Biology subjects

Marko-Varga, G.

Publications and source records attributed to Marko-Varga, G..

11 recordsLinked to original sources

bioRxiv2026

From Routine Pathology to Precision Oncology: Automated FFPE Tissue Processing for Large-Scale Molecular Studies

Melanoma incidence continues to rise globally, with formalin-fixed paraffin-embedded (FFPE) tissue archives representing an invaluable resource for large-scale retrospective proteomic studies. However, inconsistent deparaffinization remains a critical pre-analytical bottleneck limiting protein yield, reproducibility, and downstream data quality. In this study, we developed and validated a fully automated FFPE deparaffinization workflow using the Fluent(R) 780 liquid handling workstation (Tecan (C)) and evaluated its performance against a conventional manual protocol in a cohort of 54 patients with primary cutaneous melanoma, predominantly at early AJCC 8th edition stage I-II. The automated workflow achieved superior protein identification (6,146 {+/-} 860 vs. 4,941 {+/-} 1,091 proteins; p < 0.0001) with lower technical variability, while maintaining highly comparable global proteomic profiles as confirmed by principal component analysis and hierarchical clustering. A total of 8,305 proteins (96.1%) were identified by both methods, supporting the reproducibility and equivalence of the automated approach. Patients were stratified by the presence (N=21) or absence (N=33) of histological regression in the primary tumor. Proteomic comparison revealed 97 upregulated and 226 downregulated proteins in regressing melanomas, with pathway enrichment analysis demonstrating elevated mitochondrial and translational activity alongside reduced innate immune and complement pathway activation in the regression group. No statistically significant differences in overall, disease-free, or progression-free survival were observed between groups, consistent with the early-stage composition of the cohort. Digital pathology validated tissue morphology preservation across processing conditions. These findings support the integration of automated FFPE processing with proteomic and digital pathology workflows as a scalable platform for precision melanoma research. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/744404v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1a3ea5dorg.highwire.dtl.DTLVardef@6fb8edorg.highwire.dtl.DTLVardef@d60a4dorg.highwire.dtl.DTLVardef@3e47b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Proteomics-Enhanced AI-Digital Pathology in Metastatic Mucinous Colorectal Carcinoma: A Case Report

Mucinous colorectal carcinoma (CRC) is a distinct histomorphological subtype characterized by abundant extracellular mucin that may promote immune evasion and chemoresistance. We describe a metastatic mucinous CRC case integrating digital pathology and proteomics to investigate disease progression and therapy resistance. Formalin-fixed paraffin-embedded samples from the primary tumor, peritoneal metastasis, and hepatoduodenal ligament metastasis of a 56-year-old patient were analyzed. Whole-slide imaging with QuPath-based AI enabled detailed histological annotation, while data-independent acquisition mass spectrometry identified over 6,000 proteins. Digital pathology revealed extensive mucin pools, architectural evolution from heterogeneous glandular patterns in the primary tumor to cribriform morphology in advanced metastases, and immune cell exclusion from mucin-rich regions. Proteomics revealed metabolic reprogramming, suppressed antigen presentation, and stage-specific activation of inflammatory, angiogenic, EMT, and PI3K/AKT/mTOR-MYC signaling pathways, consistent with proliferative and therapy-resistant phenotypes.Integration of AI-assisted histopathology with spatial proteomics highlighted the mucin barrier as a key mediator of immune evasion and chemoresistance. These findings support a personalized therapeutic framework targeting mucin-associated mechanisms alongside pathway-directed inhibitors, suggesting that spatial multi-omics may guide precision management strategies for aggressive mucinous colorectal cancer.

pathology↗

bioRxiv2025

Phosphoproteomic profiling reveals post-translational dysregulation in Huntington's disease patient-derived neurons

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene. Although transcriptomic and proteomic changes have been characterized in patient-derived neurons, the contribution of post-translational modifications (PTMs), such as phosphorylation, remains poorly understood. Here, we present the first phosphoproteomic analysis by mass spectrometry (P-MS) of human induced neurons (iNs) directly reprogrammed from HD patient fibroblasts. We identified 177 phosphopeptides with significantly altered abundance in HD-iNs, mapping to phosphoproteins associated with key signaling pathways known to be affected in HD, such as splicing and autophagy. By integrating P-MS data with previously published proteomic and transcriptomic data from the same donors, we identified distinct subsets of ON-OFF phosphopeptides that exhibited a complete loss of phosphorylation in either HD- or Ctrl-iNs, without corresponding changes at the RNA or protein level. An exception was MXRA8, previously described in glial cells as a mediator of blood-brain barrier integrity and astrocyte-mediated neuroinflammation. This protein showed increased protein abundance despite the absence of phosphorylation in HD- iNs, suggesting a compensatory mechanism - a pattern also observed in human post mortem cortical HD tissue. Additionally, MXRA8 showed altered protein-protein interactions with lysosomal and metabolic regulators in HD-iNs, highlighting its potential role in autophagy impairment as well as in neurovascular dysfunction. These findings uncover a distinct layer of post-translational dysregulation in HD, suggesting that phospho-switch proteins such as MXRA8 may be candidate effectors of pathology and thus site-specific phosphorylation loss may contribute to impaired signaling and proteostasis in human HD neurons.

