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Primary liver cancer, mainly hepatocellular carcinoma (HCC), is the sixth most diagnosed cancer and third leading cause of cancer-related death globally. Recently, immunotherapies such as immune checkpoint inhibitors (ICIs) have made great progress in the systemic treatment of HCC. However, anti-PD-1 therapy with pembrolizumab or nivolumab as a single agent did not meet their predefined end points of overall survival in the KEYNOTE-240 and CheckMate 459 trials. It is urgent to understand the immunological rationale and explore novel ways to improve the efficacy of immunotherapy. The combination of ICIs with other therapies, such as tyrosine kinase inhibitors (TKIs), monoclonal antibodies, or local therapy, has been demonstrated to improve overall response rate and survival. In addition, modulating tumor microenvironment is a potential way to overcome the primary and secondary resistance to immunotherapies. In this review, we summarized the latest findings in the immune microenvironment, the mechanisms of their synergistic effects when combined with anti-VEGF agents or TKIs, as well as other kinds of immune treatment.

Summary Background Programmed cell death protein‐1‐targeted immunotherapy has shown promising results in phase II studies of hepatocellular carcinoma. Aim To evaluate safety and efficacy of nivolumab and pembrolizumab in an international, multicentre, real‐world cohort of patients with advanced hepatocellular carcinoma. Methods Sixty‐five patients treated with nivolumab (n = 34) or pembrolizumab (n = 31) between July 10, 2015 and December 31, 2018 (data cut‐off) across six centres in Austria and Germany were retrospectively analysed. Results Child‐Pugh class A/B/C was 32 (49%)/28 (43%)/5 (8%). Immunotherapy was used as systemic first‐/second‐/third‐/fourth‐line treatment in 9 (14%)/27 (42%)/26 (40%)/3 (5%) patients. Fifty‐four patients had at least one follow‐up imaging and were, therefore, available for radiological response assessment. The overall response and disease control rates were 12% and 49% respectively. Of 52 evaluable patients, four (8%) had hyperprogressive disease. Median time to progression was 5.5 (95% CI, 3.5‐7.4) months, median progression‐free survival was 4.6 (95% CI, 3.0‐6.2) months, and median overall survival was 11.0 (95% CI, 8.2‐13.8) months. Most common adverse events were infections (n = 7), rash (n = 6), pruritus (n = 3), fatigue (n = 3), diarrhoea (n = 3) and hepatitis (n = 3). Efficacy and safety results were comparable between Child‐Pugh A and B patients; however, median overall survival (OS) was shorter in Child‐Pugh B patients (16.7 vs 8.6 months; P = 0.065). There was no difference in terms of efficacy and adverse events between patients who received immunotherapy as first‐/second‐line and third‐/fourth‐line respectively. Conclusions Programmed cell death protein‐1‐targeted immunotherapy with nivolumab or pembrolizumab showed promising efficacy and safety in patients with advanced hepatocellular carcinoma, including subjects with Child‐Pugh stage B and patients with intensive pretreatment.

Lenvatinib is an inhibitor of multiple receptor tyrosine kinases that was recently authorized for first-line treatment of hepatocellular carcinoma (HCC). However, the clinical benefits derived from lenvatinib are limited, highlighting the urgent need to understand mechanisms of resistance. We report here that HCC cells develop resistance to lenvatinib by activating EGFR and stimulating the EGFR–STAT3–ABCB1 axis. Lenvatinib resistance was accompanied by aberrant cholesterol metabolism and lipid raft activation. ABCB1 was activated by EGFR in a lipid raft–dependent manner, which significantly enhanced the exocytosis of lenvatinib to mediate resistance. Furthermore, clinical specimens of HCC showed a correlation between the activation of the EGFR–STAT3–ABCB1 pathway and lenvatinib response. Erlotinib, an EGFR inhibitor that has also been shown to inhibit ABCB1, suppressed lenvatinib exocytosis, and combined treatment with lenvatinib and erlotinib demonstrated a significant synergistic effect on HCC both in vitro and in vivo. Taken together, these findings characterize a mechanism of resistance to a first-line treatment for HCC and offer a practical means to circumvent resistance and treat the disease. Significance: HCC cells acquire resistance to lenvatinib by activating the EGFR–STAT3–ABCB1 pathway, identifying combined treatment with erlotinib as a strategy to overcome acquired resistance and improve the clinical benefit of lenvatinib.

Purpose: To evaluate the efficacy and safety of lenvatinib in combination with transarterial chemoembolization (TACE) as an adjuvant therapy in HCC patients with high-risk of postoperative recurrence. Methods: This study was a prospective cohort study. Patients who met the eligible criteria were recruited between November 2018 to May 2021 from 8 hepatobiliary centers in China. The primary endpoint was disease-free survival (DFS). The secondary endpoints were overall survival (OS) and safety. The treatment-related adverse events (AEs) were recorded throughout the entire study period. Results: A total of 297 patients were enrolled, with 147 patients in the TACE + Lenvatinib (Len) group and 150 patients in the TACE group. The baseline characteristics were well balanced between the two groups. Patients in the TACE + Len group achieved significantly better DFS. The median DFS was 19.0 months [95% confidence interval (CI), 14.0-25.0] and 10.0 months [95% CI, 8.0-13.0] in the TACE + Len and TACE group, respectively (log-rank test p= 0.011). The adjusted hazard ratio (HR) of disease recurrence for baseline prognostic variables between the two groups was 0.68 (95% CI, 0.50–0.91, p = 0.009). Lenvatinib treatment-related AEs of grade 3 or 4 occurred in 28.6% of the patients in TACE + Len group. Conclusions: This study suggested that adjuvantTACE plus lenvatinib a promising approach for HCC patients with high-risk of postoperative recurrence, which could significantly reduce postoperative recurrence and prolong survival with a manageable safety profile.

Background Hepatocellular carcinoma (HCC) is closely associated with chronic liver diseases, particularly liver cirrhosis, which has an altered extracellular matrix (ECM) composition. The influence and its mechanism of the cirrhotic-ECM on the response of HCC to immune checkpoint inhibitor (ICI) remains less clarified.Methods In silico, proteomic and IHC assessment of alteration of cirrhotic-ECM were applied in clinical cohort. Multiple pre-clinical models with ECM manipulation were used to evaluate cirrhotic-ECM’s effect on ICI treatment. In silico, flow cytometry and IHC were applied to explore how cirrhotic-ECM affect HCC microenvironment. In vitro and in vivo experiments were carried out to identify the mechanism of how cirrhotic-ECM undermined ICI treatment.Results We defined “a pro-tumor cirrhotic-ECM” which was featured as the up-regulation of Col1. Cirrhotic-ECM/Col1 was closely related to impaired T cell function and limited aPD-1 response of HCC patients from the TCGA pan cancer cohort and the authors’ institution, as well as in multiple pre-clinical models. Mechanically, cirrhotic-ECM/Col1 orchestrated an immunosuppressive microenvironment (TME) by triggering Col1-DDR1-NFκB-CXCL8 axis, which initiated neutrophil extracellular traps (NETs) formation to shield HCC cells from attacking T cells and impede approaching T cells. Nilotinib, an inhibitor of DDR1, reversed the neutrophils/NETs dominant TME and efficiently enhanced the response of HCC to aPD-1.Conclusions cirrhotic-ECM modulated a NETs enriched TME in HCC, produced an immune suppressive TME and weakened ICI efficiency. Col1 receptor DDR1 could be a potential target synergically used with ICI to overcome ECM mediated ICI resistance. These provide a mechanical insight and novel strategy to overcome the ICI resistance of HCC.

