immune-clinical prognostic index (ICPI) was established. Performance assessment was measured with C-index and compared with seven traditional staging systems. The independent validation cohort (n = 393) was included to validate the model. RESULTS: By using the LASSO method, the TRIS were constructed on the basis of five immune features, CD3intratumoral (T), CD27T, CD68peritumoral (P), CD103T, and PD1T. Multivariate Cox analysis showed that the TRIS was an independent prognostic predictor. In the training cohort, γ-glutamyl transferase, tumor diameter, tumor differentiation, and TRIS were incorporated into the ICPI. The ICPI presented satisfactory discrimination ability, with C-index values of 0.691 and 0.686 in the training and validation cohorts, respectively. Compared with seven conventional staging systems (C-index, training cohort, 0.548-0.597; validation cohort, 0.519-0.610), the ICPI exhibited better performance for early/intermediate-stage HCCs. Further, the patients were categorized into three subgroups with X-tile software, and the stratified ICPI presented a superior corrected Akaike information criterion and homogeneity in both cohorts. CONCLUSIONS: Our ICPI was a useful and reliable prognostic tool which may offer good individualized prediction capability for HCC patients with early/intermediate stage.

SOMCL-085, a novel multi-targeted FGFR inhibitor, displays potent anticancer activity in FGFR-addicted human cancer models.

Aberrant fibroblast growth factor receptor (FGFR) activation is found across a diverse spectrum of malignancies, especially those lacking effective treatments. SOMCL-085 is a novel FGFR-dominant multi-target kinase inhibitor. Here, we explored the FGFR-targeting anticancer activity of SOMCL-085 both in vitro and in vivo. Among a panel of 20 tyrosine kinases screened, SOMCL-085 potently inhibited FGFR1, FGFR2 and FGFR3 kinase activity, with IC50 values of 1.8, 1.9 and 6.9 nmol/L, respectively. This compound simultaneously inhibited the angiogenesis kinases VEGFR and PDGFR, but without obvious inhibitory effect on other 12 tyrosine kinases. In 3 representative human cancer cell lines with different mechanisms of FGFR activation tested, SOMCL-085 (20-500 nmol/L) dose-dependently inhibited FGFR1-3 phosphorylation and the phosphorylation of their key downstream effectors PLCγ and Erk. In 7 FGFR aberrant human cancer cell lines, regardless of the mechanistic complexity of FGFR over-activation, SOMCL-085 potently inhibited FGFR-driven cell proliferation by arresting cells at the G1/S phase. In the FGFR1-amplified lung cancer cell line H1581 xenograft mice and FGFR2-amplified gastric cancer cell line SNU16 xenograft mice, oral administration of SOMCL-085 (25, 50 mg·kg-1·d-1) for 21 days substantially suppressed tumor growth without affecting their body-weight. These results suggest that SOMCL-085 is a potent multi-target FGFR inhibitor that inhibits the FGFR-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.

Perioperative blood transfusion does not affect recurrence-free and overall survivals after curative resection for intrahepatic cholangiocarcinoma: a propensity score matching analysis.

BACKGROUND: Whether perioperative blood transfusions (PBTs) adversely influence oncological outcomes for intrahepatic cholangiocarcinoma (ICC) patients after curative resection remains undetermined. METHODS: Of the 605 patients who underwent curative liver resection for ICC between 2000 and 2012, 93 received PBT. We conducted Cox regression and variable selection logistic regression analyses to identify confounding factors of PBT. Propensity score matching (PSM) and Cox regression analyses were used to compare the overall survival (OS) and disease-free survival (DFS) between the patients with or without PBT. RESULTS: After exclusion, 93 eligible patients (15.4%) received PBT, compared with 512 (84.6%) who did not receive PBT; the groups were highly biased in terms of the propensity score (PS) analysis (0.096 ± 0.104 vs. 0.479 ± 0.372, p < 0.001). PBT was associated with an increased risk of OS (HR: 1.889, 95% CI: 1.446-2.468, p < 0.001) and DFS (HR: 1.589, 95% CI: 1.221-2.067, p < 0.001) in the entire cohort. After propensity score matching (PSM), no bias was observed between the groups (PS,0.136 ± 0.117 VS. 0.193 ± 0.167, p = 0.785). In the multivariate Cox analysis, PBT was not associated with increased risks of OS (HR: 1.172, 95% CI: 0.756-1.816, p = 0.479) and DFS (HR: 0.944, 95% CI: 0.608-1.466, p = 0.799). After propensity score adjustment, PBT was still not associated with OS or DFS after ICC curative resection. CONCLUSIONS: The present study found that PBT did not affect DFS and OS after curative resection of ICC.

