Biology subjects

Roudko, V.

Publications and source records attributed to Roudko, V..

8 recordsLinked to original sources

bioRxiv2025

Integrative Omics Identifies Candidate Plasma Biomarkers and Cellular Targets Associated with Thoracic Aortic Aneurysm

ObjectivesTo define the cellular landscape of thoracic aortic aneurysm (TAA) and identify circulating biomarkers associated with disease burden. BackgroundTAA is marked by progressive aortic wall weakening and dilation, predisposing to rupture and dissection. However, its cellular architecture remains incompletely defined, and reliable circulating biomarkers are lacking. MethodsSingle-cell RNA sequencing was performed on 17 aortic tissue samples from 10 patients undergoing TAA repair and integrated with publicly available datasets to characterize disease-associated cell states. In parallel, tomographic imaging and plasma proteomics were used to identify biomarkers associated with aortic diameter. Key findings were further assessed through integration with single-cell data, external validation, and in vitro stimulation of primary human adventitial fibroblasts with fibroblast growth factor 23 (FGF-23). ResultsWe identified 25 cellular subsets, including macrophages, endothelial cells, vascular smooth muscle cells, and fibroblasts, with substantial heterogeneity in cellular composition and transcriptional state. Genome-wide association study candidate genes, including JUN and TPM3, showed cell type-specific upregulation. Plasma proteomics identified multiple biomarkers associated with aortic diameter, of which FGF-23 was independently validated in the UK Biobank as elevated in individuals with TAA. FGFR1, the receptor for FGF-23, was selectively expressed in fibroblasts and subsets of vascular smooth muscle cells, with strongest downstream signaling in fibroblasts. FGF-23 stimulation induced inflammatory and extracellular matrix remodeling programs in primary human adventitial fibroblasts. ConclusionsThese findings define the cellular landscape of TAA and identify the FGF-23-FGFR1 axis as a biomarker-linked pathway that may contribute to aneurysm progression. Condensed AbstractThoracic aortic aneurysm (TAA) is characterized by progressive aortic dilation and risk of rupture, yet its cellular architecture and circulating biomarkers remain incompletely defined. We performed single-cell RNA sequencing on 17 aortic samples from 10 patients and integrated these data with public datasets to define the cellular landscape of TAA. In parallel, imaging and plasma proteomics (n=10) were used to identify biomarkers associated with aortic diameter. We identified 25 cellular subsets, including macrophages, endothelial cells, vascular smooth muscle cells, and fibroblasts, with notable transcriptional heterogeneity and cell type-specific upregulation of GWAS-associated genes (e.g., JUN, TPM3). Plasma proteomics identified fibroblast growth factor 23 (FGF-23) as associated with aortic diameter and elevated in TAA in the UK Biobank. FGFR1, its receptor, was selectively expressed in fibroblasts and VSMCs, and FGF-23 stimulation induced inflammatory and extracellular matrix remodeling programs in fibroblasts. These findings link a circulating biomarker to stromal cell signaling in TAA. HighlightsO_LIIntegrative single-cell RNA sequencing defined a diverse cellular landscape in thoracic aortic aneurysm tissue, identifying 25 distinct cell populations. C_LIO_LICross-dataset harmonization revealed marked differences in cellular composition across studies while supporting shared stromal and immune programs in thoracic aortic aneurysm. C_LIO_LIPlasma proteomics identified FGF-23 as a candidate biomarker associated with aortic diameter and independently linked to thoracic aortic aneurysm presence in the UK Biobank C_LIO_LIFGFR1 was enriched in fibroblasts and subsets of vascular smooth muscle cells, and FGF-23 induced inflammatory and extracellular matrix remodeling programs in human primary adventitial fibroblasts. C_LI

cell biology↗

bioRxiv2025

MARQO pipeline resolves multiparametric cellular and spatial organization in cancer tissue lesions

Multiplex immunostaining analysis remains fragmented, underperforming, and labor-intensive despite tissue proteomic methodologies achieving ever-increasing marker complexity. Here we propose an open-source, semi-supervised automated pipeline that streamlines start-to-finish, single-cell resolution analysis of whole-slide tissue, named Multiplex-imaging Analysis, Registration, Quantification, and Overlaying (MARQO). We compared and validated MARQO using Multiplex Immunohistochemical Consecutive Staining on a Single Slide (MICSSS) using human tumor and adjacent normal tissue samples. Performance was compared with manually-curated pathologist determinations and quantification of multiple markers. We also optimized MARQO to analyze diverse tissue sizes (whole tissue, biopsy, tissue microarray) and staining approaches (singleplex immunohistochemistry, 20-color multiplex immunofluorescence) to determine marker co-expression patterns in multiple human solid cancer types. Lastly, we validated CD8 T cell enrichment in hepatocellular carcinoma responders to neoadjuvant cemiplimab in a phase II clinical trial, further demonstrating MARQOs ability to provide spatially-resolved in situ mechanisms by providing multiplex whole-slide single-cell resolution data.

immunology↗

bioRxiv2024

T cell epitope mapping reveals immunodominance of evolutionarily conserved regions within SARS-CoV-2 proteome.

