CDK4/6 inhibition enhances T-cell immunotherapy on hepatocellular carcinoma cells by rejuvenating immunogenicity.

Hepatocellular carcinoma (HCC) poses a significant clinical challenge, necessitating the integration of immunotherapeutic approaches. Palbociclib, a selective CDK4/6 inhibitor, has demonstrated promising efficacy in preclinical HCC models and is being evaluated as a novel therapeutic option in clinical trials. Additionally, CDK4/6 inhibition induces cellular senescence, potentially influencing the tumor microenvironment and immunogenicity of cancer cells. In this study, we conducted comprehensive bioinformatic analyses using diverse HCC transcriptome datasets, including bulk and single-cell RNA-sequencing data from public databases. We also utilized human and mouse HCC cells to investigate functional aspects. Primary T cells isolated from mouse blood were employed to assess T cell immunity against HCC cells. Results revealed that CD8+ T-cell infiltration correlates with improved outcomes in HCC patients with suppressed CDK4/6 expression. Moreover, CDK4/6 expression was associated with alterations in the immune landscape and immune checkpoint expression within the liver tumor microenvironment. Furthermore, we found that treatment with Palbociclib and Doxorubicin induces cellular senescence and a senescence-associated secretory phenotype in HCC cells. Notably, pretreatment with Palbociclib augmented T cell-mediated cytotoxicity against HCC cells, despite upregulation of PD-L1, surpassing the effects of Doxorubicin pretreatment. In conclusion, our study elucidates a novel mechanism by which CDK4/6 inhibition enhances T-cell-associated cancer elimination and proposes a potential therapeutic strategy to enhance T-cell immunotherapy on HCC.

Adaptive T-cell immunity controls senescence-prone MyD88- or CARD11-mutant B-cell lymphomas.

Aberrant B-cell receptor/NF-κB signaling is a hallmark feature of B-cell non-Hodgkin lymphomas, especially in diffuse large B-cell lymphoma (DLBCL). Recurrent mutations in this cascade, for example, in CD79B, CARD11, or NFKBIZ, and also in the Toll-like receptor pathway transducer MyD88, all deregulate NF-κB, but their differential impact on lymphoma development and biology remains to be determined. Here, we functionally investigate primary mouse lymphomas that formed in recipient mice of Eµ-myc transgenic hematopoietic stem cells stably transduced with naturally occurring NF-κB mutants. Although most mutants supported Myc-driven lymphoma formation through repressed apoptosis, CARD11- or MyD88-mutant lymphoma cells selectively presented with a macrophage-activating secretion profile, which, in turn, strongly enforced transforming growth factor ß (TGF-ß)-mediated senescence in the lymphoma cell compartment. However, MyD88- or CARD11-mutant Eµ-myc lymphomas exhibited high-level expression of the immune-checkpoint mediator programmed cell death ligand 1 (PD-L1), thus preventing their efficient clearance by adaptive host immunity. Conversely, these mutant-specific dependencies were therapeutically exploitable by anti-programmed cell death 1 checkpoint blockade, leading to direct T-cell-mediated lysis of predominantly but not exclusively senescent lymphoma cells. Importantly, mouse-based mutant MyD88- and CARD11-derived signatures marked DLBCL subgroups exhibiting mirroring phenotypes with respect to the triad of senescence induction, macrophage attraction, and evasion of cytotoxic T-cell immunity. Complementing genomic subclassification approaches, our functional, cross-species investigation unveils pathogenic principles and therapeutic vulnerabilities applicable to and testable in human DLBCL subsets that may inform future personalized treatment strategies.

circNFATC3 sponges miR-548I acts as a ceRNA to protect NFATC3 itself and suppressed hepatocellular carcinoma progression.

Circular RNAs (circRNA) have been reported as regulators involved in hepatocellular carcinoma (HCC), but their mechanism of activity remains unknown. This study performed quantitative reverse-transcription polymerase chain reaction to determine if circNFATC3 was downregulated in 46 paired HCC tissues and cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptotic, and transwell assay proved that circNFATC3 can inhibit hepatoma cell proliferation, apoptosis, and migration/invasion in vitro. Mouse xenograft assay demonstrated that circNFATC3 suppressed tumor size and weight and reduced lung metastasis in vivo, and vice versa. The RNA-seq results showed that NFATC3 itself was the most significantly differentially expressed gene when circNFATC3 was manipulated, and bioinformatics and luciferase reporter assays verified circNFATC3 regulated the expression of NFATC3 by interacting with the hsa-miR-548I. Additionally, it was also indicated that the level of NFATC3 was downregulated in HCC patients also and was significantly correlated with the staging and prognosis of HCC. Moreover, both circNFATC3 and NFATC3 were shown to inhibit the phosphorylation of JNK, c-Jun, AKT, and mTOR signaling pathways. Overall, the circNFATC3 can sponge miR-548I to protect NFATC3 itself, then it regulates hepatoma cell function via the JNK, c-Jun, AKT, and mTOR signaling pathways, and the circNFATC3 can be a tumor-repressor on HCC.