BACKGROUND: The efficacy of immune checkpoint inhibitors (ICIs), such as programmed cell death protein-1 (PD-1) or its ligand 1 (PD-L1) antibody, in hepatocellular carcinoma (HCC) is limited, and it is recommended that they be combined with other therapies. We evaluated the combination of pegylated interferon-α (Peg-IFNα) with PD-1 blockade in HCC mouse models. METHODS: We analyzed the effects of Peg-IFNα on tumor-infiltrating immune cells and PD-1 expression in the HCC immune microenvironment and examined the underlying mechanism of its unique effect on the PD-1 pathway. The in vivo efficacy of anti-PD-1 and Peg-IFNα was evaluated in both subcutaneous and orthotopic mouse models of HCC. RESULTS: The combination of Peg-IFNα with PD-1 blockade dramatically enhanced T-cell infiltration, improved the efficacy of PD-1 antibody and prolonged mouse survival compared with PD-1 antibody monotherapy. Mechanistically, Peg-IFNα could recruit cytotoxic CD8(+) T cells to infiltrate the HCC microenvironment by inducing tumor cells to secrete the chemokine CCL4. Nevertheless, the HCC microenvironment quickly overcame the immune responses by upregulating PD-1 expression in CD8(+) T cells via the IFNα-IFNAR1-JAK1-STAT3 signaling pathway. The combination of PD-1 blockade with Peg-IFNα could restore the cytotoxic capacity of CD8(+) T cells and exerted a significant synergistic effect on HCC. CONCLUSION: These results indicate that in addition to initiating the antitumor immune response itself, Peg-IFNα can also generate a microenvironment favoring PD-1 blockade. Thus, the combination of Peg-IFNα and PD-1 blockade can be a promising strategy for HCC.

Lenvatinib is an inhibitor of multiple receptor tyrosine kinases that was recently authorized for first-line treatment of hepatocellular carcinoma (HCC). However, the clinical benefits derived from lenvatinib are limited, highlighting the urgent need to understand mechanisms of resistance. We report here that HCC cells develop resistance to lenvatinib by activating EGFR and stimulating the EGFR–STAT3–ABCB1 axis. Lenvatinib resistance was accompanied by aberrant cholesterol metabolism and lipid raft activation. ABCB1 was activated by EGFR in a lipid raft–dependent manner, which significantly enhanced the exocytosis of lenvatinib to mediate resistance. Furthermore, clinical specimens of HCC showed a correlation between the activation of the EGFR–STAT3–ABCB1 pathway and lenvatinib response. Erlotinib, an EGFR inhibitor that has also been shown to inhibit ABCB1, suppressed lenvatinib exocytosis, and combined treatment with lenvatinib and erlotinib demonstrated a significant synergistic effect on HCC both in vitro and in vivo. Taken together, these findings characterize a mechanism of resistance to a first-line treatment for HCC and offer a practical means to circumvent resistance and treat the disease.Significance:HCC cells acquire resistance to lenvatinib by activating the EGFR–STAT3–ABCB1 pathway, identifying combined treatment with erlotinib as a strategy to overcome acquired resistance and improve the clinical benefit of lenvatinib.

Purpose: Hepatocellular carcinoma (HCC), a common cancer worldwide, has a dismal outcome partly due to the poor identification of early-stage HCC. Currently, one third of HCC patients present with low serum α-fetoprotein (AFP) levels, the only clinically available diagnostic marker for HCC. The aim of this study was to identify new diagnostic molecular markers for HCC, especially for individuals with low serum AFP.Experimental Design: We used the microarray technique to determine the expression profiles of 218 HCC specimens from patients with either high or low serum AFP. From the microarray study, we selected five candidate genes (i.e., GPC3, PEG10, MDK, SERPINI1, and QP-C), which were overexpressed in HCCs. Using quantitative real-time PCR analyses, we validated the expression of these five genes in 50 AFP-normal and 8 AFP-positive HCC specimens and 36 cirrhotic noncancerous hepatic specimens, which include 52 independent specimens not used in microarray analysis.Results: A significant increase in the expression of the five candidate genes could be detected in most of the HCC samples, including those with normal serum AFP and small tumors. GPC3, MDK, and SERPINI1 encode known serum proteins. Consistently, a significant increase in serum midkine, encoded by MDK, was associated with HCC patients, including those with normal serum AFP. Using prediction analysis of microarray, we showed that a combined score of these five genes can accurately classify noncancerous hepatic tissues (100%) and HCC (71%).Conclusions: We suggest that a diagnostic signature approach using a combined score of these five biomarkers rather than a single marker may improve the prediction accuracy of HCC patients, including those with normal serum AFP and smaller-sized tumors.

CCR Translation for the Article from Human Hepatocellular Carcinoma Tumor–derived Endothelial Cells Manifest Increased Angiogenesis Capability and Drug Resistance Compared with Normal Endothelial Cells

Purpose: Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype compared with endothelial cells (NEC) from adjacent normal tissue and may be able to acquire resistance to drugs. The aim of this study was to investigate the angiogenesis activity and response to drug treatment of TECs and NECs derived from human hepatocellular carcinoma (HCC).Experimental Design: TECs or NECs were isolated from HCC or adjacent normal liver tissue using anti-CD105 antibody coupled to magnetic beads. The phenotypic and functional properties of endothelial cells were characterized by testing the expression of CD105, CD31, CD144, vascular endothelial growth factor receptor-1, vascular endothelial growth factor receptor-2, and von Willebrand factor, and the ability of DiI-Ac-LDL-uptake and tube formations. CD105+ TECs were compared with CD105+ NECs and human umbilical vein endothelial cells (HUVEC) by examining their ability to proliferate, motility, ability to adhere to tumor cells, response to tumor conditioned medium, and reactions to the chemotherapy drugs Adriamycin and 5-fluorouracil and the antiangiogenic drug sorafenib.Results: Compared with CD105+ NECs and HUVECs, CD105+ TECs showed increased apoptosis resistance and motility and proangiogenic properties. Meanwhile, CD105+ TECs had a greater ability to adhere to tumor cells and survive in the tumor environment. Moreover, CD105+ TECs acquired more resistance to Adriamycin, 5-fluorouracil, and sorafenib than CD105+ NECs and HUVECs.Conclusions: TECs possessed enhanced angiogenic activity and resistance to chemotherapeutic drugs and an angiogenesis inhibitor, and may provide a better tool for studying tumor angiogenesis and antiangiogenesis drugs in HCC.

