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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2026

Chemoproteomic Characterization of GPX4 Covalent Ligands and Targeted Degradation

Despite intensive efforts, the ferroptosis gatekeeper glutathione peroxidase 4 (GPX4) remains difficult to selectively target due to stringent structural constraints surrounding its catalytic selenocysteine, which impose tight requirements on warhead reactivity and geometry. Here, leveraging a chemoproteomic approach, we characterize a potent and selective covalent GPX4 inhibitor featuring a pyrimidinylmethyl isourea warhead and define the chemical features underlying its proteome-wide selectivity. This chemotype enables tunable electrophile reactivity through steric and electronic modulation of leaving group ability, suggesting potential broader utility for targeting other recalcitrant proteins. Building on this scaffold, we further develop two selective GPX4 degraders - one CRBN-dependent and the other CRBN-independent - enabling complementary modulation of GPX4 through both inhibition and degradation. Together, these molecules expand the GPX4 chemical toolbox for more nuanced interrogation of GPX4 biology.

cancer biology↗

bioRxiv2018

Comprehensive analysis of miRNA and protein profiles within exosomes derived from canine lymphoid tumour cell lines

Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the miRNAs and proteins abundantly contained in exosomes were similar among the four cell lines. However, the profiles of miRNA within exosomes differed among the cell lines and reflected the expression pattern of miRNAs of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.

cancer biology↗

bioRxiv2026

Alternative Lengthening of Telomeres and CINSARC are interconnected toward non-translocation-related sarcomas progression

Alternative lengthening of telomeres (ALT) is a telomere elongation mechanism activated during oncogenesis and primarily acting in tumors of mesenchymal origin. Although the proteins involved in the machinery enabling ALT to elongate telomeres are becoming better understood, the underlying biology of this mechanism remains unclear. In the present study, we took advantage of a fully characterized cohort of 98 leiomyosarcomas (LMS) from the French Sarcoma Group to further our understanding of the ALT mechanism. We first compared the transcriptomic profiles of ALT+ and TERT+ LMS and demonstrated a strong enrichment of the CINSARC signature in ALT+ tumors. The establishment of an ALT+-related signature in these LMS confirmed the close association between CINSARC and ALT in two additional cohorts of non-translocation-related sarcomas. In vitro mesenchymal models of spontaneous ALT induction showed increased CINSARC expression following acquisition of the ALT mechanism. Conversely, ALT inactivation, through BLM inhibition, led to decreased CINSARC expression. These results establish CINSARC as a new hallmark of the ALT mechanism in non-translocation-related sarcomas and demonstrate the association of a cellular biological process with the CINSARC prognostic signature, namely the ALT mechanism.

cancer biology↗

bioRxiv2023

A comprehensive rRNA variation atlas in health and disease

Ribosomal DNA and RNA (rDNA and rRNA) sequences are usually discarded from sequencing analyses. But with hundreds of copies of rDNA genes it is unknown whether they possess sequence variations that form different types of ribosomes that affect human physiology and disease. Here, we developed an algorithm for variant-calling between paralog genes (termed RGA) and compared rDNA variations found in short- and long-read sequencing data from the 1,000 Genomes Project (1KGP) and Genome In A Bottle (GIAB). We additionally developed a novel protocol for long-read sequencing full-length rRNA (RIBO-RT) from actively translating ribosomes. Our analyses identified hundreds of rDNA variants, most of which, surprisingly, are short insertion-deletions (indels) and dozens of highly abundant rRNA variants that are incorporated into translationally active ribosomes. To visualize variant ribosomes at the single cell level, we developed an in-situ rRNA sequencing method (SWITCH-seq) which revealed that variants are co-expressed within individual cells. Strikingly, by analyzing rDNA, we found that variants assemble into distinct ribosome subtypes. We discovered that these subtypes acquire different rRNA structures by successfully employing dimethyl sulfate (DMS) probing of full length rRNA. With this atlas we investigated rRNA variation changes across human tissues and cancer types. This revealed tissue-specific rRNA subtype expression in endoderm/ectoderm-derived tissues. In cancer, low abundant rRNA variants can become highly expressed, which suggests the presence of cancer-specific ribosomes. Together, this study identifies and comprehensively characterizes the diversity of ribosomes at the level of rRNA variants which is dominated by indel variants, their chromosomal location and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

genetics↗

bioRxiv2025

Spatially organized lymphocytic microenvironments in high grade primary prostate tumors