Multiomics analysis reveals metabolic subtypes and identifies diacylglycerol kinase α (DGKA) as a potential therapeutic target for intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and lethal hepatobiliary tumor with few therapeutic strategies. The metabolic reprogramming of tumor cells plays an essential role in the development of tumors, while the metabolic molecular classification of iCCA is largely unknown. Here, we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients, hoping to provide a novel perspective to understand and treat iCCA. METHODS: We performed integrated multiomics analysis in 116 iCCA samples, including whole-exome sequencing, bulk RNA-sequencing and proteome analysis. Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models, the metabolic subtype of iCCA was determined. Survival and prognostic gene analyses were used to compare overall survival (OS) differences between metabolic subtypes. Cell proliferation analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinase α (DGKA) in iCCA cells. RESULTS: Three metabolic subtypes (S1-S3) with subtype-specific biomarkers of iCCA were identified. These metabolic subtypes presented with distinct prognoses, metabolic features, immune microenvironments, and genetic alterations. The S2 subtype with the worst survival showed the activation of some special metabolic processes, immune-suppressed microenvironment and Kirsten rat sarcoma viral oncogene homolog (KRAS)/AT-rich interactive domain 1A (ARID1A) mutations. Among the S2 subtype-specific upregulated proteins, DGKA was further identified as a potential drug target for iCCA, which promoted cell proliferation by enhancing phosphatidic acid (PA) metabolism and activating mitogen-activated protein kinase (MAPK) signaling. CONCLUSION: Via multiomics analyses, we identified three metabolic subtypes of iCCA, revealing that the S2 subtype exhibited the poorest survival outcomes. We further identified DGKA as a potential target for the S2 subtype.

Downregulation of GPX8 in hepatocellular carcinoma: impact on tumor stemness and migration.

PURPOSE: GPX8, which is found in the endoplasmic reticulum lumen, is a member of the Glutathione Peroxidases (GPXs) family. Its role in hepatocellular carcinoma (HCC) is unknown. METHODS: Immunohistochemical staining was used to detect the protein levels of GPX8 in HCC tissue microarrays. A short hairpin RNA lentivirus was used to knock down GPX8, and the main signaling pathways were investigated using transcriptome sequencing and a phosphorylated kinase array. The sphere formation assays, cloning-formation assays and cell migration assays were used to evaluate the stemness and migration ability of HCC cells. Identifying the GPX8-interacting proteins was accomplished through immunoprecipitation and protein mass spectrometry. RESULTS: The GPX8 protein levels were downregulated in HCC patients. Low expression of GPX8 protein was related to early recurrence and poor prognosis in HCC patients. GPX8 knockdown could enhance the stemness and migration ability of HCC cells. Consistently, Based on transcriptome analysis, multiple signaling pathways that include the PI3K-AKT and signaling pathways that regulate the pluripotency of stem cells, were activated after GPX8 knockdown. The downregulation of GPX8 could increase the expression of the tumor stemness markers KLF4, OCT4, and CD133. The in vivo downregulation of GPX8 could also promote the subcutaneous tumor-forming and migration ability of HCC cells. MK-2206, which is a small-molecule inhibitor of AKT, could reverse the tumor-promoting effects both in vivo and in vitro. We discovered that GPX8 and the 71-kDa heat shock cognate protein (Hsc70) have a direct interaction. The phosphorylation of AKT encouraged the translocation of Hsc70 into the nucleus and the expression of the PI3K p110 subunit, thereby increasing the downregulation of GPX8. CONCLUSION: The findings from this study demonstrate the anticancer activity of GPX8 in HCC by inactivating the Hsc70/AKT pathway. The results suggest a possible therapeutic target for HCC.