Endoplasmic reticulum stress induced LOX-1+ CD15+ polymorphonuclear myeloid-derived suppressor cells in hepatocellular carcinoma.

A recent study indicated that Lectin-type oxidized LDL receptor-1 (LOX-1) was a distinct surface marker for human polymorphisms myeloid-derived suppressor cells (PMN-MDSC). The present study was aimed to investigate the existence LOX-1 PMN-MDSC in hepatocellular carcinoma (HCC) patients. One hundred and twenty-seven HCC patients, 10 patients with mild active chronic hepatitis B, 10 liver cirrhosis due to hepatitis B, 10 liver dysplastic node with hepatitis B and 50 health control were included. LOX-1+  CD15+ PMN-MDSC were significantly elevated in HCC patients compared with healthy control and patients with benign diseases. LOX-1+  CD15+ PMN-MDSC in circulation were positively associated with those in HCC tissues. LOX-1+  CD15+ PMN-MDSCs significantly reduced proliferation and IFN-γ production of T cells with a dosage dependent manner with LOX-1-  CD15+ PMNs reached negative results. The suppression on T cell proliferation and IFN-γ production was reversed by ROS inhibitor and Arginase inhibitor. ROS level and activity of arginase of LOX-1 + CD15+ PMN were higher in LOX-1+  CD15+ PMN-MDSCs than LOX-1-  CD15+ PMNs, as well as the expression of the NADPH oxidase NOX2 and arginase I. RNA sequence revealed that LOX-1+ CD15+ PMN-MDSCs displayed significantly higher expression of spliced X-box -binding protein 1 (sXBP1), an endoplasmic reticulum (ER) stress marker. ER stress inducer induced LOX-1 expression and suppressive function for CD15+ PMN from health donor. For HCC patients, LOX-1+  CD15+ PMN-MDSCs were positively related to overall survival. Above all, LOX-1+  CD15+ PMN-MDSC were elevated in HCC patients and suppressed T cell proliferation through ROS/Arg I pathway induced by ER stress. They presented positive association with the prognosis of HCC patients.

Cholangiokines: undervalued modulators in the hepatic microenvironment.

The biliary epithelial cells, also known as cholangiocytes, line the intra- and extrahepatic bile ducts, forming a barrier between intra- and extra-ductal environments. Cholangiocytes are mostly known to modulate bile composition and transportation. In hepatobiliary diseases, bile duct injury leads to drastic alterations in cholangiocyte phenotypes and their release of soluble mediators, which can vary depending on the original insult and cellular states (quiescence, senescence, or proliferation). The cholangiocyte-secreted cytokines (also termed cholangiokines) drive ductular cell proliferation, portal inflammation and fibrosis, and carcinogenesis. Hence, despite the previous consensus that cholangiocytes are bystanders in liver diseases, their diverse secretome plays critical roles in modulating the intrahepatic microenvironment. This review summarizes recent insights into the cholangiokines under both physiological and pathological conditions, especially as they occur during liver injury-regeneration, inflammation, fibrosis and malignant transformation processes.

Cellular Senescence in Hepatocellular Carcinoma: The Passenger or the Driver?

With the high morbidity and mortality, hepatocellular carcinoma (HCC) represents a major yet growing burden for our global community. The relapse-prone nature and drug resistance of HCC are regarded as the consequence of varying intracellular processes and extracellular interplay, which actively participate in tumor microenvironment remodeling. Amongst them, cellular senescence is regarded as a fail-safe program, leading to double-sword effects of both cell growth inhibition and tissue repair promotion. Particularly, cellular senescence serves a pivotal role in the progression of chronic inflammatory liver diseases, ultimately leading to carcinogenesis. Given the current challenges in improving the clinical management and outcome of HCC, senescence may exert striking potential in affecting anti-cancer strategies. In recent years, an increasing number of studies have emerged to investigate senescence-associated hepatocarcinogenesis and its derived therapies. In this review, we intend to provide an up-to-date understanding of liver cell senescence and its impacts on treatment modalities of HCC.

The macrophage-associated microRNA-4715-3p / Gasdermin D axis potentially indicates fibrosis progression in nonalcoholic fatty liver disease: evidence from transcriptome and biological data.

