BioClinicum (Russia)

BackgroundThe von Hippel–Lindau (VHL) gene is frequently mutated in clear cell renal cell carcinoma (ccRCC) which results in stabilization of hypoxia-inducible factor (HIF). Despite the well-known immunosuppressive effect of HIF, ccRCC is considered an immunogenic tumor with high lymphocyte infiltration. Since NK cells have a prognostic value in ccRCC patients, it is important to understand how VHL mutations affect NK cell activity and anti-tumor immunity.MethodsTumor spheroids were generated from parental 786-O (VHL-mutated) and 786-O-pVHL (VHL-restored) ccRCC cell lines. Tumor phenotypes, proteome, and secretome were analyzed by flow cytometry, mass spectrometry, and Luminex assays, respectively. Quantitative proteomics analysis and quantitative gene ontology enrichment were used to correlate protein expression changes to ccRCC progression and immunosuppressive pathways. NK cell infiltration, activation, and cytotoxicity were assessed in co-cultures of ccRCC spheroids with NK cells from healthy donors using real-time imaging, immunostaining, and flow cytometry, respectively.ResultsVHL-mutated tumor spheroids were significantly less infiltrated by NK cells compared with VHL-restored tumor spheroids. pVHL-infiltrating NK cells showed an activated phenotype along with the ability to reduce tumor spheroid size. Proteomic analysis revealed that VHL-restored tumors express reduced levels of proteins associated with ccRCC progression and immunosuppression, including components of MHC class I processing and PD-1 signaling. Furthermore, VHL-restored tumors exhibited decreased levels of hypoxia-related and pro-tumoral cytokines, such as GROα, IL-8, IL-10, TRAIL, VEGF, and SCF. Within 768-O tumor spheroids, NK cells displayed a higher degree of hypoxia and expression of HIF1α, and inhibition of HIF1α resulted in higher NK cell infiltration into 786-O spheroids. Similarly, inhibition of the VHL-target gene, HIF2α, in 786-O spheroids resulted in increased NK cell infiltration.ConclusionsVHL mutant tumors are less infiltrated by NK cells due to immunosuppressive pathways driven by HIF stabilization. Restoration of VHL reprograms the tumor microenvironment, reducing ccRCC progression and immunosuppressive signaling while enhancing NK cell infiltration and activation. Inhibition of HIFα improves NK cell infiltration into VHL mutant tumors. Therefore, inhibition of HIFα should be explored as a therapeutic strategy in ccRCC to improve NK cell anti-tumor efficacy against VHL-mutated tumors.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04132-x.

Abstract Immune matching and rejection pose major hurdles in tissue transplantation. Here, we profileHLA-A,HLA-B, andHLA-Calleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% ofHLA-A, 43.2% ofHLA-B, and 59.2% ofHLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of populations with pre-Neolithic hunter-gatherer ancestry, such as European-American and British groups. We identified 153 double homozygotes and 51 triple homozygotes forHLA-A,HLA-B, andHLA-C. Compatibility modeling showed triple homozygous profiles match 60.5% of Lithuanians (33.3% for double homozygotes), 13.4% of British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disruptHLA-AorHLA-B, while preservingHLA-C, producing edited profiles matching over 98.1% of Lithuanians, 95.8% of European-Americans, and 95.6% of British population. Finally, we established 16 fibroblast lines from double and triple homozygotes, offering a resource for immune-compatibility studies and regenerative medicine applications.

Oral carcinogenetic is based on a variety of genomic imbalances (gross chromosome or specific gene alterations) that drive the normal oral mucosa to its neoplastic/dysplastic epithelial form and finally to a totally malignant tissue transformation. In this multi-step procedure, down-regulation of suppressor genes combined with overactivation of oncogenes are two crucial and partially early genetic events involved in the onset and progression of neoplastic/malignant epithelia transformation. More specifically, deregulation of strong transcription factors negatively affects the normal expression of a broad spectrum of genes that are involved in cell proliferation and signalling transduction to the nucleus. The purpose of the current molecular review was to explore the c-Jun (chromosome location: 1p32-p31) transcription factor transformation mechanisms to oncogene in oral squamous cell carcinoma (OSCC). A systematic review of the literature was carried out by searching in PubMed international database. The year 2010 was set as a prominent time limit for the publication date of the articles in the majority of them, whereas specific references of great importance and historical value in the field of the c-Jun gene discovery and analysis were also included. The following keywords were used: c-Jun, oncogene, signaling pathway, oral, carcinoma, transcription. A pool of 45 important articles were selected for the present study at the basis of combining molecular knowledge with new targeted therapeutic strategies. C-Jun - as a part of the c-Jun/c-Fos transcription factors' complex -critically regulates the expression levels in a variety of genes inside the cellular microenvironment. A broad spectrum of malignancies, including OSCC, demonstrate c-Jun alterations driving the gene to its oncogenic phenotype. Interestingly, c-Jun oncogenic activation is mediated by high-risk human papilloma virus (HR-HPV) persistent infection in significant subsets of these malignancies. C-Jun was the first oncogene - acting as a strong transcription factor - that was discovered and cloned 35 years ago. C-Jun is the living history of oncogenes and its discovery marks a significant step in the evolution of molecular biology.