Supplementary Data from Inhibition of EGFR Overcomes Acquired Lenvatinib Resistance Driven by STAT3–ABCB1 Signaling in Hepatocellular Carcinoma

Supplementary Materials and Methods, Figures, and Tables

PDF file - 589K, Supplementary Fig. S1 Elevated MDK levels in the culture medium of HCC cell lines compared with normal liver cell lines. Supplementary Fig. S2 The prognostic values of AFP and MDK levels for HCC patients. Supplementary Fig. S3.Expression of serum MDK in another independent cohort (cohort B) from Egypt. Supplementary Fig. S4. Logistic Regression Model to combine information of MDK and AFP for prediction of HCC in the learning cohort A (n=252): Comparison of AUROC for diagnosis HCC through logist regression model I, serum MDK and AFP. Supplementary Fig. S5. Positive predictive value (PPV) and negative predictive value (NPV) for identifying HCC patients through serum MDK according to different prevalence. Supplementary Fig. S6. Correlation between serum AFP and MDK levels in HCC patients (n=252). Supplementary Fig. S7. Serum MDK is elevated in different HCC subsets, No significant difference in serum MDK levels was found between the learning cohort A and the external validation cohort of HCC patients(P=0.398), or between the early stage HCC patients in learning and validation cohort (P=0.295). Table S1. Relationship between MDK expression and clinicopathological features of HCC patients. Table S2. Clinical and biochemical characteristics of liver cirrhosis (LC) patients in cohort A. Table S3. Baseline characteristics of the hospital controls with different etiologies from learning Cohort A. Table S4. Clinical characteristics of HCC patients according to different cohorts in this study. Table S5. MDK protein levels in cancer-free cirrhotic liver and HCC tissues. Table S6. Comparison of AFP and MDK with clinicopathological parameters in learning cohort A. Table S7．Univariate Cox-regression Analyses of Factors Associated With OS and TTR. Table S8. Sensitivities and specificities of AFP and MDK according to various cutoff values. Table S9. Cross-validation of the diagnostic ability of serum MDK. Table S10. Comparison of MDK with clinicopathological parameters in validation cohort C.

Purpose: To evaluate the value of serum midkine (MDK) as a diagnostic biomarker in hepatocellular carcinoma, particularly for those with negative alpha-fetoprotein (AFP) and at an early stage.Experimental Design: MDK expression in tumors was assessed by immunohistochemistry from 105 patients with hepatocellular carcinomas or liver cirrhosis. Serum MDK levels were detected by ELISA in 933 participants including hepatocellular carcinomas and hospital controls from different medical centers. Sensitivities and specificities of serum MDK in diagnosing hepatocellular carcinoma according to AFP level and Barcelona Clinic Liver Cancer (BCLC) stage were analyzed.Results: MDK levels were significantly elevated in hepatocellular carcinoma tissues as well as serum samples. The sensitivity of serum MDK for hepatocellular carcinoma diagnosis was much higher than that of AFP (86.9% vs. 51.9%) with similar specificities (83.9% vs. 86.3%). Notably, serum MDK had an outstanding performance in distinguishing AFP-negative hepatocellular carcinomas from different controls: In those AFP-negative hepatocellular carcinomas, the sensitivity could reach as high as 89.2%. Moreover, receiver operating characteristic (ROC) curve analysis also showed that serum MDK had a better performance compared with AFP in distinguishing early-stage hepatocellular carcinomas as well as small hepatocellular carcinomas. Even in very early-stage hepatocellular carcinomas, MDK showed an obviously higher sensitivity compared with AFP (80% vs. 40%). Furthermore, serum MDK level was significantly decreased in patients with hepatocellular carcinomas after curative resection and re-elevated when tumor relapse occurred.Conclusions: Serum MDK is significantly elevated in most hepatocellular carcinomas, including those with negative AFP and at an early stage, which may serve as a novel diagnostic marker in early diagnosis and postoperative monitoring of hepatocellular carcinomas. Clin Cancer Res; 19(14); 3944–54. ©2013 AACR.

Hepatocellular carcinoma (HCC) is a genetically heterogeneous disease for which a dominant actionable molecular driver has not been identified. Patients with the stem cell–like EpCAM+AFP+ HCC subtype have poor prognosis. Here, we performed a genome-wide RNAi screen to identify genes with a synthetic lethal interaction with EpCAM as a potential therapeutic target for the EpCAM+AFP+ HCC subtype. We identified 26 candidate genes linked to EpCAM/Wnt/β-catenin signaling and HCC cell growth. We further characterized the top candidate PMPCB, which plays a role in mitochondrial protein processing, as a bona fide target for EpCAM+ HCC. PMPCB blockage suppressed EpCAM expression and Wnt/β-catenin signaling via mitochondria-related reactive oxygen species production and FOXO activities, resulting in apoptosis and tumor suppression. These results indicate that a synthetic lethality screen is a viable strategy to identify actionable drivers of HCC and identify PMPCB as a therapeutically vulnerable gene in EpCAM+ HCC subpopulations.Significance:This study identifies PMPCB as critical to mitochondrial homeostasis and a synthetic lethal candidate that selectively kills highly resistant EpCAM+ HCC tumors by inactivating the Wnt/β-catenin signaling pathway.

Supplementary figures as related to the manuscript. Data represent schematic RNAi screen workflow, additional survival analyses, and experiments performed in different hepatocellular carcinoma cell lines.

Supplementary Data from Human Hepatocellular Carcinoma Tumor–derived Endothelial Cells Manifest Increased Angiogenesis Capability and Drug Resistance Compared with Normal Endothelial Cells

Supplementary Tables 1-4, Figure Legend from Association of Specific Genotypes in Metastatic Suppressor HTPAP with Tumor Metastasis and Clinical Prognosis in Hepatocellular Carcinoma

Supplementary Text, Figures 1-3, Tables 1-6 from EpCAM and α-Fetoprotein Expression Defines Novel Prognostic Subtypes of Hepatocellular Carcinoma

The phosphatidic acid phosphatase HTPAP has been defined as a metastatic suppressor of hepatocellular carcinoma (HCC), but little is known about its function or potential applications as a prognostic marker. In this study, we analyzed patterns of HTPAP genetic variation and gene expression in 864 patients who underwent HCC resection, assessing these patterns for correlations to tumor metastasis potential. Focusing on two tagSNPs that were selected (+357G/C and +1838A/G), we found that only the +357G/C genotype was significantly associated with HTPAP mRNA and protein expression levels and the probability of metastasis. In an independent cohort of 665 HCC patients, we determined that the +357G/C genotype was associated with shorter time to recurrence and overall survival. Together, these results indicated that the HTPAP tagSNP +357 GG+GC genotypes may influence HCC metastatic potential and clinical prognosis by down-regulating HTPAP expression. Extending these results, a global expression profiling analysis identified 41 genes including the pro-inflammatory genes IL-8 and TLR2 that were significantly overexpressed in the +357 GG+GC group, as possible coregulated markers with HTPAP. Together, our findings identify an HTPAP genotype and associated gene expression pattern that favors metastasis progression and that could be used to predict tumor metastasis and prognosis in HCC patients. Cancer Res; 71(9); 3278–86. ©2011 AACR.