The spatial arrangement of immune cells in the tumor microenvironment (TME) varies widely, from dispersed to clustered and tumor excluded to infiltrating. Multiplexed spatial profiling is an effective means of characterizing tumor-infiltrating lymphocytes (TILs) and immune complexes such as tertiary lymphoid structures (TLS) in the TME. However, few approaches have been described for objectively parametrizing patterns of immune organization and assessing their association with biological or clinical variables. This makes it difficult to evaluate whether a set of tumors is relatively immunologically cold or hot. Here we describe an intuitive set of statistical tools (available in the R package, tlsR) for characterizing lymphocyte patterns in the TME of solid cancers. We apply tlsR to primary prostate cancer (PCa), which is often described as immunologically cold. Using a cohort of 29 radical prostatectomy specimens stratified into low Gleason-grade (LGG; n=15) and high Gleason-grades (HGG; n =14) we show that HGG PCa is significantly more infiltrated than LGG PCa with lymphocytes organized into B cell or T cell enriched immune clusters (BICs and TICs). A subset of these ICs have the B and T cell zonation and follicular dendritic cells characteristic of a bona fide TLS. HGGs are also enriched with ICs containing precursor exhausted T cells (Tpex) and proliferating B cells and their tumor compartments harbor granzyme-B+ cytotoxic T cells in contact with cancer cells. Thus, far from being cold, a subset of HGG PCa has features associated with active immune surveillance, a finding with implications for emerging PCa immunotherapies.

cancer biology↗

bioRxiv2025

Comparison of volumetric dynamic optical coherence tomography with biological methods for evaluation of radiation effects in prostate tumor spheroids

Significance3D tumor spheroids are more physiologically representative of in vivo patient tumors compared to 2D monolayer culture. However, their 3D nature challenges the use of conventional biological techniques like proliferation assays, fluorescence microscopy, and the clonogenic assay, which is the gold standard method for assessing cell survival following radiation. However, clonogenic assay requires spheroid disaggregation. AimNon-invasive volumetric imaging with dynamic optical coherence tomography (dOCT) enables cellular activity to be visualized with spatial resolution within 3D tumor spheroids. Cellular activity observed via dOCT in irradiated prostate tumor spheroids was quantified for comparison with conventional biological techniques. ApproachA Varian TrueBeam linear accelerator was used to irradiate spheroid and monolayer cultures with a 6 MV beam. Cellular activity was estimated from dOCT images generated via frequency banding and compared to clonogenic assay, proliferation assay, fluorescence microscopy, and 3D cell simulation. ResultsProstate cancer cells cultured as spheroids demonstrated improved radio-resistance via clonogenic assay compared to monolayer culture. The dOCT method demonstrated quantitative and qualitative agreement with proliferation assay and fluorescence microscopy, respectively. ConclusionsA longer duration of repeated dOCT measurement in tumor spheroids following radiation treatment could offer a non-invasive alternative to the clonogenic assay.

cancer biology↗

bioRxiv2019

Integrated Phosphoproteomics and Transcriptional Classifiers Reveal Hidden RAS Signaling Dynamics in Multiple Myeloma

A major driver of multiple myeloma is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employ an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in multiple myeloma to inform precision medicine strategies. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. Transcriptional analysis suggests that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs. WT RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes whereas RAS mutations overall carry no survival impact. We further develop an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. These results may lead to improved stratification of MM patients in clinical trials while also revealing unexplored modes of Ras biology.

cancer biology↗

bioRxiv2021

Emerging single cell endothelial heterogeneity supports sprouting tumour angiogenesis and growth

