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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 19 recordsLinked to original sources

bioRxiv2026

Ex vivo human tumor slices more accurately predict patient responses to an oncolytic virus than in vivo mouse models

Immunotherapies, including oncolytic viruses (OV), are promising therapies that can enhance anti-tumor immune responses. However, preclinical success of immunotherapies in mouse models has not always translated to clinical benefit in cancer patients. This study compared preclinical efficacy and mechanism of action for ASP9801, a vaccinia virus expressing IL-7 and IL-12, using mouse models of colorectal cancer (CRC) in vivo and in human organotypic tumor slice models ex vivo. The murine surrogate for ASP9801 significantly reduced tumor volumes in treated and abscopal tumors in two different CRC models in vivo (MC38 and RO100). Treatment efficacy was accentuated when combined with anti-PD1 treatment, and single-cell RNA sequencing analysis revealed depletion of tumor cells and increased T cell infiltration and activation in both treated and abscopal tumors. However, human tissue analysis ex vivo (E-slices) using PDX models and patient samples showed that ASP9801 is not effective in CRC, consistent with clinical trial results. On the other hand, ASP9801 was highly effective in GBM, indicating indication-specific efficacy of ASP9801, and how E-slice assays can be used to identify treatment-sensitive indications. This study demonstrates the superiority of E-slices over mouse models for predicting clinical response and its utility in planning clinical trials.

cancer biology↗

bioRxiv2026

Immune-cell depleted diffuse large B-cell lymphomas have reduced expression of MHC class I

Immunotherapy has transformed treatment for many cancers. In the aggressive and genetically heterogeneous diffuse large B-cell lymphoma (DLBCL), CD19 CAR T-cell therapy is highly effective, whereas immune checkpoint blockade has shown limited benefit. Loss of MHC expression is a common mechanism to escape T-cell cytotoxicity, and loss of MHC class I (MHC-I) and II are frequent in DLBCL. We applied imaging mass cytometry to diagnostic biopsies from younger, high-risk DLBCL patients to map the tumor microenvironment (TME) spatial architecture in relation to tumor cell MHC expression, mutational status, transcriptomic and proteomic profiles. Neighborhood analyses identified four TME subtypes: immune-cell depleted and three immune-infiltrated types (mixed, CD4 T cell-rich, CD8 T-cell/macrophage-rich). Depleted cases had shorter overall survival (p = 0.033) and increased expression of proteins involved in DNA replication and proliferation markers compared to infiltrated cases. Tumor cell MHC-I expression was heterogeneous. Cases with low frequency of MHC-I-pos tumor cells were enriched for the depleted TME type. MHC-I-pos tumor cells were surrounded by CD4 and CD8 T cells and M1 macrophages, whereas MHC-I-neg tumor cells were closer to other MHC-I-neg tumor cells. These findings suggest that TME-based classification incorporating tumor cell MHC-I status may improve individualized immunotherapy selection.

cancer biology↗

bioRxiv2026

Cross-species analysis links cell-cell communication rewiring to NOTCH2 during serous endometrial carcinogenesis

Cell-cell interactions shape the fate of mutant cells during cancer initiation but how these interactions evolve during progression to pathologically recognizable lesions remain poorly understood. Here, we investigated cell-cell communication during serous endometrial carcinoma (SEC; also known as uterine serous carcinoma) development using a lineage-traceable mouse model and cross-species analyses of the mouse and human neoplastic endometrium. In mice, the early, pre-dysplastic stage was marked by a global decrease in inferred cell-cell interactions, followed by extensive communication network rewiring during neoplastic progression. Pathway-specific analysis revealed a similar pattern for NOTCH signaling, with NOTCH2 emerging as the dominant NOTCH receptor in Trp53/Rb1-mutant immature epithelial cells. Functionally, NOTCH2 promoted the outgrowth of more proliferative mutant organoids. Cross-species transcriptomic analysis identified conserved immature epithelial states in mouse and human neoplastic endometrial epithelium. In human tissues, NOTCH2 was overexpressed in serous endometrial intraepithelial carcinoma, a precursor of SEC, and in overt SEC. Furthermore, elevated NOTCH2 expression was associated with poor patient survival. These findings link cell-cell communication rewiring during experimental SEC development to conserved neoplastic epithelial states and identify NOTCH2 as an early marker and a potential target of disease interception.