Nonalcoholic fatty liver disease (NAFLD) is highly possible to progress to cirrhosis, malignancy, and liver failure through fibrogenesis. The enormous potential of pathogenetic and therapeutic targets in NAFLD has been revealed. This study aimed to explore novel factors potentially indicating or mediating NAFLD progression. Multiple bulk and single-cell RNA sequencing datasets were used, in which landscapes of cell populations were clarified to characterize immune cell infiltration. Significantly high infiltration of macrophages (MPs) was discovered during NAFLD progression. Samples in bulk NASH datasets were regrouped by MP level. Highly differentially expressed genes (DEGs) were identified in the Ctrl vs. NASH comparison, low MP vs. high MP comparison, and the weighted gene co-expression network analysis (WGCNA) clusters. Eight hub genes were identified as promising targets by protein-protein interaction analysis and validated in fibrosis progression, microRNA (miR)-protein interactions were predicted, and the hub genes were verified in a free fatty acid (FFA)-induced macrophage injury model. The results showed that Gasdermin D (GSDMD) was upregulated with fibrosis progression in NAFLD and was associated with macrophage infiltration. In addition, a potential regulator (miR-4715-3p) was correlated with GSDMD. The miR-4715-3p/GSDMD axis potentially modulates macrophage-associated immunity and indicates fibrosis progression in NAFLD.

Efficacy of cytokine-induced killer cell-based immunotherapy for hepatocellular carcinoma.

In attempts to delay tumor progression after surgery or minimally invasive local treatments, multidisciplinary strategies have been broadly studied in patients with hepatocellular carcinoma (HCC). The objective of this present study was to evaluate the efficacy of autologous transplantations of cytokine-induced killer (CIK) cells as an adjuvant therapy for patients with HCC. A total of 264 patients with HCC were enrolled in this retrospective study. Of these patients, 165 received either CIK cell therapy alone or as adjuvant therapy to surgery, transcatheter arterial chemoembolization (TACE), or TACE-based comprehensive treatments (CT). The remaining 99 patients received only surgery or TACE. Kaplan-Meier analysis and the Chi-squared test were used to analyze the overall survival (OS), progression-free survival (PFS), and clinical characteristics of the patients in the different treatment subgroups. Kaplan-Meier analysis suggested that patients in the Surgery+CIK group had a significantly improved OS compared with those in the other three groups (P < 0.001). Furthermore, patients who developed a fever after the CIK cell treatments manifested a likely better OS (P = 0.028). Subgroup analysis indicated that patients in the Surgery+CIK group likely had an improved PFS but a similar OS compared with the patients in the Surgery-alone group (P = 0.055 for PFS, and P = 0.746 for OS). Further subgroup analysis showed that the OS in both the TACE+CIK and CT+CIK groups was prolonged significantly compared with that in the TACE-alone group (P = 0.015 and P = 0.018, respectively). However, similar OS was observed between the TACE+CIK and CT+CIK groups (P = 0.686). Autologous transplantation of CIK cells as an adjuvant therapy was associated with better survival for patients with HCC, especially for those who had also undergone TACE. A fever reaction might be a potential event for assessing the curative effect of the CIK treatment.

The Predictive Value of Albumin-to-Alkaline Phosphatase Ratio for Overall Survival of Hepatocellular Carcinoma Patients Treated with Trans-Catheter Arterial Chemoembolization Therapy.