Supplementary Figure 1 from Association of Specific Genotypes in Metastatic Suppressor HTPAP with Tumor Metastasis and Clinical Prognosis in Hepatocellular Carcinoma

Supplementary Figures 1-3, Table 1 from A Unique Metastasis Gene Signature Enables Prediction of Tumor Relapse in Early-Stage Hepatocellular Carcinoma Patients

The heterogeneous nature of hepatocellular carcinoma (HCC) and the lack of appropriate biomarkers have hampered patient prognosis and treatment stratification. Recently, we have identified that a hepatic stem cell marker, epithelial cell adhesion molecule (EpCAM), may serve as an early biomarker of HCC because its expression is highly elevated in premalignant hepatic tissues and in a subset of HCC. In this study, we aimed to identify novel HCC subtypes that resemble certain stages of liver lineages by searching for EpCAM-coexpressed genes. A unique signature of EpCAM-positive HCCs was identified by cDNA microarray analysis of 40 HCC cases and validated by oligonucleotide microarray analysis of 238 independent HCC cases, which was further confirmed by immunohistochemical analysis of an additional 101 HCC cases. EpCAM-positive HCC displayed a distinct molecular signature with features of hepatic progenitor cells including the presence of known stem/progenitor markers such as cytokeratin 19, c-Kit, EpCAM, and activated Wnt-β-catenin signaling, whereas EpCAM-negative HCC displayed genes with features of mature hepatocytes. Moreover, EpCAM-positive and EpCAM-negative HCC could be further subclassified into four groups with prognostic implication by determining the level of α-fetoprotein (AFP). These four subtypes displayed distinct gene expression patterns with features resembling certain stages of hepatic lineages. Taken together, we proposed an easy classification system defined by EpCAM and AFP to reveal HCC subtypes similar to hepatic cell maturation lineages, which may enable prognostic stratification and assessment of HCC patients with adjuvant therapy and provide new insights into the potential cellular origin of HCC and its activated molecular pathways. [Cancer Res 2008;68(5):1451–61]

Cabozantinib, mainly targeting cMet and vascular endothelial growth factor receptor 2, is the second-line treatment for patients with advanced hepatocellular carcinoma (HCC). However, the lower response rate and resistance limit its enduring clinical benefit. In this study, we found that cMet-low HCC cells showed primary resistance to cMet inhibitors, and the combination of cabozantinib and mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exhibited a synergistic inhibitory effect on the in vitro cell proliferation and in vivo tumor growth of these cells. Mechanically, the combination of rapamycin with cabozantinib resulted in the remarkable inhibition of AKT, extracellular signal-regulated protein kinases, mTOR, and common downstream signal molecules of receptor tyrosine kinases; decreased cyclin D1 expression; and induced cell cycle arrest. Meanwhile, rapamycin enhanced the inhibitory effects of cabozantinib on the migration and tubule formation of human umbilical vascular endothelial cells and human growth factor-induced invasion of cMet inhibitor-resistant HCC cells under hypoxia condition. These effects were further validated in xenograft models. In conclusion, our findings uncover a potential combination therapy of cabozantinib and rapamycin to combat cabozantinib-resistant HCC.

The advent of immune checkpoint inhibitors (ICIs) has revolutionized the therapeutic options of hepatobiliary malignancies. However, the clinical benefit provided by immunotherapy seems limited to a small subgroup of patients with hepatobiliary malignancies. The identification of reliable predictors of the response to immunotherapy is urgently needed.Literature search was conducted in PubMed for relevant articles published up to May 2022. Information of clinical trials was obtained from https://clinicaltrials.gov/.Biomarkers for ICI response of hepatobiliary malignancies remain in the exploration stage and lack compelling evidence. Tumor programmed death-ligand 1 (PD-L1) expression is the most widely studied biomarker in hepatocellular carcinoma (HCC) and biliary tract cancers (BTCs), but there are conflicting results on its predictive potential. Tumor mutational burden (TMB) is generally low both in HCC and BTCs, and the clinical trials of TMB are rare in hepatobiliary malignancies. Promisingly, mismatch repair deficiency (dMMR)/high microsatellite instability (MSI-H) may be a predictive biomarker of response to anti-PD-1 therapy in BTCs. Furthermore, some emerging biomarkers, such as gut microbiota, show predictive potential in the preliminary studies. Radiomics and liquid-biopsy biomarkers, including circulating tumor cells, circulating tumor DNA (ctDNA) and exosomal PD-L1 provide a quick and non-invasive approach for monitoring the ICI response, showing a new promising direction.Multiple potential biomarkers for predicting ICI response of hepatobiliary malignancies have been explored and tried to apply in clinic. Yet there is no robust evidence to prove their clinical value in predicting immunotherapeutic response for patients with hepatobiliary malignancies. The identification of predictors for response to ICIs is an urgent need and major challenge. Further studies are warranted to validate the role of emerging biomarkers in predicting immunotherapeutic responses.

Dysregulated lipid metabolism represents an important metabolic alteration in cancer. Fatty acids, cholesterol, and phospholipid are the three most prevalent lipids that act as energy producers, signaling molecules, and source material for the biogenesis of cell membranes. The enhanced synthesis, storage, and uptake of lipids contribute to cancer progression. The rewiring of lipid metabolism in cancer has been linked to the activation of oncogenic signaling pathways and cross talk with the tumor microenvironment. The resulting activity favors the survival and proliferation of tumor cells in the harsh conditions within the tumor. Lipid metabolism also plays a vital role in tumor immunogenicity via effects on the function of the noncancer cells within the tumor microenvironment, especially immune‐associated cells. Targeting altered lipid metabolism pathways has shown potential as a promising anticancer therapy. Here, we review recent evidence implicating the contribution of lipid metabolic reprogramming in cancer to cancer progression, and discuss the molecular mechanisms underlying lipid metabolism rewiring in cancer, and potential therapeutic strategies directed toward lipid metabolism in cancer. This review sheds new light to fully understanding of the role of lipid metabolic reprogramming in the context of cancer and provides valuable clues on therapeutic strategies targeting lipid metabolism in cancer., Lipid metabolic reprogramming‐mediated crosstalk between cancer cells and tumor microenvironment contributes to cancer progression. Stromal cells, such as adipocyte, CAF, epithelia cells contribute to lipid metabolic reprogramming in cancer cells. Cancer cells undergo lipid metabolism rewiring to generate building materials for membrane, lipid second messenger and energy supply. Cancer cells can also secret lipid metabolites to affect immune cell functions, creating a tumor‐favoring immune microenvironment.