cancer biology↗

bioRxiv2026

Patient-Derived Glioma Models Preserve Tumor Heterogeneity and Identify Stearoyl-CoA Desaturase1 (SCD1) as a Candidate Biomarker for Precision Immunotherapy

Background: Pediatric and adult brain tumors, including glioblastoma, astrocytoma, ependymoma, and medulloblastoma, remain associated with poor prognosis despite advances in surgery, radiation, and chemotherapy. Therapeutic resistance, tumor heterogeneity, and treatment-related toxicity highlight the need for clinically relevant models that enable precision medicine and immunotherapy development. Methods: Freshly dispersed tumors (FDTs), low-passage patient-derived brain tumor (PBT) spheroid lines, and matched patient-derived xenograft (PDX) models were established from patients with primary brain tumors. Models were characterized using single-cell and bulk RNA sequencing, whole-exome sequencing, multiparameter flow cytometry, and immunohistochemistry. PBTs were compared with matched FDTs to evaluate model fidelity. Results: PBT lines were established in approximately 68% of cases and retained key patient-specific genomic alterations, including IDH1, MGMT, TP53, and PTEN, with expression of therapeutically relevant targets, including IL13R2, EGFR, HER2, WNT1, JAK1/2, and NOTCH1-4. Gene expression profiles of PBTs closely correlated with matched FDTs (R = 0.38, P = 0.0038). PBT and PDX models preserved intratumoral heterogeneity and non-clonal populations, enabling identification of therapy-resistant subclones during in vitro selection. Molecular analyses identified Stearoyl-CoA Desaturase1 (SCD1) as an overexpressed biomarker across glioma PBTs and matched patient tumors. Ingenuity Pathway Analysis identified SCD1 as an upstream regulator of EGFR-, TP53-, and CYCS-associated signaling networks implicated in tumor progression and immune suppression. Conclusions: Clinically relevant PBT and matched PDX models recapitulate molecular, transcriptional, and histopathological characteristics of primary brain tumors. These platforms provide tools for biomarker discovery, therapeutic testing, and precision immunotherapy development, while identifying SCD1 as a biomarker and therapeutic target in glioma.

cancer biology↗

bioRxiv2026

Rewiring of Integrin Signaling and Cell-cycle Deregulation Drive SMARCB1-Deficient Epithelioid Sarcoma

Epithelioid sarcoma (EPS) is an aggressive soft-tissue sarcoma characterized by loss of the chromatin-remodeling subunit SMARCB1. The oncogenic programs driving EPS remain poorly understood. Through CRISPR loss-of-function screens, we identified conserved dependencies on integrin signaling components and cyclin-dependent kinases (CDKs). Genetic disruption of integrin subunit alpha V (ITGAV)-centered signaling impaired epithelioid cluster formation and reduced MYC expression. SMARCB1 re-expression phenocopied these effects and revealed that SMARCB1 loss selectively represses context-dependent integrin subunits while preserving an ITGAV-centered pro-survival axis, associated with altered BAF complex occupancy. Analysis of EPS cell lines and primary tumors revealed frequent genetic or epigenetic inactivation of CDKN2A/p16, indicating that loss of cell-cycle control is a key cooperating event in EPS development and providing a mechanistic rationale for targeting CDK4/6. Together, these findings establish integrin-driven oncogenic signaling coupled with disruption of cell-cycle control as a central oncogenic program in EPS and identify actionable therapeutic vulnerabilities.

cancer biology↗

bioRxiv2026

Systemic hypoxia drives glycogen-fueled progression of lung adenocarcinoma

In advanced stages, lung adenocarcinoma obstructs airways and disrupts ventilation-perfusion relationships in the lung, causing systemic hypoxemia and enabling a feed-forward loop that accelerates malignancy. Systemic hypoxemia is also experienced due to common respiratory comorbidities such as chronic obstructive pulmonary disease (COPD) and obstructive sleep apnea (OSA), potentially accelerating malignancy. In a statewide electronic health record network, pre-existing COPD (598 matched pairs) or sleep apnea (235 matched pairs) independently predicted worse survival following incident lung cancer diagnosis. Since the mechanistic basis of the link between malignancy and hypoxia is not well understood, we created systemic hypoxia in KrasLSL-G12D/+;Trp53fl/fl (KP) mice by delivering low inspired oxygen concentrations (8% inspired oxygen; 8 h daily). Hypoxia nearly doubled tumor multiplicity and selectively remodeled cancer central carbon metabolism. Spatially resolved metabolomics revealed marked tumor-compartment glycogen accumulation, elevated tricarboxylic-acid cycle intermediates, and depleted glycolytic pools. Quantitative proteomics across cellular models and autochthonous tumors demonstrated that systemic hypoxia drives glycogen mobilization selectively through the lysosomal enzyme acid -glucosidase (GAA). Tumor-cell-autonomous deletion of GAA eliminated the hypoxia-driven growth advantage and disrupted downstream anabolic biosynthetic pathways. Thus, systemic hypoxia drives lung adenocarcinoma expansion by mobilizing lysosomal glycogen reserves through GAA to sustain proliferative growth.