Blood vessels supplying tumors are often dysfunctional and generally heterogeneous. The mechanisms underlying this heterogeneity remain poorly understood. Here, using multicolor lineage tracing, in vivo time-lapse imaging and single cell RNA sequencing in a mouse glioma model, we identify tumour-specific blood endothelial cells that originate from cells expressing the receptor for colony stimulating factor 1, Csf1r, a cytokine which controls macrophage biology. These Csf1r lineage endothelial cells (CLECs) form up to 10% of the tumour vasculature and express, besides classical blood endothelial cell markers, a gene signature that is distinct from brain endothelium but shares similarities with lymphatic endothelial cell populations. in silico analysis of pan-cancer single cell RNAseq datasets highlights the presence of a comparable subpopulation in the endothelium of a wide spectrum of human tumours. We show that CLECs actively contribute to sprouting and remodeling of tumour blood vessels and that selective depletion of CLECs reduces vascular branching and tumour growth. Our findings indicate that a non-tumour resident Csf1r-positive population is recruited to tumours, differentiates into blood endothelial cells to contribute to vascularization and, thereby, tumour growth.

cancer biology↗

bioRxiv2022

Trade-off between conservation of biological variation and batch effect removal in deep generative modeling for single-cell transcriptomics

Single-cell RNA sequencing (scRNA-seq) technology has contributed significantly to diverse research areas in biology, from cancer to development. Since scRNA-seq data is high-dimensional, a common strategy is to learn low-dimensional latent representations better to understand overall structure in the data. In this work, we build upon scVI, a powerful deep generative model which can learn biologically meaningful latent representations, but which has limited explicit control of batch effects. Rather than prioritizing batch effect removal over conservation of biological variation, or vice versa, our goal is to provide a birds eye view of the trade-offs between these two conflicting objectives. Specifically, using the well established concept of Pareto front from economics and engineering, we seek to learn the entire trade-off curve between conservation of biological variation and removal of batch effects. A multi-objective optimisation technique known as Pareto multi-task learning (Pareto MTL) is used to obtain the Pareto front between conservation of biological variation and batch effect removal. Our results indicate Pareto MTL can obtain a better Pareto front than the naive scalarization approach typically encountered in the literature. In addition, we propose to measure batch effect by applying a neural-network based estimator called Mutual Information Neural Estimation (MINE) and show benefits over the more standard Maximum Mean Discrepancy (MMD) measure. The Pareto front between conservation of biological variation and batch effect removal is a valuable tool for researchers in computational biology. Our results demonstrate the efficacy of applying Pareto MTL to estimate the Pareto front in conjunction with applying MINE to measure the batch effect.

bioinformatics↗

bioRxiv2019

PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways

Murine xenografts of pediatric leukemia are known to accurately recapitulate genomic aberrations. How this translates to the functional capacity of the proteome is unknown. Here, we studied global protein abundance, phosphorylation, and proteolytic processing in 11 pediatric B- and T-cell acute lymphoblastic leukemia patients and 19 corresponding xenografts. Protein level differences that stratified pediatric disease subtypes at diagnostic and relapse stages were largely recapitulated in xenograft models. Patient xenografts lacked multiple human leukocyte antigens, and complement proteins, and presented incomplete response mechanisms to the host immune system which is absent in the murine model. The dominant expression of MKI67 and cell cycle proteins indicated a high proliferative capacity of xenografted cells residing in the spleen. Structural genomic changes and mutations found in patients were reflected at the protein level. The post-translational modification landscape is shaped by leukemia type and host and only to a limited degree by the patient of origin. This study portrays how genomic and host factors shape protein and post-translational modification landscapes differently, and confirms murine patient-derived xenograft as competent model system while highlighting important areas of diverging biology.

cancer biology↗

bioRxiv2025

Transcriptomic Analysis Of The Relationship Between Pancreatic Ductal Adenocarcinoma Stromal Subtypes And Neural Invasion