Methyltransferase like 3 inhibition limits intrahepatic cholangiocarcinoma metabolic reprogramming and potentiates the efficacy of chemotherapy.

N6-methyladenosine (m6A) RNA methylation and its associated methyltransferase like 3 (METTL3) are involved in the development and maintenance of various tumors. The present study aimed to evaluate the cross-talk of METTL3 with glucose metabolism and reveal a novel mechanism for intrahepatic cholangiocarcinoma (ICC) progression. Real-time quantitative PCR, western blotting, and immunohistochemistry analyses suggested that METTL3 was highly expressed in ICC, which was correlated with poor patient prognosis. Immunoprecipitation sequencing of m6A-RNA showed that METTL3 upregulated m6A modification of NFAT5, which recruited IGF2BP1 for NFAT5 mRNA stabilization. Elevated expression of NFAT5 increased the expression of the gluconeogenesis-related genes GLUT1 and PGK1, resulting in enhanced aerobic glycolysis, proliferation, and tumor metastasis of ICC. Moreover, higher METTL3 expression was observed in tumor tissues of ICC patients with activated ICC glucose metabolism. Importantly, STM2457, a highly potent METTL3 inhibitor, which inhibited METTL3 activity and acted synergistically with gemcitabine, suggests that reprogramming RNA epigenetic modifications may serve as a potential therapeutic strategy. Overall, our findings highlighted the role of METTL3-mediated m6A modification of NFAT5 in activating glycolytic reprogramming in ICC and proposed that the METTL3/NFAT5 axis was a clinical target for the management of ICC chemoresistance by targeting cancer glycolysis.

Development of a deep pathomics score for predicting hepatocellular carcinoma recurrence after liver transplantation.

BACKGROUND AND PURPOSE: Tumor recurrence after liver transplantation (LT) impedes the curative chance for hepatocellular carcinoma (HCC) patients. This study aimed to develop a deep pathomics score (DPS) for predicting tumor recurrence after liver transplantation using deep learning. PATIENTS AND METHODS: Two datasets of 380 HCC patients who underwent LT were enrolled. Residual convolutional neural networks were used to identify six histological structures of HCC. The individual risk score of each structure and DPS were derived by a modified DeepSurv network. Cox regression analysis and Concordance index were used to evaluate the prognostic significance. The cellular exploration of prognostic immune biomarkers was performed by quantitative and spatial proximity analysis according to three panels of 7-color immunofluorescence. RESULTS: The overall classification accuracy of HCC tissue was 97%. At the structural level, immune cells were the most significant tissue category for predicting post-LT recurrence (HR 1.907, 95% CI 1.490-2.440). The C-indices of DPS achieved 0.827 and 0.794 in the training and validation cohorts, respectively. Multivariate analysis for recurrence-free survival (RFS) showed that DPS (HR 4.795, 95% CI 3.017-7.619) was an independent risk factor. Patients in the high-risk subgroup had a shorter RFS, larger tumor diameter and a lower proportion of clear tumor borders. At the cellular level, a higher infiltration of intratumoral NK cells was negatively correlated with recurrence risk. CONCLUSIONS: This study established an effective DPS. Immune cells were the most significant histological structure related to HCC recurrence. DPS performed well in post-LT recurrence prediction and the identification of clinicopathological features.

A RIPK3-independent role of MLKL in suppressing parthanatos promotes immune evasion in hepatocellular carcinoma.

Mixed lineage kinase domain-like (MLKL) is widely accepted as an executioner of necroptosis, in which MLKL mediates necroptotic signaling and triggers cell death in a receptor-interacting protein kinase 3 (RIPK3)-dependent manner. Recently, it is increasingly noted that RIPK3 is intrinsically silenced in hepatocytes, raising a question about the role of MLKL in hepatocellular carcinoma (HCC). This study reports a previously unrecognized role of MLKL in regulating parthanatos, a programmed cell death distinct from necroptosis. In HCC cells with intrinsic RIPK3 deficiency, knockout of MLKL impedes the orthotopic tumor growth, activates the anti-tumor immune response and enhances the therapeutic effect of immune checkpoint blockade in syngeneic HCC tumor models. Mechanistically, MLKL is required for maintaining the endoplasmic reticulum (ER)-mitochondrial Mg2+ dynamics in HCC cells. MLKL deficiency restricts ER Mg2+ release and mitochondrial Mg2+ uptake, leading to ER dysfunction and mitochondrial oxidative stress, which together confer increased susceptibility to metabolic stress-induced parthanatos. Importantly, pharmacological inhibition of poly(ADP-ribose) polymerase to block parthanatos restores the tumor growth and immune evasion in MLKL-knockout HCC tumors. Together, our data demonstrate a new RIPK3-independent role of MLKL in regulating parthanatos and highlight the role of MLKL in facilitating immune evasion in HCC.