cancer biology↗

bioRxiv2026

Complementary cytotoxicity of GD2-targeted photoimmunotherapy and 5-aminolevulinic acid photodynamic therapy in neuroblastoma and osteosarcoma

Phototherapy, a light-activated anticancer treatment, enables localized tumor-cell killing with distinct mechanisms of action. Photoimmunotherapy (PIT) produces immunogenic tumor cell death upon near-infrared light activation of a photoabsorber through antigen-specific targeting. Photodynamic therapy (PDT) produces reactive oxygen species through red-light activation of intracellular protoporphyrin IX generated from 5-aminolevulinic acid uptake and metabolism. PIT may have limited activity in antigen-low cells, whereas PDT has less precise tumor selectivity. We combined these modalities to define their interaction, broaden cytotoxicity, and determine whether dual treatment could reduce light-dose requirements. We conjugated dinutuximab, which targets the GD2 antigen, to IRDye 700DX and characterized plasma-membrane localization by confocal and widefield microscopy. PIT and PDT monotherapies were evaluated across agent and light doses in neuroblastoma (NB) and osteosarcoma (OS) cell lines. Combination matrices were tested using interaction, highest-single-agent, and Bliss analyses. Both monotherapies demonstrated significant light-dose-dependent effects in NB and OS. PIT produced no measurable cytotoxicity in antigen-blunted control cells, whereas PDT remained effective, confirming antigen-dependence of PIT and antigen-independence of PDT. The combination interaction was significant in SK-N-BE(2) but not LM7. At selected combinations, however, dual treatment produced greater killing than the more effective matched monotherapy in both SK-N-BE(2) and LM7 (Padj<0.022). Notably, lowest combination of PIT 10 J/cm2 plus PDT 10 J/cm2 achieved 90.3% killing in SK-N-BE(2), exceeding higher light-dose PIT or PDT monotherapy, suggesting a light-dose sparing effect. These findings establish potent and complementary PIT-PDT activity, supporting dual phototherapy to broaden cytotoxicity and reduce light-dose requirements in GD2-expressing tumor phototherapy.

cancer biology↗

bioRxiv2026

Microbial valerate is associated with CAR T dysbiosis and its supplementation enhances CAR T function in B-cell lymphoma

Anaerobe-depleting antibiotic exposure is associated with inferior progression-free survival after CD19 CAR T-cell therapy in large B-cell lymphoma, yet the cellular mechanisms linking gut dysbiosis to the CAR T-cell product and whether this imprint is reversible have remained undefined. In two independent CAR-T candidate cohorts, low stool valerate at the time of CAR-T eligibility identified a multi-metabolite-deficient dysbiotic gut microbiome state marked by depletion of fiber-fermenting commensals and loss of carbohydrate-fermentation, SCFA-biosynthesis, and amino-acid metabolism pathways. Reanalysis of single-cell RNA sequencing from 42 lymphoma patients stratified by piperacillin-tazobactam/imipenem/meropenem (PIM) exposure revealed that PIM-exposed CAR T-cell products were CD4-skewed, with significantly elevated AP-1/immediate-early gene (IEG) and cellular activation signatures that together predicted inferior progression-free survival. Ex vivo conditioning of CAR T-cells with valerate produced a chromatin and transcription factor program distinct from butyrate or propionate, characterized by KLF/SP/EGR family engagement, KLF4 promoter opening, and broad induction of AP-1/IEG and MHC class II transcripts, whereas butyrate drove broader chromatin remodeling with TBX21/EOMES/NF-{kappa}B gains and KLF2 promoter closure, and propionate induced an NFY-centered program with preferential commitment to low-mitochondrial-content states. Untargeted metabolomics confirmed valerate uptake and mitochondrial {beta}-oxidation in CAR T-cells, while dietary sodium valerate supplementation in meropenem-treated mice bearing A20 lymphoma significantly reduced tumor burden and extended survival compared with CAR T-cells alone. These findings identify stool valerate as a bedside-deployable biomarker of dysbiosis-imprinted CAR T-cell dysfunction and support ex vivo or dietary valerate supplementation as a clinically tractable strategy to improve CAR-T anti-tumor function in patients with disrupted gut microbiomes.