We investigated the association between pancreatic ductal adenocarcinoma (PDAC) subtypes and neural invasion (NI) status in order to assess what factors influence the inter-tumor heterogeneity of NI. We built single-sample classifiers for prominent subtype schemes using multiple approaches. We predicted the subtype labels of samples with known NI status using the classifiers based on pairwise gene expression comparisons, which were consistently the best-performing models. These classifiers were made available in a Shiny app (https://salihaey.shinyapps.io/pcsubtypes/). Stromal subtypes were found to be most significantly associated with NI, with activated stroma being enriched in neuro-invasive samples. We compared the results of differential expression and gene set enrichment analyses between NI and stromal subtypes from both bulk and single-cell RNA-seq data, which revealed a prominent role of myCAFs and common signatures between activated stroma and NI (e.g., ECM remodeling and TGF{beta} signaling) as well as signatures that changed in divergent directions for activated stroma and NI (e.g., iCAF markers). Our study presents support for the clinical implications of PDAC stromal subtypes by revealing their association with NI incidence, while providing biological insights into the interactions between diverse genetic programs that influence NI emergence.

cancer biology↗

bioRxiv2026

In vivo intercellular CRISPR screens using viral proximity barcoding reveal regulators of tumor-immune interactions

Cell-cell interactions shape tumor growth, immune evasion, and therapeutic response, but systematically dissecting their genetic regulators in vivo remains challenging. Existing cell-cell interaction tracing technologies rely largely on protein labels or enzymatic reactions, limiting their information content and scalability. To overcome these limitations, we developed match-seq, an imaging-free, sequencing-based cell proximity tracing system that uses virus-like particles to transmit barcodes between neighboring cells. Barcoded mRNAs are transmitted from sender cells to nearby receiver cells, thereby establishing a spatial linkage that can be computationally reconstructed by barcode sequencing after tissue dissociation. We apply this system to an in vivo syngeneic murine tumor model, where we observe robust labeling of all immune cell lineages, and leverage barcode labeling to reconstruct cell type niches that recapitulate known tumor spatial biology. Furthermore, we couple this system with CRISPR perturbations and single-cell RNA sequencing to perform genetic screens in vivo on tumor-immune interactions at single-cell resolution. We use this method to infer cell-cell spatial relationships and uncover genetic dependencies in cancer cells that change the composition and cell state of their local microenvironments. By calculating a local immune activation signature, we are able to prioritize targets whose deletion enhances anti-tumor immunity through distinct effector cell types: loss of Tgfb1 engages CD8 T cells and macrophages, Traf7 loss elicits a CD4 T cell response, and Nectin3 loss promotes NK cell-mediated immunity, relationships that we validate by in vivo immune cell depletion. Notably, Tgfb1 and Nectin3 deletion had little effect on cancer cell fitness in the pooled screen yet suppressed tumor growth when deleted throughout the tumor, demonstrating the potential of match-seq to reveal functionally important tumor-immune interactions that would otherwise remain hidden. Together, these results establish a general framework for pooled genetic dissection of cell-cell interactions in vivo, extending CRISPR screening from cell-intrinsic phenotypes to the mechanisms by which cells shape and respond to their local microenvironments.

cancer biology↗

bioRxiv2023

IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes

In diffuse large B-cell lymphoma (DLBCL), the transcription factor IRF8 is the target of a series of potentially oncogenic events, including, chromosomal translocation, focal amplification, and super-enhancer perturbations. IRF8 is also frequently mutant in DLBCL, but how these variants contribute to lymphomagenesis is unknown. We modeled IRF8 mutations in DLBCL and found that they did not meaningfully impact cell fitness. Instead, IRF8 mutants, mapping either to the DNA-binding domain (DBD) or c-terminal tail, displayed diminished transcription activity towards CIITA, a direct IRF8 target. In primary DLBCL, IRF8 mutations were mutually exclusive with mutations in genes involved in antigen presentation. Concordantly, expression of IRF8 mutants in murine B cell lymphomas uniformly suppressed CD4, but not CD8, activation elicited by antigen presentation. Unexpectedly, IRF8 mutation did not modify MHC CII expression on the cell surface, rather it downmodulated CD74 and HLA- DM, intracellular regulators of antigen peptide processing/loading in the MHC CII complex. These changes were functionally relevant as, in comparison to IRF8 WT, mice harboring IRF8 mutant lymphomas displayed a significantly higher tumor burden, in association with a substantial remodeling of the tumor microenvironment (TME), typified by depletion of CD4, CD8, Th1 and NK cells, and increase in T-regs and Tfh cells. Importantly, the clinical and immune phenotypes of IRF8-mutant lymphomas were rescued in vivo by ectopic expression of CD74. Deconvolution of bulk RNAseq data from primary human DLBCL recapitulated part of the immune remodeling detected in mice and pointed to depletion of dendritic cells as another feature of IRF8 mutant TME. We concluded that IRF8 mutations contribute to DLBCL biology by facilitating immune escape.