Single-cell and spatial architecture of primary liver cancer.

Primary liver cancer (PLC) poses a leading threat to human health, and its treatment options are limited. Meanwhile, the investigation of homogeneity and heterogeneity among PLCs remains challenging. Here, using single-cell RNA sequencing, spatial transcriptomic and bulk multi-omics, we elaborated a molecular architecture of 3 PLC types, namely hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular-cholangiocarcinoma (CHC). Taking a high-resolution perspective, our observations revealed that CHC cells exhibit internally discordant phenotypes, whereas ICC and HCC exhibit distinct tumor-specific features. Specifically, ICC was found to be the primary source of cancer-associated fibroblasts, while HCC exhibited disrupted metabolism and greater individual heterogeneity of T cells. We further revealed a diversity of intermediate-state cells residing in the tumor-peritumor junctional zone, including a congregation of CPE+ intermediate-state endothelial cells (ECs), which harbored the molecular characteristics of tumor-associated ECs and normal ECs. This architecture offers insights into molecular characteristics of PLC microenvironment, and hints that the tumor-peritumor junctional zone could serve as a targeted region for precise therapeutical strategies.

Serial circulating tumor DNA to predict early recurrence in patients with hepatocellular carcinoma: a prospective study.

We studied the value of circulating tumor DNA (ctDNA) in predicting early postoperative tumor recurrence and monitoring tumor burden in patients with hepatocellular carcinoma (HCC). Plasma-free DNA, germline DNA, and tissue DNA were isolated from 41 patients with HCC. Serial ctDNAs were analyzed by next-generation sequencing before and after operation. Whole-exome sequencing was used to detect the DNA of HCC and adjacent tissues. In total, 47 gene mutations were identified in the ctDNA of the 41 patients analyzed before surgery. ctDNA was detected in 63.4% and 46% of the patient plasma pre- and postoperation, respectively. The preoperative ctDNA positivity rate was significantly lower in the nonrecurrence group than in the recurrence group. With a median follow-up of 17.7 months, nine patients (22%) experienced tumor recurrence. ctDNA positivity at two time-points was associated with significantly shorter recurrence-free survival (RFS). Tumors with NRAS, NEF2L2, and MET mutations had significantly shorter times to recurrence than those without mutations and showed high recurrence prediction performance by machine learning. Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in preoperative ctDNA was a strong independent predictor of RFS. ctDNA is a real-time monitoring indicator that can accurately reflect tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of RFS in individuals with HCC.

Conversion therapy for initially unresectable hepatocellular carcinoma using a combination of toripalimab, lenvatinib plus TACE: real-world study.

BACKGROUND: Combination conversion therapies afforded curative surgery chance for initially unresectable hepatocellular carcinoma (uHCC). This study aimed to evaluate the conversion rate and clinical outcomes of a first-line conversion regimen of lenvatinib combined with transarterial chemoembolization (TACE) plus immunotherapy for initial uHCC by interpreting real-world data. METHODS: Conversion therapy data of patients with uHCC from November 2018 to January 2021 were analysed. The regimens included triple combination therapy (t-CT: lenvatinib, TACE, plus toripalimab) and dual combination therapy (d-CT: lenvatinib plus TACE). Another study population diagnosed with hepatocellular carcinoma of macrovascular invasion disease were included as the upfront surgery cohort. Treatment responses and conversion rate were primary outcomes. Survival and adverse events were analysed. RESULTS: Fifty-one patients receiving t-CT (n = 30) and d-CT (n = 21) were enrolled. Higher overall response rates (76.7 per cent versus 47.6 per cent, P = 0.042) and disease control rates (90.0 per cent versus 57.1 per cent, P = 0.042) were observed via t-CT than d-CT. Both median overall survival and event-free survival were not reached in the t-CT cohort. A higher rate of curative conversion resection was achieved through t-CT than d-CT (50.0 per cent versus 19.0 per cent, P = 0.039). The disease-free survival of patients undergoing conversion resection in the t-CT cohort (n = 15) was higher than that in the upfront surgery cohort (n = 68, P = 0.039). Both t-CT and d-CT regimens were tolerable. CONCLUSIONS: Better treatment responses and conversion rate for patients with uHCC were obtained with first-line t-CT. Neoadjuvant t-CT before surgery should be recommended for patients with macrovascular invasion.