cancer biology↗

bioRxiv2026

Attenuated Salmonella-Mediated Delivery of GSDMD Potentiates PD-1 Blockade Therapy against Melanoma

Immunotherapy has emerged as a core therapeutic strategy for melanoma. Programmed death protein 1 (PD-1) is a critical immune checkpoint molecule that restrains host anti-tumor immunity, and therapeutic agents blocking the PD-1 signaling pathway have been widely deployed in clinical practice. Nevertheless, single-agent PD-1 blockade fails to elicit robust clinical responses in the majority of patients. Therefore, there is an urgent unmet need to develop combinatorial regimens capable of augmenting the anti-tumor efficacy of PD-1 inhibition. Gasdermin D (GSDMD), a pore-forming effector protein that orchestrates pyroptosis, exerts inherent anti-tumor activities upon overexpression. However, whether GSDMD can synergize with PD-1 blockade to enhance therapeutic outcomes against melanoma remains poorly defined. To address this question, we established an attenuated Salmonella engineered strain for targeted delivery of GSDMD, and further investigated the anti-melanoma therapeutic efficacy of combining this engineered bacterium with anti-PD-1 antibody via immunofluorescence staining, flow cytometry and other analytical approaches. Our in vivo results demonstrated that combinatorial treatment markedly suppressed melanoma progression in tumor-bearing mice relative to monotherapy with either GSDMD-expressing bacteria or anti-PD-1 antibody alone. Mechanistically, co-treatment upregulated intratumoral expression of GSDMD and the pro-apoptotic protein BAX, while simultaneously downregulating PD-1 expression. In addition, the GSDMD/anti-PD-1 combination significantly elevated the proportions of CD4 and CD8 T lymphocytes in both peripheral blood and splenic tissues, and facilitated robust tumor infiltration by these two T cell subsets. Compared with phosphate-buffered saline (PBS) and scramble control groups, combinatorial therapy promoted tumor infiltration of M1-type tumor-associated macrophages (TAMs) and repolarized TAMs away from the immunosuppressive M2 phenotype. Consistently, serum levels of the pro-inflammatory cytokines TNF- and IFN-{gamma} were markedly elevated following combined intervention. Collectively, this study verifies that attenuated Salmonella carrying GSDMD synergizes with anti-PD-1 antibody to elicit potent anti-tumor effects in melanoma-bearing mice by amplifying systemic and intratumoral anti-tumor immune responses, which provides a preclinical rationale for novel combinatorial therapeutic strategies against melanoma.

cancer biology↗

bioRxiv2026

Profiling Siglec-7 and Siglec-9 ligands across the LuCaP PDX series: Implications for glyco-immune checkpoint inhibition in advanced prostate cancer

Advanced prostate cancer exhibits profound cellular and molecular heterogeneity, frequently becoming resistant to androgen receptor (AR) targeting through lineage plasticity and neuroendocrine differentiation. Immunotherapies have shown limited efficacy in prostate cancer, largely due to its immunosuppressive tumour microenvironment. Hypersialylation contributes to immune evasion by engaging sialic acid-binding immunoglobulin-like lectins (Siglecs) on immune cells, forming glyco-immune checkpoints. Although this pathway represents a promising therapeutic target, the distribution of Siglec ligands across diverse prostate cancer phenotypes and their response to standard-of-care hormone therapy remain poorly understood. Here, we utilised high-affinity engineered sialoglycan-binding reagents (HYDRA) to perform comprehensive immunohistochemical profiling of Siglec-7 and Siglec-9 ligands across a panel of 40 Washington Carcinoma of the Prostate (LuCaP) patient-derived xenograft (PDX) models. Ligand expression was evaluated in relation to AR status and neuroendocrine phenotype. To determine the impact of androgen deprivation on the tumour glycome, ligand expression was compared between matched PDX lines grown in intact and castrated mice. Our findings reveal widespread but heterogeneous expression of Siglec-7 and Siglec-9 ligands across the LuCaP cohort. Expression levels were comparable between AR-positive adenocarcinoma models and AR-negative neuroendocrine variants, demonstrating that this glyco-immune checkpoint is maintained across distinct prostate cancer lineages. Under castration conditions, glycan remodelling occurred in a model-dependent manner. A subset of PDX models exhibited reduced Siglec ligand expression following castration, suggesting partial AR dependence. In contrast, other models displayed increased ligand expression, consistent with adaptive immune evasion in response to therapeutic stress, while a third group remained largely unchanged. Collectively, our study demonstrates that the Siglec-7/9 glyco-immune checkpoint axis is broadly maintained across the spectrum of prostate cancer lineage plasticity but is dynamically remodelled by androgen deprivation in a patient-specific manner. These findings support the sialoglycan-Siglec axis as a lineage-independent immunotherapeutic target and suggest that strategies aimed at disrupting Siglec-mediated immune suppression, such as tumour desialylation, may be most effective when combined with androgen deprivation therapy to enhance anti-tumour immunity.