Background: We have previously reported the prognostic value of the albumin-to-alkaline phosphatase ratio (AAPR) for advanced hepatocellular carcinoma (HCC) patients who are not receiving any standard anticancer therapy. However, the prognostic value of the AAPR for HCC patients treated with trans-catheter arterial chemoembolization therapy (TACE) was not investigated. Methods: We retrospectively analysed 372 HCC patients treated with TACE (the training cohort) and applied receiver operating characteristic curves (ROC curves) to identify the best cut-off value for the AAPR in this cohort. Then, univariate analyses by the Kaplan-Meier method and multivariate analysis by a Cox proportional hazards regression model were conducted. Both comparisons of the ROC curves and the likelihood ratio test (LRT) were employed to evaluate the abilities of different factors in predicting the survival of patients in this cohort. Finally, the prognostic value of the AAPR was validated in two cohorts: one included 202 HCC patients treated with supportive care (validation cohort I), and the other included 82 HCC patients treated with TACE (validation cohort II). Results: We identified 0.439 as the best cut-off value of the AAPR by ROC curve analysis. An AAPR > 0.439 was significantly correlated with a lower frequency of Child-Pugh grade B, portal vein tumour thrombus (PVTT), T3-4 and lymph node metastasis (P < 0.05). The median overall survival (OS) of the patients with an AAPR > 0.439 was significantly longer than that of those with an AAPR ≤ 0.439 (58.4 m vs 17.8 m, respectively, P < 0.001). The AAPR was identified as an independent prognostic factor after univariate and multivariate analyses (HR = 0.636, P = 0.003). The independent prognostic value of the AAPR was also confirmed in validation cohorts I and II. Additionally, we substituted the AAPR for the Child-Pugh grade in the CLIP system and integrated the AAPR into the TNM system. We found that the area under the curve (AUC) of the AAPR-CLIP system was significantly larger than that of the CLIP and the TNM when predicting 3-month, 6-month, 1-year and 2-year survival (P < 0.05). There was no significant difference between the AUCs for the AAPR-CLIP and the AAPR-TNM. The LRT suggested that both AAPR-CLIP and AAPR-TNM had significantly larger χ2 values and smaller AIC values than that of their corresponding primary system (P < 0.05). Conclusions: The AAPR was an independent prognostic index for the HCC patients treated with TACE. Both AAPR-CLIP and AAPR-TNM outperformed their corresponding primary system in predicting OS in the current study.

Modified CLIP score with the albumin-bilirubin grade retains prognostic value in HBV-related hepatocellular carcinoma patients treated with trans-catheter arterial chemoembolization therapy.

Background: The Cancer of the Liver Italian Program (CLIP) score is commonly used for prognosis prediction of hepatocellular carcinoma (HCC). The CLIP includes the Child-Pugh grade, which is relatively subjective, for hepatic encephalopathy assessment. A newly developed scoring system called albumin-bilirubin grade (ALBI grade), consists of albumin and bilirubin to assess liver function reserve objectively. Here, we substituted the ALBI grade for the Child-Pugh grade to establish the ALBI-CLIP scoring system and validated its prognostic value in hepatitis B virus (HBV)-related HCC patients treated with trans-catheter arterial chemoembolization (TACE) therapy. Methods: We retrospectively analyzed HBV-related HCC patients who received TACE therapy. Baseline characteristics were collected and evaluated to classify patients according to ALBI-CLIP, CLIP and TNM systems. Univariate analyses using the Kaplan-Meier method and the log-rank test, as well as multivariate analysis using the Cox proportional hazards regression model, were conducted to detect independent prognostic factors for overall survival. Receiver operating characteristic (ROC) curves and a likelihood ratio test (LRT) were both utilized to compare the values of ALBI-CLIP, CLIP and TNM staging systems in predicting survival. Results: With a total of 389 patients included in the current study, 301 (77.4%) and 88 (22.6%) were classified as Child-Pugh grade A and B, respectively. However, 152 (39.1%), 227 (58.4%) and 10 (2.5%) patients were correspondingly classified into ALBI grade 1, 2 and 3. The areas under the curves of ALBI-CLIP, CLIP and TNM systems were 0.804, 0.778 and 0.734, respectively, for predicting 3-month survival; 0.796, 0.778 and 0.733, respectively, for 6-month survival; 0.697, 0.687 and 0.644, respectively, for 1-year survival; and 0.618, 0.612 and 0.569, respectively, for 2-year survival. The LRT indicated that the ALBI-CLIP and the CLIP had similar values of χ2 and Akaike information criterion (AIC) while the TNM system had the smallest χ2 value (χ2 = 12.1, 11.9, 10.5; AIC = 2620.2, 2620.5, 2621.1 for ALBI-CLIP, CLIP and TNM, respectively). Conclusions: In conclusion, our present study suggested that the ALBI-CLIP scoring system retained the prognostic value of the CLIP in HBV-related HCC treated with TACE therapy.

Albumin-to-Alkaline Phosphatase Ratio as an Independent Prognostic Factor for Overall Survival of Advanced Hepatocellular Carcinoma Patients without Receiving Standard Anti-Cancer Therapies.