Tumor-associated macrophages (TAMs) are crucial components of the tumor microenvironment. They play vital roles in hepatocellular carcinoma (HCC) progression. However, the interactions between TAMs and HCC cells have not been fully characterized. In this study, TAMs were induced using human monocytic cell line THP-1 cells in vitro to investigate their functions in HCC progression. S100 calcium-binding protein A9 (S100A9), an inflammatory microenvironment-related secreted protein, was identified to be significantly upregulated in TAMs. S100A9 expression in tumor tissues was associated with poor survival of HCC patients. It could enhance the stem cell-like properties of HepG2 and MHCC-97H cells by activating nuclear factor-kappa B signaling pathway through advanced glycosylation end product-specific receptor in a Ca2+ -dependent manner. Furthermore, we found that, after treatment with S100A9, HepG2 and MHCC-97H cells recruited more macrophages via chemokine (CC motif) ligand 2, which suggests a positive feedback between TAMs and HCC cells. Taken together, our findings reveal that TAMs could upregulate secreted protein S100A9 and enhance the stem cell-like properties of HCC cells and provide a potential therapeutic target for combating HCC.

Metastasis and metabolism reprogramming are two major hallmarks of cancer. In the initiation and progression of cancer, tumor cells are known to undergo fundamental metabolic changes to sustain their development and progression. In recent years, much more attentions have been drawn to their important roles in facilitating cancer metastasis through regulating the biological properties. In this review, we summarized the recent progresses in the studies of metabolism reprogramming of cancer metastasis, particularly of primary liver cancer, and highlight their potential applications.

We assess the safety and feasibility of the left hepatic vein preferential approach (LHVPA) based on left hepatic vein (LHV) anatomy for living donor laparoscopic left lateral sectionectomy (LLLS). Data from 50 donors who underwent LLLS in Huashan Hospital from October 2016 to November 2019 were analyzed retrospectively. On the basis of the classification of the LHV anatomy, the vein was defined as the direct import type, upper branch type, or indirect import type. A subgroup analysis was performed to compare the outcomes between the LHVPA and non-LHVPA groups. All 50 patients underwent pure LLLS. The mean operative duration was 157.5 ± 29.7 minutes. The intraoperative blood loss was 160.4 ± 97.5 mL. No complications more severe than grade 3 occurred. LHVPA was applied in 13 patients, whereas non-LHVPA was applied in 10 patients with the direct import type and upper branch type anatomy. The operative duration was shorter in the LHVPA group than the non-LHVPA group (142.7 ± 22.0 versus 173.0 ± 22.8 minutes; P = 0.01). Intraoperative blood loss was reduced in the LHVPA group compared with the non-LHVPA group (116.2 ± 45.6 versus 170.0 ± 63.3 mL; P = 0.02). The length of the LHV reserved extrahepatically in the LHVPA group was longer than in the non-LHVPA group (4.3 ± 0.2 versus 3.3 ± 0.3 mm; P = 0.01). Fewer reconstructions of the LHV in the direct import type anatomy were required for the LHVPA group than for the non-LHVPA group (0/8 versus 4/6). LHVPA based on the LHV anatomy is recommended in LLLS because it can further increase the safety and the efficiency of surgery for suitable donors.

Objective: Epigenetic aberration plays an important role in the development and progression of hepatocellular carcinoma (HCC). However, the alteration of RNA N6-methyladenosine (m6A) modifications and its role in HCC progression remain unclear. We therefore aimed to provide evidence using bioinformatics analysis. Methods: We comprehensively analyzed the m6A regulator modification patterns of 605 HCC samples and correlated them with metabolic alteration characteristics. We elucidated 390 gene-based m6A-related signatures and defined an m6Ascore to quantify m6A modifications. We then assessed their values for predicting prognoses and therapeutic responses in HCC patients. Results: We identified 3 distinct m6A modification patterns in HCC, and each pattern had distinct metabolic characteristics. The evaluation of m6A modification patterns using m6Ascores could predict the prognoses, tumor stages, and responses to sorafenib treatments of HCC patients. A nomogram based on m6Ascores showed high accuracy in predicting the overall survival of patients. The area under the receiver operating characteristic curve of predictions of 1, 3, and 5-year overall survivals were 0.71, 0.69, and 0.70 in the training cohort, and in the test cohort it was 0.74, 0.75, and 0.71, respectively. M6Acluster C1, which corresponded to hypoactive mRNA methylation, lower expression of m6A regulators, and a lower m6Ascore, was characterized by metabolic hyperactivity, lower tumor stage, better prognosis, and lower response to sorafenib treatment. In contrast, m6Acluster C3 was distinct in its hyperactive mRNA methylations, higher expression of m6A regulators, and higher m6Ascores, and was characterized by hypoactive metabolism, advanced tumor stage, poorer prognosis, and a better response to sorafenib. The m6A cluster, C2, was intermediate between C1 and C3. Conclusions: HCCs harbored distinct m6A regulator modification patterns that contributed to the metabolic heterogeneity and diversity of HCC. Development of m6A gene signatures and the m6Ascore provides a more comprehensive understanding of m6A modifications in HCC, and helps predict the prognosis and treatment response.

Aberrant activation of receptor tyrosine kinases (RTKs) and the subsequent metabolic reprogramming play critical roles in cancer progression. Our previous study has shown that Golgi membrane protein 1 (GOLM1) promotes hepatocellular carcinoma (HCC) metastasis by enhancing the recycling of RTKs. However, how this RTK recycling process is regulated and coupled with RTK degradation remains poorly defined. Here, we demonstrate that cholesterol suppresses the autophagic degradation of RTKs in a GOLM1-dependent manner. Further mechanistic studies reveal that GOLM1 mediates the selective autophagy of RTKs by interacting with LC3 through an LC3-interacting region (LIR), which is regulated by a cholesterol-mTORC1 axis. Lowering cholesterol by statins improves the efficacy of multiple tyrosine kinase inhibitors (TKIs) in vivo. Our findings indicate that cholesterol serves as a signal to switch GOLM1-RTK degradation to GOLM1-RTK recycling and suggest that lowering cholesterol by statin may be a promising combination strategy to improve the TKI efficiency in HCC.

Wound healing and tumor progression share some common biological features; however, how variations in wound healing patterns affect hepatocellular carcinoma (HCC) prognosis remains unclear.We analyzed the wound healing patterns of 594 HCC samples from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) and correlated them with immune infiltration and the expression levels of immune checkpoint genes. A risk score, which we named the "heal.immune" score, was established via stepwise Cox estimation. We constructed a nomogram based on age, sex, TNM stage, and heal.immune score and explored its predictive value for HCC prognosis. Seventy-four clinical patients were enrolled in this study, and all were from Huashan Hospital of Fudan University between 2015 and 2017 to serve as an independent validation group.We identified two distinct wound healing patterns in HCC. The biological processes of healing cluster 1 (C1) are related to metabolism, while those of healing cluster 2 (C2) are related to the inflammatory response and immune cell accumulation. A total of 565 wound healing-related genes (based on Gene Ontology) and 25 immune checkpoint genes were considered. By analyzing differentially expressed genes and implementing a stepwise Cox estimation analysis, six genes withWe established a prognostic nomogram based on the heal.immune gene signature, which includes six wound healing- and immunity-related genes. This nomogram accurately predicts the OS of HCC patients.