neuroscience↗

bioRxiv2025

A proteomic study of the dual oncogenic and tumor- suppressive roles of SIRT3 in lung and breast cancer cell lines

Mitochondria play a crucial role in metabolism and energy production by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They also help maintain intracellular calcium levels, facilitate communication between the nucleus and cytoplasm, detoxify reactive oxygen species (ROS), and regulate apoptosis (programmed cell death). Reversible acetylation of mitochondrial proteins is a key post-translational modification influencing these processes, with the NAD+-dependent deacetylase SIRT3 being a major regulator. While SIRT3 has been described as a tumor suppressor in some contexts and as a tumor promoter in others, its role appears to be tissue- and metabolism-specific. Here, we compared the proteomic and acetylomic responses of lung adenocarcinoma (A549) and breast adenocarcinoma (MCF7) cell lines to SIRT3 inhibition by 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP). The two lines were selected based on distinct metabolic phenotypes and reported differences in basal SIRT3 abundance. Total proteome and mitochondrial-enriched fractions were analyzed separately for each cell line to avoid cross-line normalization bias. We identified 6,457 proteins and 4,199 acetylated peptides, revealing divergent pathway enrichments and acetylation patterns following SIRT3 inhibition. A549 cells showed enrichment of oxidative metabolism pathways, whereas MCF7 cells exhibited features consistent with metabolic reprogramming. These findings suggest that the cellular context determines the proteomic consequences of SIRT3 modulation and highlight the importance of metabolism in shaping SIRT3-associated phenotypes. All raw mass spectrometry data are publicly available in [repository accession number]. SignificanceSIRT3s impact on cancer metabolism is context-dependent. By separately profiling lung (A549) and breast (MCF7) cancer cells, we demonstrate that SIRT3 inhibition leads to distinct proteomic and acetylomic outcomes in each cell type. Our findings underscore the need for cell-type-specific strategies when targeting SIRT3 in cancer therapy and provide an open-access proteomic resource for further mechanistic studies.

cancer biology↗

bioRxiv2024

Predicting immune checkpoint therapy response in three independent metastatic melanoma cohorts

While Immune checkpoint inhibition (ICI) therapy shows significant efficacy in metastatic melanoma, only about 50% respond, lacking reliable predictive methods. We introduce a panel of six proteins aimed at predicting response to ICI therapy. Evaluating previously reported proteins in two untreated melanoma cohorts, we used a published predictive model (EaSIeR score) to identify potential proteins distinguishing responders and non-responders. Six proteins initially identified in the ICI cohort correlated with predicted response in the untreated cohort. Additionally, three proteins correlated with patient survival, both at the protein, and at the transcript levels, in an independent immunotherapy treated cohort. Our study identifies predictive biomarkers across three melanoma cohorts, suggesting their use in therapeutic decision-making. O_FIG O_LINKSMALLFIG WIDTH=164 HEIGHT=200 SRC="FIGDIR/small/592032v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@754668org.highwire.dtl.DTLVardef@1616263org.highwire.dtl.DTLVardef@827009org.highwire.dtl.DTLVardef@1899ba7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Histopathology-assisted proteogenomics provides foundations for stratification of melanoma metastases

Here we describe the histopathology-driven proteogenomic landscape of 142 treatment-naive metastatic melanoma samples. We identified five proteomic subtypes that integrate the immune and stroma microenvironment components, and associate with clinical and histopathological parameters, providing foundations for an in-depth molecular classification of melanoma. Our study shows that BRAF V600 mutated melanomas display heterogeneous biology, where the presence of an oncogene-induced senescence-like phenotype improves patient survival. Therefore, we propose a mortality-risk-based stratification, which may contribute to a more personalized approach to patient treatment. We also found a strong association between tumor microenvironment composition, disease progression, and patient outcome supported by single-cell omic signatures that point to straightforward histopathological connective tissue-to-tumor ratio assessment for better informed medical decisions. A melanoma-associated signature of single amino acid variants (SAAV) responsible for remodeling the extracellular matrix was uncovered together with SAAV-derived neoantigen candidates as targets of anti-tumor immune responses. Overall, this study offers comprehensive stratifications of melanoma metastases that may help develop tailored strategies for diagnosing and treating the disease.