cancer biology↗

bioRxiv2026

Membrane voltage and connexin expression work together to enhance tumor growth and metastasis in cancer

There is strong evidence of tumors manipulating their resting membrane potential (Vmem). While most fully-differentiated cells have a Vmem of roughly -70mV, tumor cells are generally depolarized, with Vmem {approx}-30mV, which more closely resembles the Vmem of stem cells. This is often believed to serve the purpose of accelerating the cell cycle and hence advantaging tumor proliferation. But when the tumor becomes invasive, its cells sometimes revert to a hyperpolarized Vmem with no obvious reason why. Separately, it is well accepted that solid tumors that are not yet invasive greatly underexpress connexins relative to healthy tissue; connexins, for our purpose, form gap junctions (GJs), small connecting tubes between nearby cells. Tumors that are invasive, by contrast, overexpress connexins. There is very little explanation for the paradox that connexins are first underexpressed and then overexpressed. However, it has long been known that Vmem electrically gates GJs; specifically, that homotypic GJs conduct best when the two cells they connect have a similar Vmem. Our in-silico model results explain this phenomenon, showing that when considered together, tumors' electrical and connexin-expression behaviors form a unified and effective strategy to control communication between the tumor and its healthy neighbor cells. This has implications for the emerging field of cancer bioelectrics, potentially leading to more precisely-targeted therapies.

cancer biology↗

bioRxiv2026

Cholesterol and p53 promote senescence and systemic fibrosis in metabolic dysfunction-associated steatohepatitis

Background & aims: TP53 (p53) coordinates diverse cellular stress response programmes including pro-survival activities, senescence, and cell death. During tissue damage, p53 can shape both the local cellular response to injury, including the fibrotic response, and influence distal organ biology. Fibrosis in the liver is a major driver of hepatocellular carcinoma (HCC) risk within metabolic dysfunction-associated steatohepatitis (MASH). It is also an important determinant of dysfunction in multiple distal tissues including the kidneys, lungs, and heart. Despite significant clinical burden, our understanding of the molecular determinants of fibrotic MASH and its relationship to multiorgan fibrosis remain incomplete. Here, we investigate local and systemic effects of hepatocellular p53 activity and cholesterol during MASH development, with implications for disease prevention. Methods: This study utilised a genetic model of stabilised p53, diet-induced MASH models with varying cholesterol compositions, and an in vitro obesogenic system to investigate p53 activity during liver disease development. Non-invasive imaging and histopathological analyses were employed to monitor p53 activity, MASH, and multiorgan fibrosis in vivo. Complementary approaches, including in vitro human multicomponent liver spheroids, cytokine arrays, and analyses of human MASH transcriptomic and proteomic datasets, were used to examine molecular drivers and patient relevance. Results: Using an inducible mouse model of MDM2 E3 ubiquitin ligase deficiency to stabilise p53, we report that hepatocellular MDM2 E3 loss results in progressive fibrotic damage, robust hepatocellular expression of the p53 target gene CDKN1A/p21 (p21),and induces p21 and fibrosis in the kidneys of male mice in a sex-specific manner. In diet-induced MASH, we observe cholesterol and p53-dependent development of liver fibrosis, high expression of hepatocellular p21, and induction of p21 and fibrosis in the kidneys of male mice-reminiscent of features observed in MDM2 E3-deficient mice. We also observe fibrosis in the lungs and heart of male MASH mice. Both a cholesterol-free obesogenic diet and liver-specific loss of p53 mitigate hepatic fibrosis and systemic induction of p21 and fibrosis. Mechanistically, p53 induces hepatic expression of senescence-associated secretory phenotype (SASP) factors, including GDF15, in vivo. A human multicomponent LiverACE spheroid model showed a concordant trend towards increased GDF15 protein abundance under steatotic stress, while in humans, elevated circulating GDF15 levels in advanced MASH correlate with increased TNFRSF1A and EPHA2, circulating markers linked to kidney injury. Conclusions: Our work identifies undue p53 activity within the liver as a driver of multiorgan fibrosis in a sex-specific manner, affecting male but not female mice. We implicate cholesterol in promoting this pro-fibrotic environment in vivo and highlight circulating factors that could identify at-risk patients for multiorgan fibrosis in MASH.