Background Mitochondrial dynamics plays an important role in tumour progression. However, how these dynamics integrate tumour metabolism in hepatocellular carcinoma (HCC) metastasis is still unclear. Methods The mitochondrial fusion protein mitofusin-1 (MFN1) expression and its prognostic value are detected in HCC. The effects and underlying mechanisms of MFN1 on HCC metastasis and metabolic reprogramming are analysed both in vitro and in vivo. Results Mitochondrial dynamics, represented by constant fission and fusion, are found to be associated with HCC metastasis. High metastatic HCC displays excessive mitochondrial fission. Among genes involved in mitochondrial dynamics, MFN1 is identified as a leading downregulated candidate that is closely associated with HCC metastasis and poor prognosis. While promoting mitochondrial fusion, MFN1 inhibits cell proliferation, invasion and migration capacity both in vitro and in vivo. Mechanistically, disruption of mitochondrial dynamics by depletion of MFN1 triggers the epithelial-to-mesenchymal transition (EMT) of HCC. Moreover, MFN1 modulates HCC metastasis by metabolic shift from aerobic glycolysis to oxidative phosphorylation. Treatment with glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) significantly suppresses the effects induced by depletion of MFN1. Conclusions Our results reveal a critical involvement of mitochondrial dynamics in HCC metastasis via modulating glucose metabolic reprogramming. MFN1 may serve as a novel potential therapeutic target for HCC.

Hepatocellular carcinoma (HCC) is a genetically heterogeneous disease for which a dominant actionable molecular driver has not been identified. Patients with the stem cell–like EpCAM+AFP+ HCC subtype have poor prognosis. Here, we performed a genome-wide RNAi screen to identify genes with a synthetic lethal interaction with EpCAM as a potential therapeutic target for the EpCAM+AFP+ HCC subtype. We identified 26 candidate genes linked to EpCAM/Wnt/β-catenin signaling and HCC cell growth. We further characterized the top candidate PMPCB, which plays a role in mitochondrial protein processing, as a bona fide target for EpCAM+ HCC. PMPCB blockage suppressed EpCAM expression and Wnt/β-catenin signaling via mitochondria-related reactive oxygen species production and FOXO activities, resulting in apoptosis and tumor suppression. These results indicate that a synthetic lethality screen is a viable strategy to identify actionable drivers of HCC and identify PMPCB as a therapeutically vulnerable gene in EpCAM+ HCC subpopulations. Significance: This study identifies PMPCB as critical to mitochondrial homeostasis and a synthetic lethal candidate that selectively kills highly resistant EpCAM+ HCC tumors by inactivating the Wnt/β-catenin signaling pathway.

BACKGROUND As an important aspect of tumor heterogeneity, genetic variation may influence susceptibility and prognosis in different types of cancer. By exploring the prognostic value of genetic variation, this study aimed to establish a model for predicting postoperative survival and assessing the impact of variation on clinical outcomes in patients with hepatocellular carcinoma (HCC). MATERIAL AND METHODS A genome-wide association study of 367 patients with HCC was conducted to identify single nucleotide polymorphisms (SNPs) associated with prognosis. Identified predictors were further evaluated in 758 patients. Two prognostic models were established using Cox proportional hazards regression and Nomogram strategy, and validated in another 316 patients. The effect of the SNP rs2431 was analyzed in detail. RESULTS A prognostic model including 5 SNPs (rs10893585, rs2431, rs34675408, rs6078460, and rs6766361) was established and exhibited high predictive accuracy for HCC prognosis. The panel combined with tumor node metastasis (TNM) stage resulted in a significantly higher c-index (0.723) than the individual c-index values. Stratified by the Nomogram prediction model, the median overall survival for the low-risk and high-risk groups were 100.1 versus 30.8 months (P

ObjectiveIn the tumour microenvironment, critical drivers of immune escape include the oncogenic activity of the tumour cell-intrinsic osteopontin (OPN), the expression of programmed death ligand 1 (PD-L1) and the expansion of tumour-associated macrophages (TAMs). We investigated the feasibility of targeting these pathways as a therapeutic option in hepatocellular carcinoma (HCC) mouse models.DesignWe analysed the number of tumour-infiltrating immune cells and the inflammatory immune profiles in chemically induced liver tumour isolated from wild-type and OPNknockout (KO) mice. In vitro cell cocultures were further conducted to investigate the crosstalk between TAMs and HCC cells mediated by OPN, colony stimulating factor-1 (CSF1) and CSF1 receptor (CSF1R). The in vivo efficacy of anti-PD-L1 and CSF1/CSF1R inhibition was evaluated in OPN overexpressing subcutaneous or orthotopic mouse model of HCC.ResultsThe numbers of TAMs, as well as the expression levels of M2 macrophage markers and PD-L1 were significantly decreased, but the levels of cytokines produced by T-helper 1 (Th1) cells were upregulated in tumour tissues from OPN KO mice compared with that from the controls. In addition, we observed a positive association between the OPN and PD-L1 expression, and OPN expression and TAM infiltration in tumour tissues from patients with HCC. We further demonstrated that OPN facilitates chemotactic migration, and alternative activation of macrophages, and promotes the PD-L1 expression in HCC via activation of the CSF1-CSF1R pathway in macrophages. Combining anti-PD-L1 and CSF1R inhibition elicited potent antitumour activity and prolonged survival of OPNhigh tumour-bearing mice. Histological, flow cytometric and ELISA revealed increased CD8+ T cell infiltration, reduced TAMs and enhanced Th1/Th2 cytokine balance in multiple mouse models of HCC.ConclusionsOPN/CSF1/CSF1R axis plays a critical role in the immunosuppressive nature of the HCC microenvironment. Blocking CSF1/CSF1R prevents TAM trafficking and thereby enhances the efficacy of immune checkpoint inhibitors for the treatment of HCC.