cancer biology↗

bioRxiv2022

Proteogenomic Characterization Reveals Therapeutic Opportunities Related to Mitochondrial Function in Melanoma

The dynamics of more than 1900 mitochondrial proteins was explored through quantitative proteomics in 151 melanoma-related tissue samples of both surgical and autopsy origin. Dysregulation of mitochondrial pathways in primary tumors, metastases, and peritumoral tissues was correlated with age and survival of patients, as well as with tumor cell proliferation and the BRAF mutation status of the tumors. The outlined proteomic landscape confirmed the central role of a pathologically upregulated mitochondrial translation machinery and oxidative phosphorylation (OXPHOS) in the development, proliferation, and progression of melanomas. Our results from different melanoma cell lines confirmed our findings and we could document that treatments with selected OXPHOS inhibitors and antibiotics successfully impaired tumor cell proliferation. In addition, we provided proteomic evidence on the mechanism-of-action of the different treatments. These observations could contribute to the development of therapeutic approaches targeting the mitochondrial pathology in melanoma. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/513481v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@11f21e2org.highwire.dtl.DTLVardef@e98c64org.highwire.dtl.DTLVardef@100145forg.highwire.dtl.DTLVardef@399284_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMitochondrial proteome landscape outlined in 151 melanoma-related samples C_LIO_LIMitochondrial Translation and OXPHOS impact disease severity and survival C_LIO_LIBRAF V600E mutation correlates with upregulation of mitochondrial energy production C_LIO_LITargeting the mitochondrial OXPHOS and ribosomes impairs tumor cell proliferation C_LIO_LITherapeutic opportunities complementary to the standard of care are proposed C_LI In briefMitochondrial proteome profiling of melanomas reveals dysregulation in major metabolic pathways, suggesting a central role of the mitochondria within the development and progression of melanoma. Targeting mitochondrial pathways has the potential to impact the course of the disease, which provides opportunities for complementary drug interventions.

cancer biology↗

bioRxiv2022

Age-associated Differences in the Human Lung Extracellular Matrix

IntroductionExtracellular matrix (ECM) remodelling has been associated with chronic lung diseases. However, information about specific age-associated differences in lung ECM is currently limited. In this study we aimed to identify and localize age-associated ECM differences in human lung using comprehensive transcriptomic, proteomic and immunohistochemical analyses. MethodsOur previously identified age-associated gene expression signature of the lung was re-analysed limiting it to an aging signature based on 270 control patients (37-80 years) and focused on the Matrisome core geneset using geneset enrichment analysis. To validate the age-associated transcriptomic differences on protein level, we compared the age-associated ECM genes (F <0.05) with a profile of age-associated proteins identified from a lung tissue proteomics dataset from 9 control patients (49-76 years) (FDR<0.05). Extensive immunohistochemical analysis was used to localize the age-associated ECM differences in lung tissues from control patients (9-82 years). ResultsComparative analysis of transcriptomic and proteomic data identified 7 ECM proteins with higher expression with age at both gene and protein level: COL1A1, COL6A1, COL6A2, COL14A1, FBLN2, LTBP4 and LUM. With immunohistochemistry we demonstrated higher protein expression with age for COL6A2 in whole tissue, parenchyma, airway wall and blood vessel, for COL14A1 in bronchial epithelium and blood vessel, and for FBLN2 and COL1A1 in lung parenchyma. ConclusionOur study revealed that higher age is associated with lung ECM remodelling, with specific differences occurring in defined regions within the lung. These differences may affect lung structure and physiology with aging and as such may increase susceptibility for developing chronic lung diseases. Key messagesO_ST_ABSWhat is already known on this topicC_ST_ABSsummarise the state of scientific knowledge on this subject before you did your study and why this study needed to be done. In animal models, it has been demonstrated that aging alters the composition of the lung ECM, with more deposition of collagen and degradation of elastin. Similar ECM differences have been observed in age-associated chronic lung diseases, including COPD; moreover, we observed in lung tissue that several ECM genes associate differently with age in COPD patients compared to non-COPD controls(1). Detailed knowledge on age-associated changes in specific ECM proteins as well as regional differences within the lung is lacking. What this study addssummarise what we now know as a result of this study that we did not know before. We identified 7 age-associated ECM proteins i.e. COL1A1, COL6A1, COL6A2 COL14A1, FBLN2, LTBP4 and LUM with higher transcript and protein levels in human lung tissue with age. Extensive immunohistochemical analysis revealed significant age-associated differences for 3 of these ECM proteins in specific compartments of the lung, with the most notable differences in the blood vessels and parenchyma. How this study might affect research, practice, or policysummarise the implications of this study. The identification of age-associated differences in specific human lung ECM proteins lays a new foundation for the investigation of ECM differences in age-associated chronic lung diseases. Additionally, examining the function of these age-associated ECM proteins and their cellular interactions in lung injury and repair responses may provide novel insight in mechanisms underlying chronic lung diseases.

