3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFßRII-SMADS pathway.

BACKGROUND: Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. RESULTS: In vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFß-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFßRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFßRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFßRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis. CONCLUSIONS: Our results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFßRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.

Prognostic 7-SLC-Gene Signature Identified via Weighted Gene Co-Expression Network Analysis for Patients with Hepatocellular Carcinoma.

Background: Solute carrier (SLC) proteins play an important role in tumor metabolism. But SLC-associated genes' prognostic significance in hepatocellular carcinoma (HCC) remained elusive. We identified SLC-related factors and developed an SLC-related classifier to predict and improve HCC prognosis and treatment. Methods: From the TCGA database, corresponding clinical data and mRNA expression profiles of 371 HCC patients were acquired, and those of 231 tumor samples were derived from the ICGC database. Genes associated with clinical features were filtered using weighted gene correlation network analysis (WGCNA). Next, univariate LASSO Cox regression studies developed SLC risk profiles, with the ICGC cohort data being used in validation. Result: Univariate Cox regression analysis revealed that 31 SLC genes (P < 0.05) were related to HCC prognosis. 7 (SLC22A25, SLC2A2, SLC41A3, SLC44A1, SLC48A1, SLC4A2, and SLC9A3R1) of these genes were applied in developing a SLC gene prognosis model. Samples were classified into the low-andhigh-risk groups by the prognostic signature, with those in the high-risk group showing a significantly worse prognosis (P < 0.001 in the TCGA cohort and P=0.0068 in the ICGC cohort). ROC analysis validated the signature's prediction power. In addition, functional analyses showed enrichment of immune-related pathways and different immune status between the two risk groups. Conclusion: The 7-SLC-gene prognostic signature established in this study helped predict the prognosis, and was also correlated with the tumor immune status and infiltration of different immune cells in the tumor microenvironment. The current findings may provide important clinical indications for proposing a novel combination therapy consists of targeted anti-SLC therapy and immunotherapy for HCC patients.

Establishment of a prognostic model based on m6A regulatory factors and stemness of hepatocellular carcinoma using RNA-seq data and scRNA-seq data.

BACKGROUND: Hepatocellular carcinoma (HCC) with high incidence and mortality is one of the most common malignant cancers worldwide. Increasing evidence has reported that N6-methyladenosine (m6A) modification has been considered as a major contribution to the occurrence and development of tumors. METHOD: In our study, we comprehensively analyzed the connection between m6A regulatory factors and cancer stem cells (CSCs) of HCC to establish a clinical tool for predicting its outcome. First, we concluded that the expression level of m6A regulatory factors was related with the stemness of hepatocellular carcinoma. Subsequently, we gained a ten hub regulatory factors that were associated with prognosis of hepatocellular carcinoma by overall survival (OS) analysis using ICGC and TCGA datasets, and these regulatory factors included YTHDF1, IGF2BP1, METTL3, IGF2BP3, HNRNPA2B1, IGF2BP2, RBM15B, HNRNPC, RBMX, and LRPPR. Next, we found that these ten hub m6A regulatory factors were highly expressed in CSCs, and CSCs related pathways were also enriched by the gene set variation analysis (GSVA). Then, correlation, consensus clustering and PCA analysis were performed to reveal potential therapeutic benefits of HCC. Moreover, univariate Cox regression (UNICOX), LASSON and multivariate Cox regression (MULTICOX) analyses were adopted to establish HCC prognosis prediction signature. RESULTS: Four regulatory factors RBM15B, LRPPRC, IGF2BP1, and IGF2BP3 were picked as valuable prognostic indicators. CONCLUSION: In summary, these ten hub regulatory factors would be useful therapeutic targets for HCC treatment, and RBM15B/LRPPRC/IGF2BP1/IGF2BP3 prognostic indicators can be used to guide therapy for HCC patients.

Effective management of advanced colon cancer genotyping microsatellite stable/microsatellite instable-low with Kirsten rat sarcoma viral oncogene mutation using nivolumab plus ipilimumab combined with regorafenib and irinotecan: A case report.

Microsatellite stable /microsatellite instable-low is the most common colorectal cancer genotype, counting for approximately 85% of common colorectal cancer patients. Treatment of advanced microsatellite stable/microsatellite instable-low colorectal cancer is difficult and successful pharmacological treatment options are currently lacking. Here, we report a case of a 37-year-old man with advanced colorectal cancer genotyping microsatellite stable/microsatellite instable-low with a Kirsten rat sarcoma viral oncogene (G12V) mutation. Following palliative surgery, the patient did not response to the common recommended chemotherapy FOLFIRI regimen and other chemotherapy options. Finally, the patient was successfully treated using a unique combinational immunotherapy, using nivolumab plus ipilimumab combined with regorafenib and irinotecan. Significant improvement in the Karnofsky Performance Status scores, liver function and well-being, reduction in serum tumor biomarkers, and reduction in the size of multiple liver metastatic tumors was evident. This report provides a rare case in which a unique and effective combinational immunotherapy for refractory advanced colon cancer patients is discussed. It encourages further research into combined immunotherapy for immuno-insensitive colon cancer patients.

Establishment of a 3D model of tumor-driven angiogenesis to study the effects of anti-angiogenic drugs on pericyte recruitment.

Hepatocellular carcinoma (HCC), as a well-vascularized tumor, has attracted increasing attention in antiangiogenic therapies. Notably, emerging studies reveal that the long-term administration of antiangiogenic drugs induces hypoxia in tumors. Pericytes, which play a vital role in vascular stabilization and maturation, have been documented to be associated with antiangiogenic drug-induced tumor hypoxia. However, the role of antiangiogenic agents in regulating pericyte behavior still remains elusive. In this study, by using immunostaining analysis, we first demonstrated that tumors obtained from HCC patients were highly angiogenic, in which vessels were irregularly covered by pericytes. Therefore, we established a new 3D model of tumor-driven angiogenesis by culturing endothelial cells, pericytes, cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) with microcarriers in order to investigate the effects and mechanisms exerted by antiangiogenic agents on pericyte recruitment during tumor angiogenesis. Interestingly, microcarriers, as supporting matrices, enhanced the interactions between tumor cells and the extracellular matrix (ECM), promoted malignancy of tumor cells and increased tumor angiogenesis within the 3D model, as determined by qRT-PCR and immunostaining. More importantly, we showed that zoledronic acid (ZA) reversed the inhibited pericyte recruitment, which was induced by sorafenib (Sora) treatment, through fostering the expression and activation of ErbB1/ErbB2 and PDGFR-ß in pericytes, in both an in vitro 3D model and an in vivo xenograft HCC mouse model. Hence, our model provides a more pathophysiologically relevant platform for the assessment of therapeutic effects of antiangiogenic compounds and identification of novel pharmacological targets, which might efficiently improve the benefits of antiangiogenic treatment for HCC patients.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin ß1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.