BackgroundFibroblast activation protein (FAP) is commonly expressed in activated stromal fibroblasts in various epithelial tumours. Recently, 68Ga-FAPI-04 has been used for tumour imaging in positron emission tomography/computed tomography (PET/CT). This study aimed to compare the diagnostic performances of 68Ga-FAPI-04 PET/CT and 18F-FDG PET/CT in hepatocellular carcinoma (HCC), and to assess factors associated with 68Ga-FAPI-04 uptake in HCC.Materials and MethodsTwenty-nine patients with suspiciously HCC who received both 18F-FDG and 68Ga-FAPI-04 PET/CT were included in this retrospective study. The results were interpreted by two experienced nuclear medicine physicians independently. The maximum and mean standardized uptake values (SUVmax and SUVmean) were measured in the lesions and liver background, respectively. The tumour-to-background ratio (TBR) was then calculated as lesion’s SUVmax divided by background SUVmean.ResultsA total of 35 intrahepatic lesions in 25 patients with HCC were finally involved in the statistical analysis. 68Ga-FAPI-04 PET/CT showed a higher sensitivity than 18F-FDG PET/CT in detecting intrahepatic HCC lesions (85.7% vs. 57.1%, P = 0.002), including in small (≤ 2 cm in diameter; 68.8% vs. 18.8%, P = 0.008) and well- or moderately-differentiated (83.3% vs. 33.3%, P = 0.031) tumors. SUVmax was comparable between 68Ga-FAPI-04 and 18F-FDG (6.96 ± 5.01 vs. 5.89 ± 3.38, P > 0.05), but the TBR was significantly higher in the 68Ga-FAPI-04 group compared with the 18F-FDG group (11.90 ± 8.35 vs. 3.14 ± 1.59, P < 0.001). SUVmax and the TBR in 68Ga-FAPI-04 positive lesions were associated with tumour size (both P < 0.05), but not the remaining clinical and pathological features (all P > 0.05).Conclusions68Ga-FAPI-04 PET/CT is more sensitive than 18F-FDG PET/CT in detecting HCC lesions, and 68Ga-FAPI-04 uptake is correlated mainly with tumour size.

Background & aim Recently, the clinical trials of lenvatinib plus pembrolizumab in hepatocellular carcinoma (HCC) displayed an impressive objective response rate (ORR). This study aimed to clarify the mechanism for optimal patient selection. Approach & results First, in patients with HCC, lenvatinib-treated recurrent tumors had lower programmed death ligand 1 (PD-L1) expression and regulatory T cell (Treg) infiltration compared with matched primary tumors. Consistently, in C57BL/6 wild-type mice receiving anti-PD-1 therapy, PD-L1 expression and Treg infiltration in subcutaneous tumors reduced when adding lenvatinib to the scheme. Mechanistically, on one hand, fibroblast growth factor receptor 4 (FGFR4) was the most pivotal target in PD-L1 down-regulation by lenvatinib in vitro. Furthermore, lenvatinib reinforced the proteasomal degradation of PD-L1 by blocking the FGFR4-GSK3β axis and rescued the sensitivity of interferon-γ-pretreated HCC cells to T-cell killing by targeting FGFR4. On the other hand, the level of interleukin 2 (IL-2) increased after anti-PD-1 treatment, but IL-2-mediated Treg differentiation was blocked by lenvatinib through targeting FGFR4 to restrain signal transducer and activator of transcription 5 (STAT5) phosphorylation. By regulating the variations in the number of Tregs and tumor FGFR4 level in C57BL/6-Foxp3DTR (DEREG) mice, we found that high levels of FGFR4 and Tregs infiltration sensitized tumor to the combination treatment. Finally, high levels of FGFR4 and forkhead box protein P3 (Foxp3) conferred immune tolerance but better response to the combined therapy in patient cohorts. Conclusions Lenvatinib reduced tumor PD-L1 level and Treg differentiation to improve anti-PD-1 efficacy by blocking FGFR4. Levels of FGFR4 expression and Tregs infiltration in tumor could serve as biomarkers for screening HCC patients with lenvatinib plus anti-PD-1 combination therapy.

Exosomes are a subset of extracellular vesicles that carry specific combinations of proteins, nucleic acids, metabolites, and lipids. Mounting evidence suggests that exosomes participate in intercellular communication and act as important molecular vehicles in the regulation of numerous physiological and pathological processes, including cancer development. Exosomes are released by various cell types under both normal and pathological conditions, and they can be found in multiple bodily fluids. Moreover, exosomes carrying a wide variety of important macromolecules provide a window into altered cellular or tissue states. Their presence in biological fluids renders them an attractive, minimally invasive approach for liquid biopsies with potential biomarkers for cancer diagnosis, prediction, and surveillance. Due to their biocompatibility and low immunogenicity and cytotoxicity, exosomes have potential clinical applications in the development of innovative therapeutic approaches. Here, we summarize recent advances in various technologies for exosome isolation for cancer research. We outline the functions of exosomes in regulating tumor metastasis, drug resistance, and immune modulation in the context of cancer development. Finally, we discuss prospects and challenges for the clinical development of exosome-based liquid biopsies and therapeutics.

Rationale: Metastasis, the development of secondary malignant growth at a distance from a primary tumor, is the main cause of cancer-associated death. However, little is known about how metastatic cancer cells adapt to and colonize in the new organ environment. Here we sought to investigate the functional mechanism of cholesterol metabolic aberration in colorectal carcinoma (CRC) liver metastasis. Methods: The expression of cholesterol metabolism-related genes in primary colorectal tumors (PT) and paired liver metastases (LM) were examined by RT-PCR. The role of SREBP2-dependent cholesterol biosynthesis pathway in cell growth and CRC liver metastasis were determined by SREBP2 silencing in CRC cell lines and experimental metastasis models including, intra-splenic injection models and liver orthotropic injection model. Growth factors treatment and co-culture experiment were performed to reveal the mechanism underlying the up-regulation of SREBP2 in CRC liver metastases. The in vivo efficacy of inhibition of cholesterol biosynthesis pathway by betulin or simvastatin were evaluated in experimental metastasis models. Results: In the present study, we identify a colorectal cancer (CRC) liver metastasis-specific cholesterol metabolic pathway involving the activation of SREBP2-dependent cholesterol biosynthesis, which is required for the colonization and growth of metastatic CRC cells in the liver. Inhibiting this cholesterol biosynthesis pathway suppresses CRC liver metastasis. Mechanically, hepatocyte growth factor (HGF) from liver environment activates SREBP2-dependent cholesterol biosynthesis pathway by activating c-Met/PI3K/AKT/mTOR axis in CRC cells. Conclusion: Our findings support the notion that CRC liver metastases show a specific cholesterol metabolic aberration. Targeting this cholesterol biosynthesis pathway could be a promising treatment for CRC liver metastasis.