Background Albumin-to-Alkaline Phosphatase Ratio (ALB/ALP ratio, AAPR), a newly developed index of liver function, has been rarely discussed about its prognostic value in malignancies. The current study attempted to evaluate the prognostic prediction of AAPR in advanced HCC. Methods 237 advanced HCC patients who refused any standard anti-cancer therapies were retrospectively analyzed. The threshold value of AAPR was determined by receiver operating characteristic (ROC) curve. Univariate analyses using Kaplan-Meier method and log-rank test, and multivariate analysis using Cox proportional hazards regression model were conducted. Comparisons of ROC curves and likelihood ratio test (LRT) were utilized to compare the value of different factors in predicting survival. Results ROC curve analysis confirmed 0.38 as the optimal cutoff value of AAPR in evaluating overall survival (OS). Patients with an AAPR > 0.38 exhibited significantly lower frequencies of ascites, portal vein tumor thrombus, Child-Pugh grade B & C, and KPS < 70 (all P < 0.05). These patients also displayed a longer median survival time than those with an AAPR ≤ 0.38 (5.8 m vs 2.4 m, P < 0.01). Univariate and multivariate analyses identified AAPR as an independent prognostic indicator (HR = 0.592, P = 0.007). Furthermore, we integrated AAPR with TNM system and found that area under curve of AAPR-TNM system was significantly larger than that of TNM system when predicting 3-month survival (0.670 vs 0.611, P < 0.01). Moreover, LRT indicated that AAPR-TNM system had a significantly larger χ2 (26.4 vs 16.4, P < 0.01) and a significantly smaller Akaike information criterion value (1936 vs 1948, P < 0.01) comparing with TNM system. Conclusions Our study implied that AAPR was a potentially valuable prognostic index for advanced HCC patients without receiving any standard anti-cancer therapies. AAPR-TNM system preceded TNM system in predicting overall survival in this study.

Autologous transplantation of cytokine-induced killer cells as an adjuvant therapy for hepatocellular carcinoma in Asia: an update meta-analysis and systematic review.

BACKGROUND: High recurrence rate after curative treatment is the major problem for hepatocellular carcinoma (HCC). Cytokine-induced killer cells (CIKs) therapy was extensively studied among HCC patients. However, the value of CIKs therapy was controversial. A meta-analysis was performed to investigate the efficacy of adjuvant CIKs after invasive treatments among HCC patients. METHODS: We searched online for literatures studying sequential CIKs therapy for HCC patients. Recurrence-free survival (RFS), progress-free survival (PFS) and overall survival (OS) were set as the main endpoints. Both overall and subgroup analysis were accomplished. RESULTS: A total of 12 clinical trials with 1,387 patients were included. The pooled analysis showed a significant improvement of RFS, PFS and OS in CIK group (HR 0.56, 95% CI 0.47-0.67, p<0.00001 for RFS; HR 0.53, 95% CI 0.40-0.69, p<0.00001 for PFS; HR 0.59, 95% CI 0.46-0.77, p<0.0001 for OS). The proportion of CD4+ T cells increased significantly, while CD8+ T cells decreased significantly after CIKs therapy (WMD 4.07, 95% CI 2.58-5.56, p<0.00001; WMD -2.84, 95% CI -4.67 to -1.01, p=0.002, respectively). No significant differences of adverse events between CIK and non-CIK group existed. CONCLUSIONS: Conventionally invasive therapies combined with CIKs therapy could improve the prognosis of HCC patients, especially for RFS and PFS, with mild side effects. Optimizing patient selection shall be the direction in future studies.

Identification of the prognostic value of lymphocyte-to-monocyte ratio in patients with HBV-associated advanced hepatocellular carcinoma.

The inflammatory microenvironment serves an important function in the progression of hepatocellular carcinoma (HCC). Peripheral blood lymphocyte-to-monocyte ratio (LMR), as a novel inflammatory biomarker combining an estimate of host immune homeostasis with the tumor microenvironment, has been identified to be a predictor of clinical outcomes in a number of malignancies. The present study aimed at investigating the prognostic value of LMR in patients with hepatitis B virus (HBV)-associated advanced HCC. A total of 174 patients with HBV-associated advanced HCC, without fever or signs of infections, were analyzed. Clinicopathological parameters, including LMR, were evaluated to identify predictors of overall survival time. Univariate and multivariate analysis was performed using Cox's proportional hazards model. A threshold value was determined using a time-dependent receiver operating characteristic curve. Univariate and multivariate analysis identified LMR as an independent prognostic factor in overall survival (OS) time in patients with HBV-associated advanced HCC (P<0.05). The threshold value of LMR was 2.22. All patients were divided into either a low LMR group (≤2.22) or a high LMR group (>2.22). The OS time of the high LMR group was significantly longer compared with the low LMR group (P<0.001). Patients in the high LMR group exhibited a significantly increased 3-month and 6-month OS rate, compared with that of the patients within the low LMR group (P<0.001). An increased level of LMR was significantly associated with the presence of metastasis, ascites and increased tumor size (P<0.01). LMR is an independent prognostic factor of HBV-associated advanced HCC patients and an increased baseline LMR level indicates an improved prognosis.