In an analysis of HCC samples from 956 patients, we found almost 25% to express markers of an inflammatory response. We identified 2 subclasses, characterized by adaptive or exhausted immune responses. These findings indicate that some HCCs might be susceptible to therapeutic agents designed to block the regulatory pathways in T cells, such as programmed death-ligand 1, programmed cell death 1, or transforming growth factor beta 1 inhibitors.

HB patients GENOMIC STUDY TRANSCRIPTOMIC STUDY METHYLATION STUDY CytoScan HD ®-array RNA-sequencing/ ddPCR HTA ®-array/ RT-qPCR 850K (EPIC)-array/ QUAlu Dysregulation of global RNA & BLCAP editing Overexpression of 14q32 DLK1-DIO3 genes 16 + VIM-gene signature (C1/C2/C2B) 2 epigenomic HB subtypes (Epi-CA & Epi-CB) CLINICAL PARAMETERS: prognostic marker identification Poor prognostic factors:-4q,-18, 17q11.2 AI (NF1) CHKA new therapeutic target Molecular risk stratification MRS1 MRS2 MRS3 Strong 14q32 Epi-CB Time Survival Highlights Hepatoblastoma (HB) involves global dysregulation of RNA editing, including in the tumor suppressor BLCAP. Overexpression of a 300 kb region within the 14q32 DLK1/DIO3 locus is a new hallmark of HB. We identified 2 epigenomic HB subtypes-Epi-CA and Epi-CB-with distinct degrees of DNA hypomethylation and CpG island hypermethylation. The molecular risk stratification of HB, based on the 14q32-signature and epigenomic subtypes, is associated with patient outcomes. The enzyme CHKA could be a novel therapeutic target for patients with HB.

Interfering with tumor metabolism is an emerging strategy for treating cancers that are resistant to standard therapies. Featuring a rapid proliferation rate and exacerbated glycolysis, hepatocellular carcinoma (HCC) creates a highly hypoxic microenvironment with excessive production of lactic and carbonic acids. These metabolic conditions promote disease aggressiveness and cancer-related immunosuppression. The pH regulatory molecules work as a bridge between tumor cells and their surrounding milieu. Herein, we show that the pH regulatory molecules CAIX, CAXII and V-ATPase are overexpressed in the HCC microenvironment and that interfering with their pathways exerts antitumor activity. Importantly, the V-ATPase complex was expressed by M2-like tumor-associated macrophages. Blocking ex vivo V-ATPase activity established a less immune-suppressive tumor microenvironment and reversed the mesenchymal features of HCC. Thus, targeting the unique cross-talk between tumor cells and the tumor microenvironment played by pH regulatory molecules holds promise as a strategy to control HCC progression and to reduce the immunosuppressive pressure mediated by the hypoxic/acidic metabolism, particularly considering the potential combination of this strategy with emerging immune checkpoint-based immunotherapies.

The onset of cancer is unavoidably accompanied by suppression of antitumor immunity. This occurs through mechanisms ranging from the progressive accumulation of regulatory immune cells associated with chronic immune stimulation and inflammation, to the expression of immunosuppressive molecules. Some of them are being successfully exploited as therapeutic targets, with impressive clinical results achieved in patients, as in the case of immune checkpoint inhibitors. To limit immune attack, tumor cells exploit specific pathways to render the tumor microenvironment hostile for antitumor effector cells. Local acidification might, in fact, anergize activated T cells and facilitate the accumulation of immune suppressive cells. Moreover, the release of extracellular vesicles by tumor cells can condition distant immune sites contributing to the onset of systemic immune suppression. Understanding which mechanisms may be prevalent in specific cancers or disease stages, and identifying possible strategies to counterbalance would majorly contribute to improving clinical efficacy of cancer immunotherapy. Here, we intend to highlight these mechanisms, how they could be targeted and the tools that might be available in the near future to achieve this goal.

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Actual issues during tissue regeneration are to ensure the survival of transplanted cells at the site of their application and further activity, especially in case of local pathological alterations such as inflammation and ischemia. For this purpose, the matrices that can not only fill the defects of tissues, but also be scaffolds for cells are developed.The aim of this study was to evaluate the effectiveness of 3D cultivation of murine adipose-derived multipotent mesenchymal stromal cells (MSMCs) in hydrogel based on carbomer 974P.Materials and methods. MSMCs were obtained from the adipose tissue of FVB-Cg-Tg(GFPU)5Nagy/J mice transgenic for GFP gene. The cells were phenotyped by flow cytometry and directly differentiated into osteogenic and adipogenic direction to confirm multipotent phenotype. MMSCs were cultured and directly differentiated into osteogenic direction in three-dimensional hydrogel scaffolds. For hydrogel preparation we used carbomer 974P with composition of glycerol, propylene glycol, triethylamine and agarose in original proportion.Results. The three-dimensional hydrogel based on carbomer 974P for the further engraftment with MMSCs was obtained. Modified protocols for the preparation of hydrogels based on carbomer and agarose and their rehydration by culture media for the 3D cultivation of adipose-derived MMSCs have been developed. The optimal concentration of MSMCs and the injection method for engraftment of hydrogels of the required form and size are selected. It was shown that adipose-derived MMSCs in 3D carbomer hydrogel preserve the potential of directed osteogenic differentiation.Conclusion. Three-dimensional hydrogel based on carbomer 974P is capable to support cells, provide the necessary cytoarchitectonics, maintain intercellular interactions, which can promote further long-term survival and specialization of graft.

Background. Adipose-derived stem cells (ADSCs) are a promising source for the regeneration of bone tissue injuries. At the same time, three-dimensional cultures provide spatial organization of stem cells for optimal intercellular signaling, contact interaction and increase the efficiency of directed osteogenic differentiation prior to further transplantation. The aim of the study was to establish the regenerative potential of mouse adipose-derived stem cells in micromass grafts differentiated into the osteogenic direction to restore the bone injury in mice. Methods. Three-dimensional micromass cultures of murine ADSCs with further differentiation into osteogenic direction were obtained. The migration potential of cells from micromass in vitro and the effectiveness of differentiation by staining for alkaline phosphatase were evaluated. Mice with the model of femoral bone injury were transplanted with ADSCs micromass grafts and 21 days later the lesion site was examined by histological and morphometric methods. Results. The protocols for the cultivation and directed osteogenic differentiation of ADSCs in the three-dimensional micromass culture have been developed. Alkaline phosphatase production was demonstrated in cells that migrated from micromass, confirming the effectiveness of differentiation. In macroscopic examination 21 days after graft transplantation, the defect sites in femur were filled with dense tissue, while in control bones without the use of transplants, the size of the defect by 80 ± 6 % corresponded to the initial diameter and depth of injury. Histological examination of femoral bone lesions in the area of transplantation of micromass grafts revealed the formation of granulation tissue followed by the replacement of defects with newly formed bone tissue with thickening of periosteum and compact bone substance, similar to callus in fracture regeneration. In animals that underwent transplantation of micromass without prior osteogenic differentiation, the diameter of the zone of active regeneration of the diaphysis at the site of injury was 1.3 ± 0.2 mm while in the group with transplantation of directed differentiated graft it was significantly lower (0.37 ± 0.12 mm, p ≤ 0.05). Conclusions. Three-dimensional grafts of adipose-derived multipotent mesenchymal stromal cells cultured in micromass are able to improve bone tissue regeneration in a model of bone injury in mice. In this case, the grafts differentiated into osteogenic direction, provide better morphological indicators of bone recovery, compared with the micromass without prior differentiation.

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