Disseminated tumor cells often fall into a long term of dormant stage, characterized by decreased proliferation but sustained survival, in distant organs before awakening for metastatic growth. However, the regulatory mechanism of metastatic dormancy and awakening is largely unknown. Here, we show that the epithelial-like and mesenchymal-like subpopulations of breast cancer stem-like cells (BCSCs) demonstrate different levels of dormancy and tumorigenicity in lungs. The long non-coding RNA (lncRNA) NR2F1-AS1 (NAS1) is up-regulated in the dormant mesenchymal-like BCSCs, and functionally promotes tumor dissemination but reduces proliferation in lungs. Mechanistically, NAS1 binds to NR2F1 mRNA and recruits the RNA-binding protein PTBP1 to promote internal ribosome entry site (IRES)-mediated NR2F1 translation, thus leading to suppression of ΔNp63 transcription by NR2F1. Furthermore, ΔNp63 downregulation results in epithelial-mesenchymal transition, reduced tumorigenicity and enhanced dormancy of cancer cells in lungs. Overall, the study links BCSC plasticity with metastatic dormancy, and reveals the lncRNA as an important regulator of both processes., Disseminated tumor cells often become dormant before awakening for metastatic growth. Here, the authors report that the lncRNA, NR2F1-AS1, is upregulated in dormant mesenchymal-like breast cancer stem-like cells and promotes dissemination but inhibits proliferation, leading to metastatic dormancy.

Background and aims Pyruvate kinase M2 (PKM2) is an essential regulator of the Warburg effect, but its biological function promoting immune escape of hepatocellular carcinoma (HCC) is unclear. Methods GEPIA web tool and immunohistochemistry (IHC) analysis were employed to evaluate the clinical relevance of PKM2 in HCC patients. Both in vitro CCK-8, colony formation, and transwell assays, and in vivo xenografts were performed to evaluate the malignancy of HCC cells. PKM2 and PD-L1 levels were examined by Western blot, qRT-PCR, and IHC. The role of PKM2 on in vivo immune response was also investigated. Results PKM2 was significantly upregulated in HCC and associated with a poor prognosis of HCC patients. Knockdown of PKM2 inhibited in vitro proliferation, migration, and invasion of HCC cells, as well as in vivo tumor growth. Strikingly, PKM2 showed a strong correlation with the expression of immune inhibitory cytokines and lymphocyte infiltration in HCC. The overexpression of PKM2 sensitized HCC to immune checkpoint blockade, which enhanced IFN-γ positive CD8 T cells in HCC mice models. Conclusion PKM2 might be a predictor and a potential therapeutic target for immune checkpoint inhibitors in HCC.

Vasculogenic mimicry (VM), a vessel-like network formed by highly aggressive tumor cells, plays an important role in accelerating cancer progression. This special vascularization pattern is closely associated with a poor prognosis in various cancers. As yet, however, the regulatory mechanism of VM formation is largely unknown. In this study, we assess whether the long noncoding RNA PVT1 is involved in VM generation in gastric cancer. VM formation was determined by immunohistochemistry using PAS/CD31 double staining in gastric cancers and Matrigel tube formation in vitro. qRT-PCR and Western blotting were used to assess mRNA and protein expression. Interaction between PVT1 and STAT3 was determined using a RNA pull-down assay. Luciferase reporter and chromatin immunoprecipitation assays were performed to evaluate transcriptional activity of STAT3 on the Slug gene promoter. We found that PVT1 can induce VM generation both in vitro and in vivo. Mechanistically, we found that PVT1 interacted with and activated STAT3 through a 850–1770 nt fragment. PVT1 facilitated STAT3 recruitment to the Slug promoter and transcriptionally enhanced Slug expression, thereby triggering epithelial-to-mesenchymal transition (EMT) and VM capillary formation. STAT3 inhibition effectively blocked PVT1-mediated VM. In primary gastric cancer samples, a positive correlation was found between PVT1 and Slug upregulation, and patients with a high PVT1 and Slug expression exhibited markedly shorter survival times. Our results shed light on the role of PVT1 in gastric cancer cell-dependent VM formation. Our findings provide valuable clues for the design of new anti-angiogenic therapeutic strategies. The PVT1/STAT3 axis may serve as a potential target in gastric cancer treatment.

Background The propensity of the activated neutrophils to form extracellular traps (NETs) is demonstrated in multiple inflammatory conditions. In this study, we investigated the roles of NETs in metastasis of hepatocellular carcinoma (HCC) and further explored the underlying mechanism of how NETs affect metastasis as well as the therapeutic value. Methods The neutrophils were isolated from the blood of human HCC patients and used to evaluate the formation of NETs. The expression of NET markers was detected in tumor specimens. A LPS-induced NET model was used to investigate the role of NETs on HCC metastasis. RNA-seq was performed to identify the key molecular event triggered by NETs, and their underlying mechanism and therapeutic significance were explored using both in vitro and in vivo assays. Results NET formation was enhanced in neutrophils derived from HCC patients, especially those with metastatic HCCs. NETs trapped HCC cells and subsequently induced cell-death resistance and enhanced invasiveness to trigger their metastatic potential, which was mediated by internalization of NETs into trapped HCC cells and activation of Toll-like receptors TLR4/9-COX2 signaling. Inhibition of TLR4/9-COX2 signaling abrogated the NET-aroused metastatic potential. A combination of DNase 1 directly wrecking NETs with anti-inflammation drugs aspirin/hydroxychloroquine effectively reduced HCC metastasis in mice model. Conclusions NETs trigger tumorous inflammatory response and fuel HCC metastasis. Targeting NETs rather than neutrophils themselves can be a practice strategy against HCC metastasis.

Additional file 1. Supplementary methods.

Additional file 3: Table S1. Clinicopathological characteristics of HCC patients for NETs pathological analysis (n = 104). Table S2. Clinicopathological characteristics of HCC patients for serum MPO-DNA detection (n = 73). Table S3. Real-time PCR primers used in the study. Table S4. Primary antibodies used in the study.

In the hepatocellular carcinoma (HCC) microenvironment, chemokine receptors play a critical role in tumorigenesis and metastasis. Our previous studies have found that osteopontin (OPN) is a promoter for HCC metastasis. However, the role of chemokine receptors in OPN-induced HCC metastasis remains unclear. In this study, we demonstrate that OPN is dramatically elevated in HCC tissues with metastasis and that high expression of OPN correlates with poorer overall survival and higher recurrence rate. OPN upregulates chemokine receptor expression, migration, invasion and pulmonary metastasis in HCC. We find that C-C chemokine receptor type 1 (CCR1) and C-X-C chemokine receptor type 6 (CXCR6) are the most upregulated chemokine receptors induced by OPN. CCR1 knockdown results in reduction of migration, invasion and pulmonary metastasis induced by OPN in vitro and in vivo, whereas CXCR6 knockdown does not reverse OPN-promoted migration and invasion. Moreover, OPN upregulates the expression of CCR1 through activating phosphoinositide 3-kinase (PI3K)/AKT and hypoxia-inducible factor 1α (HIF-1α) in HCC cells. Furthermore, blockade of OPN-CCR1 axis with CCR1 antagonist significantly restrains the promoting effects of OPN on HCC progression and metastasis. In human HCC tissues, OPN expression shows significantly positive correlation with CCR1 expression, and the patients with high levels of both OPN and CCR1 have the most dismal prognosis. Collectively, our results indicate that the OPN-CCR1 axis in HCC is important for accelerating tumor metastasis and that CCR1 is a potential therapeutic target for controlling metastasis in HCC patients with high OPN.