As SARS-CoV-2 variants continue to emerge capable of evading neutralizing antibodies, it has become increasingly important to fully understand the breadth and functional profile of T cell responses to determine their impact on the immune surveillance of variant strains. Here, sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains of SARS-CoV-2 suggested that epitope immunodominance and cross-reactivity are major predictive determinants of T cell immunity. To identify immunodominant epitopes across the viral proteome, we generated a comprehensive map of CD4+ and CD8+ T cell epitopes within non-spike proteins that induced polyfunctional T cell responses in convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by mutations in emerging variants. Conservation analysis across historical human coronaviruses combined with in silico alanine scanning mutagenesis of non-spike proteins underscored the functional importance of mutationally-constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the mutationally-constrained SARS-CoV-2 proteome, potentially providing immune surveillance against emerging variants, and inform the design of next-generation vaccines targeting antigens throughout SARS-CoV-2 proteome for broader and more durable protection. One Sentence SummaryPolyfunctional CD8+ and CD4+ T cells directed against SARS-CoV-2 target mutationally constrained regions of the viral proteome.

immunology↗

bioRxiv2024

Informing biologically relevant signal from spatial transcriptomic data

Visium is a spatial sequencing technology that utilizes messenger RNA (mRNA) to spatially map gene expression within tissues. Despite its potential, research utilizing deconvolution tools and exploring microenvironment dynamics remains challenging. We address this gap by benchmarking deconvolution tools across diverse biological contexts, identifying optimal methodologies. Subsequently, we introduce a novel pipeline integrating advanced deconvolution techniques and novel tools for reproducible tissue microenvironment analysis. Through this approach, we uncover intricate immune aggregate biology, highlighting the power of our methodology in unraveling complex biological phenomena.

bioinformatics↗

bioRxiv2024

Functional impairment of "helpless" CD8+ memory T cells is transient and driven by prolonged but finite cognate antigen presentation

Generation of functional CD8+ T cell memory typically requires engagement of CD4+ T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (TE) and subsequent memory (TM) CD8+ T cell formation appear impervious to a lack of CD4+ T cell help during priming. Nonetheless, such "helpless" CD8+ TM respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8+ T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8+ TE differentiation is largely normal but a multiplicity of helpless CD8 TM defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8+ TM potential and recall capacity. Our findings provide a unified explanation for helpless CD8+ T cell memory and emphasize an unexpected CD8+ TM plasticity with implications for vaccination strategies and beyond.

immunology↗

bioRxiv2021

Perturb-map enables CRISPR genomics with spatial resolution and identifies regulators of tumor immune composition

The cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGF{beta} expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGF{beta} on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

immunology↗

bioRxiv2020

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain prostate cancer cell fitness

Cancer cells are differentially dependent on the splicing machinery compared to normal untransformed cells. The splicing machinery thus represents a potential therapeutic target in cancer. To identify splicing factors important for prostate cancer cell (PCa) cell growth, we performed a parallel pooled shRNA screen on in vitro passaged cells and in vivo xenografted PCa tumor lines. Our screen revealed HNRNPM as a potential regulator of PCa cell growth. RNA- and eCLIP-sequencing data suggest that HNRNPM is bound to transcripts of key homeostatic genes and that loss of HNRNPM binding in a subset of these genes results in aberrant exon inclusion and exon back-splicing events in target transcripts. In both linear and circular mis-spliced transcripts, HNRNPM appears to preferentially bind to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM dependent linear splicing events using splice-switching antisense oligonucleotides (SSOs) was sufficient to inhibit cell growth in HNRNPM expressing cells. This suggests that prostate cancer cell dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Taken together, our data reveal a role for HNRNPM in supporting prostate cancer cell fitness, and also as a potential therapeutic target in PCa.

genomics↗

bioRxiv2019

Widespread immunogenic poly-epitope frameshift mutations in microsatellite unstable tumors

Microsatellite instability-high (MSI-H) tumors are an important model system for evaluating neoantigen-based immunotherapies given their high tumor mutation burden and response to checkpoint blockade. We identified tumor-specific, frameshift peptides, encoding multiple epitopes that originated from indel mutations shared among patients with MSI-H endometrial, colorectal and stomach cancers. Epitopes derived from these shared frameshifts have high population occurrence rates, wide presence in many tumor subclones and are predicted to bind to the most frequent HLA alleles in the TCGA MSI-H patient cohorts. Neoantigens arising from these mutations are more dissimilar to both self and viral antigens, indicating the creation of peptides, that, when translated, can present truly novel antigens to the immune system. Finally, we validated the immunogenicity of common frameshift peptides from MSI-H endometrial patients in an array of T cell stimulation experiments, using peripheral blood mononuclear cells isolated from healthy donors. Our study describes for the first time the widespread occurrence and strong immunogenicity of tumor-specific antigens, derived from shared frameshift mutations in MSI-H cancer and Lynch syndrome patients, suitable for the design of common preventive \"off-the-shelf\" cancer vaccines.

cancer biology↗