molecular biology↗

bioRxiv2021

Proteogenomics Reveals how Metastatic Melanoma Modulates the Immune System to Allow Immune Evasion

Malignant melanoma (MM) develops from the melanocytes and in its advanced stage is the most aggressive type of skin cancer. Here we report a comprehensive analysis on a prospective cohort study, including non-tumor, primary and metastasis tissues (n=77) with the corresponding plasma samples (n=56) from patients with malignant melanoma. The tumors and surrounding tissues were characterized with a combination of high-throughput analyses including quantitative proteomics, phosphoproteomics, acetylomics, and whole exome sequencing (WES) combined with in-depth histopathology analysis. Melanoma cell proliferation highly correlates with dysregulation at the proteome, at the posttranslational- and at the transcriptome level. Some of the changes were also verified in the plasma proteome. The metabolic reprogramming in melanoma includes upregulation of the glycolysis and the oxidative phosphorylation, and an increase in glutamine consumption, while downregulated proteins involved in the degradation of amino acids, fatty acids, and the extracellular matrix (ECM) receptor interaction. The pathways most dysregulated in MM including the MAP kinases-, the PI3K-AKT signaling, and the calcium homeostasis, are among the most affected by mutations, thus, dysregulation in these pathways can be manifested as drivers in melanoma development and progression. The phosphoproteome analysis combined with target-based prediction mapped 75% of the human kinome. Melanoma cell proliferation was driven by two key factors: i) metabolic reprogramming leading to upregulation of the glycolysis and oxidative phosphorylation, supported by HIF-1 signaling pathway and mitochondrial translation; and ii) a dysregulation of the immune system response, which was mirrored by immune system processes in the plasma proteome. Regulation of the melanoma acetylome and expression of deacetylase enzymes discriminated between groups based on tissue origin and proliferation, indicating a way to guide the successful use of HDAC inhibitors in melanoma. The disease progression toward metastasis is driven by the downregulation of the immune system response, including MHC class I and II, which allows tumors to evade immune surveillance. Altogether, new evidence is provided at different molecular levels to allow improved understanding of the melanoma progression, ultimately contributing to better treatment strategies. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/439245v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@937db9org.highwire.dtl.DTLVardef@1230b57org.highwire.dtl.DTLVardef@1bd889eorg.highwire.dtl.DTLVardef@547d90_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Distinct sub-cellular autophagy impairments occur independently of protein aggregation in induced neurons from patients with Huntington's disease

Huntingtons disease (HD) is a neurodegenerative disorder caused by CAG expansions in the huntingtin (HTT) gene. Modelling HD has remained challenging, as rodent and cellular models poorly recapitulate the disease. To address this, we generated induced neurons (iNs) through direct reprogramming of human skin fibroblasts, which retain age-dependent epigenetic characteristics. HD-iNs displayed profound deficits in autophagy, characterised by reduced transport of late autophagic structures from the neurites to the soma. The neurite-specific alterations in autophagy resulted in shorter, thinner and fewer neurites presented by HD-iNs. CRISPRi-mediated silencing of HTT did not rescue this phenotype but rather resulted in additional autophagy alterations in ctrl-iNs, highlighting the importance of wild type HTT in neuronal autophagy. In summary, our work identifies a distinct subcellular autophagy impairment in aged patient derived HD-neurons and provides a new rational for future development of autophagy activation therapies.

neuroscience↗

bioRxiv2019

Post-transcriptional mechanisms distinguish human and chimp forebrain progenitor cells

The forebrain has expanded in size and complexity during hominoid evolution. The contribution of post-transcriptional control of gene expression to this process is unclear. Using in-depth proteomics in combination with bulk and single-cell RNA sequencing, we analyzed protein and RNA levels of almost 5,000 genes in human and chimpanzee forebrain neural progenitor cells. We found that species differences in protein expression level was often independent of RNA levels, and more frequent than transcriptomic differences. Low-abundant proteins were more likely to show species-specific expression levels, while proteins expressed at a high level appeared to have evolved under stricter constraints. Our study implicates a previously unappreciated broad and important role for post-transcriptional regulatory mechanisms in the evolution of the human forebrain.

neuroscience↗