cancer biology↗

bioRxiv2023

Intronic miR-6741-3p is involved in oral squamous cell carcinoma pathogenesis by targeting the oncogene SRSF3

Epigenetic silencing through methylation is one of the major mechanisms for downregulation of tumor suppressor miRNAs in various malignancies. The aim of this study was to identify novel tumor suppressor miRNAs which are silenced by DNA hypermethylation and investigate the role of at least one of these in oral squamous cell carcinoma (OSCC) pathogenesis. We treated cells from an OSCC cell line SCC131 with 5-Azacytidine, a DNA methyltransferase inhibitor, to reactivate tumor suppressor miRNA genes silenced/downregulated due to DNA methylation. At 5-day post-treatment, total RNA was isolated from the 5-Azacytidine and vehicle control-treated cells. The expression of 2,459 mature miRNAs was analysed between 5-Azacytidine and control-treated OSCC cells by the microRNA microarray analysis. Of the 50 miRNAs which were found to be upregulated following 5-Azacytidine treatment, we decided to work with miR-6741-3p in details for further analysis, as it showed a mean fold expression of >4.0. The results of qRT-PCR, Western blotting, and dual-luciferase reporter assay indicated that miR-6741-3p directly targets the oncogene SRSF3 at the translational level only. The tumor-suppressive role of miR-6741-3p was established by various in vitro and in vivo assays. Our results revealed that miR-6741-3p plays a tumor-suppressive role in OSCC pathogenesis, in part, by directly regulating SRSF3. Based on our observations, we propose that miR-6741-3p may serve as a potential biological target in tumor diagnostics, prognostic evaluation, and treatment of OSCC and perhaps other malignancies.

cancer biology↗

bioRxiv2023

Automated workflow for the cell cycle analysis of non-adherent and adherent cells using a machine learning approach

Understanding the details of the cell cycle at the level of individual cells is critical for both cellular biology and cancer research. While existing methods using specific fluorescent markers have advanced our ability to study the cell cycle in cells that adhere to surfaces, there is a clear gap when it comes to non-adherent cells. In this study, we combine a specialized surface to improve cell attachment, the genetically-encoded FUCCI(CA)2 sensor, an automated image processing and analysis pipeline, and a custom machine-learning algorithm. This combined approach allowed us to precisely measure the duration of different cell cycle phases in non-adherent cells. Our method provided detailed information from hundreds of cells under different experimental conditions in a fully automated manner. We validated this approach in two different Acute Myeloid Leukemia (AML) cell lines, NB4 and Kasumi-1, which have unique cell cycle characteristics. Additionally, we tested the impact of drugs affecting the cell cycle in NB4 cells. Importantly, our cell cycle analysis system is freely available and has also been validated for use with adherent cells. In summary, this report introduces a comprehensive, automated method for studying the cell cycle in both adherent and non-adherent cells, offering a valuable tool for cancer research and drug development.

cell biology↗

bioRxiv2024

Targeting the sphingolipid rheostat in IDH1mut glioma alters cholesterol homeostasis and triggers apoptosis via membrane degradation