Emotion recognition and the resulting responses are important for survival and social functioning. However, how socially derived information is processed for reliable emotion recognition is incompletely understood. Here, we reveal an evolutionarily conserved long-range inhibitory/excitatory brain network mediating these socio-cognitive processes. Anatomical tracing in mice revealed the existence of a subpopulation of somatostatin (SOM) GABAergic neurons projecting from the medial prefrontal cortex (mPFC) to the retrosplenial cortex (RSC). Through optogenetic manipulations and Ca2+ imaging fiber photometry in mice and functional imaging in humans, we demonstrate the specific participation of these long-range SOM projections from the mPFC to the RSC, and an excitatory feedback loop from the RSC to the mPFC, in emotion recognition. Notably, we show that mPFC-to-RSC SOM projections are dysfunctional in mouse models relevant to psychiatric vulnerability and can be targeted to rescue emotion recognition deficits in these mice. Our findings demonstrate a cortico-cortical circuit underlying emotion recognition.

Most drugs are metabolized in the liver, which can lead to their activation or inactivation with a change in the parent compound pharmacology, as well as liver damage by active metabolites. Preclinical animal studies of drug safety do not always predict its effect on humans due to species specificity. Thus, for the rapid drug screening, and especially prodrugs, an in vitro system is required that allows predicting xenobiotic cytotoxicity with consideration of their metabolism in liver cells. The use of a microfluidic chip (BioClinicum) made it possible to cultivate a 2D culture of human HaCaT keratinocytes with spheroids of human hepatoma HepaRG cells. After incubation in a specially selected universal serum-free medium containing 3.8 mM cyclophosphamide, pronounced death of HaCaT cells was observed in comparison with culturing in the absence of liver cells.

Structurally similar catalytic subunits A of ricin (RTA) and viscumin (MLA) exhibit cytotoxic activity through ribosome inactivation. Ricin is more cytotoxic than viscumin, although the molecular mechanisms behind this difference are still poorly understood. To shed more light on this problem, we used a combined biochemical/molecular modeling approach to assess possible relationships between the activity of toxins and their structural/dynamic properties. Based on bioassay measurements, it was suggested that the differences in activity are associated with the ability of RTA and MLA to undergo structural/hydrophobic rearrangements during trafficking through the endoplasmic reticulum (ER) membrane. Molecular dynamics simulations and surface hydrophobicity mapping of both proteins in different media showed that RTA rearranges its structure in a membrane-like environment much more efficiently than MLA. Their refolded states also drastically differ in terms of hydrophobic organization. We assume that the higher conformational plasticity of RTA is favorable for the ER-mediated translocation pathway, which leads to a higher rate of toxin penetration into the cytoplasm.

While head and neck squamous cell carcinomas (HNSCC) are marginally decreasing due to the reduction in exposure to the major risk factors, tobacco and alcohol, the incidence of high-risk human papillomavirus (HPV)-positive oropharynx squamous cell carcinomas (OPSCC), especially those in the tonsil and base of tongue subsites, are increasing. Patients with the latter are younger, display a longer overall survival, and show a lower recurrence rate after standard-of-care treatment than those with HPV-negative OPSCC. This may reflect an important role for immune surveillance and control during the natural history of the virally driven tumour development. Immune deviation through acquisition of immune-suppressive factors in the tumour microenvironment (TME) is discussed in relation to treatment response. Understanding how the different immune factors are integrated in the TME battleground offers opportunities for identifying prognostic biomarkers as well as novel therapeutic strategies. OPSCC generally receive surgery or radiotherapy for early-stage tumour treatment, but many patients present with locoregionally advanced disease requiring multimodality therapies which can involve considerable complications. This review focuses on the utilization of newly emerged immune checkpoint inhibitors (PD-1/PD-L1 pathway) for treatment of HNSCC, in particular HPV-positive OPSCC, since they could be less toxic and more efficacious. PD-1/PD-L1 expression in the TME has been extensively investigated as a biomarker of patient response but is yet to provide a really effective means for stratification of treatment. Extensive testing of combinations of therapeutic approaches by types and sequencing will fuel the next evolution of treatment for OPSCC.