Cystathionine ß-synthase mediated PRRX2/IL-6/STAT3 inactivation suppresses Tregs infiltration and induces apoptosis to inhibit HCC carcinogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by inflammation and immunopathogenesis. Accumulating evidence has shown that the cystathionine ß-synthase/hydrogen sulfide (CBS/H2S) axis is involved in the regulation of inflammation. However, roles of CBS in HCC development and immune evasion have not been systematically investigated, and their underlying mechanisms remain elusive. Here, we investigated the roles of CBS in tumor cells and tumor microenvironment of HCC. METHODS: 236 HCC samples were collected to detect the expression of CBS, cleaved Caspase-3 and paired related homeobox 2 (PRRX2) and the number of immune cells. HCC cell lines were employed to examine the effects of CBS on cellular viability, apoptosis and signaling in vitro. Cbs heterozygous knockout mice, C57BL/6 mice, nude mice and non-obese diabetic severe combined immunodeficiency mice were used to investigate the in vivo functions of CBS. RESULTS: Downregulation of CBS was observed in HCC, and low expression of CBS predicted poor prognosis in HCC patients. CBS overexpression dramatically promoted cellular apoptosis in vitro and inhibited tumor growth in vivo. Activation of the Cbs/H2S axis also reduced the abundance of tumor-infiltrating Tregs, while Cbs deficiency promoted Tregs-mediated immune evasion and boosted tumor growth in Cbs heterozygous knockout mice. Mechanistically, CBS facilitated the expression cleaved Caspase-3 in tumor cells, and on the other hand, suppressed Foxp3 expression in Tregs via inactivating IL-6/STAT3 pathway. As a transcription factor of IL-6, PRRX2 was reduced by CBS. Additionally, miR-24-3p was proven to be an upstream suppressor of CBS in HCC. CONCLUSIONS: Our results indicate the antitumor function of CBS in HCC by inactivation of the PRRX2/IL-6/STAT3 pathway, which may serve as a potential target for HCC clinical immunotherapy.

Age-adjusted Charlson Comorbidity Index predicts survival in intrahepatic cholangiocarcinoma patients after curative resection.

BACKGROUND: Comorbidity among cancer patients is prevalent and influential to prognosis after operation. Limited data are available on comorbidity evaluations in patients with intrahepatic cholangiocarcinoma (ICC). This study aimed to assess the comorbidity distribution in ICC patients and to adapt the Charlson Comorbidity Index (CCI) or the age-adjusted CCI (ACCI) for survival prediction. METHODS: The study cohort included 268 ICC patients treated with curative surgery from January 2000 to December 2007 at the Department of Liver Surgery, Zhongshan Hospital. The association between the comorbidity index and overall survival (OS) or disease-free survival (DFS). was analyzed by the Kaplan-Meier method. Multivariable analysis was established to select the determinant parameters. RESULTS: Major comorbid conditions of ICC patients included liver disease, hypertension, diabetes and ulcer. The median follow-up time was 25.5 months in the whole data set. Among the entire cohort, the 1-, 3- and 5-year OS rates were 55.3%, 26.0% and 15.6%, respectively. In multivariate analysis, the ACCI correlated with OS, and higher scores were associated with poorer prognosis (hazard ratio =1.134, 95% confidence interval: 1.015-1.267 and P value =0.026). CCI was not an independent predictive factor for OS or DFS. CONCLUSIONS: In contrast to CCI, ACCI was a more promising model to accurately predict OS in ICC patients who underwent liver resection. Further research should be focused on the impact of comorbidity therapies.