The cross-regulation of metabolism and trafficking is not well understood for the vital sphingolipids and cholesterol constituents of cellular compartments. While reports are starting to surface on how sphingolipids like sphingomyelin (SM) dysregulate cholesterol levels in different cellular compartments (Jiang et al., 2022), limited research is available on the mechanisms driving the relationship between sphingolipids and cholesterol homeostasis, or its biological implications. Previously, we have identified sphingolipid metabolism as a unique vulnerability for IDH1mut gliomas via a rational drug design. Herein, we show how modulating sphingolipid levels affects cholesterol homeostasis in brain tumors. However, we unexpectedly discovered for the first time that C17 sphingosine and NDMS addition to cancer cells alters cholesterol homeostasis by impacting its cellular synthesis, uptake, and efflux leading to a net decrease in cholesterol levels and inducing apoptosis. Our results reflect a reverse correlation between the levels of sphingosines, NDMS, and unesterified, free cholesterol in the cells. We show that increasing sphingosine and NDMS (a sphingosine analog) levels alter not only the trafficking of cholesterol between membranes but also the efflux and synthesis of cholesterol. We also demonstrate that despite the effort to remove free cholesterol by ABCA1-mediated efflux or by suppressing machinery for the influx (LDLR) and biosynthetic pathway (HMGCR), apoptosis is inevitable for IDH1mut glioma cells. This is the first study that shows how altering sphingosine levels directly affects cholesterol homeostasis in cancer cells and can be used to manipulate this relationship to induce apoptosis in IDH1mut gliomas.

cancer biology↗

bioRxiv2024

Dicarbonyl stress debilitates mesothelial defense against metastasizing ovarian cancer

Chronic metabolic disorders and aging result in accumulation of active dicarbonyls that glycate biomolecules rendering them dysfunctional. Although metabolic aberrations are known to be epidemiologically associated with faster cancer progression, cell biological determinants of such associations remain elusive. The formation of micro-metastases in epithelial ovarian cancer involves its colonization of visceral peritonea through clearance of mesothelia that line the coelom. In this study, we observe that cocultures of immortalized human coelomic MeT-5A mesothelia with human ovarian cancer cells OVCAR-3 and SK-OV-3 show greater infiltration by the latter when exposed to increasing concentrations of the dicarbonyl methylglyoxal (MG). Treatment with increasing concentrations of MG caused death and senescence within human and murine serosal mesothelia. Cells showed higher levels of advanced glycation end products, dysregulated occludens junction protein ZO-1, and disrupted localization of cortical filamentous actin and its regulator ezrin, indicating poor inter-cell adhesion. Time lapse imaging also showed impaired migration for MG-treated single mesothelia and for their collective monolayers. Agent-based computer modeling of coculture dynamics predicted that a combined effect of confluence and migration allows inter-adherent mesothelia to contain the spread of colonizing cancer cells, which was confirmed through coculture time lapses of cancer colonization within higher and lower mesothelial densities. We found ovarian cancer cells showed higher levels of glyoxalase-1 (GLO-1) enzyme, which catabolizes MG, suggesting how they escaped its cytotoxic effects. Consistent with this, treatment of OVCAR-3 with MG concurrently with pharmacological inhibition of GLO-1 showed greater cell death. Our results suggest dicarbonyl stress helps colonizing cancer cells overcome the resistance of natural homoeostatic barriers and its inhibition may, in supplementation with chemotherapy, stem metastasis.

cancer biology↗

bioRxiv2025

A Unified Single-Cell Atlas of HNSCC: Uncovering HPV and Sex Variability in the Tumor Microenvironment

Head and neck squamous cell carcinoma (HNSCC) is highly heterogeneous, with variations driven by HPV status and sex. However, existing single-cell RNA sequencing (scRNA-seq) studies are often limited in sample size and lack standardized methodologies, limiting cross-study comparisons. To address this, we integrated scRNA-seq data from 78 patients (274,911 cells) across multiple studies, creating a unified HNSCC atlas that harmonizes annotations and enables robust tumor microenvironment (TME) analyses. Using STACAS for semi-supervised integration and automated annotation tools such as Ikarus and scGate, we improved tumor and immune cell classification. Leveraging our atlas, we identified HPV- and sex-specific shifts in immune and stromal composition, with HPV+ tumors enriched in adaptive immune cells and HPV- tumors showing more stromal and myeloid populations. Preliminary sex-stratified analyses suggested distinct microenvironmental patterns, warranting further investigation. This publicly available atlas provides a comprehensive framework for reproducibly studying HNSCC biology, improving patient stratification, and may help informing personalized therapies.

cancer biology↗