cancer biology↗

bioRxiv2026

Vitamin D counters bone invasion by mammary cancer through inhibition of inflammation and epithelial-to-mesenchymal transition

Vitamin D deficiency is associated with poor outcome in several cancers in humans, and administration of vitamin D or analogs has been shown to decrease tumor progression and metastasis in animal mammary cancer models. We previously demonstrated significant acceleration of carcinogenesis in vitamin D-deficient mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mammary cancer model as well as of its spontaneous metastasis to lungs. While vitamin D also plays a role in skeletal metastasis, detailed mechanisms of its promotion of bone invasion and metastatic events are not completely elucidated. In the present study we used tibially-injected MMTV-PyMT mammary tumor cells to analyse how dietary-induced vitamin D deficiency in non-immunodeficient FVB mice accelerates bone invasion. Mechanistically, we observed vitamin D deficiency to increase pro-inflammation cytokines and nestin expression in internal bone surface and marrow, and to increase epithelial-to-mesenchymal transition (EMT) through Zeb1 transcription factor. In vitro, treatment of MMTV-PyMT tumor cells with CXCL12 was observed to stimulate Zeb1 expression, and this effect was efficiently countered by 1,25(OH)2D treatment. Analysis of cytokines in MMTV-PyMT mammary tumor cells in vitro showed significant reduction in several pro-inflammatory cytokines with 1,25(OH)2D treatment (GM-CSF, ICAM-1, IL-1ra, IP-10, JE, MCP-5, MIP-1, MIP-1{beta}, MIP-2, RANTES and CXCL12), a crucial observation in view of the current evidence that inflammation is one of the hallmarks of cancer. Furthermore, vitamin D repleteness is associated with very high expression of Socs1 (suppressor of cytokine signalling 1), an inhibitor of JAK/STAT pathway which prevents excessive inflammatory responses and has a tumor-suppressive role. These findings provide a strong link between vitamin D deficiency and acceleration of inflammation-driven bone invasion, and nestin and EMT. The evidence suggests that vitamin D-repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing invasion of skeletal sites.

cancer biology↗

bioRxiv2026

Nuclear Cathepsin L Remodels the Replication Machinery to Create a Therapeutic Vulnerability in Ovarian Cancer

Abstract Therapeutic resistance in ovarian cancer is frequently driven by persistent replication stress, yet the molecular mechanisms that convert replication stress into a therapeutically exploitable vulnerability remain incompletely understood. Here, we identify drug-induced nuclear cathepsin L (nCTSL) as a previously unrecognized regulator of replication stress and DNA repair. Clofarabine (CLF) combined with the ATR inhibitor AZD6738, or the CHK1 inhibitor prexasertib promoted nuclear accumulation of CTSL, where it remodeled the replication machinery through degradation of CCNE1, MCM3, MCM6, and geminin, accompanied by loss of RAD51 and 53BP1 and increased {gamma}H2AX and phospho-RPA2. DNA fiber analysis demonstrated marked inhibition of replication fork progression following CLF-based combinations, whereas CTSL depletion accelerated fork progression and abolished therapy-induced replication stress. Reconstitution with the nuclear M1F CTSL isoform restored replication restraint, confirming a direct role for nuclear CTSL in regulating replication dynamics. GFP-based DNA repair reporter assays further revealed that CLF-based combinations suppress DNA repair competence in a CTSL-dependent manner, indicating that nuclear CTSL couples replication stress amplification with functional inhibition of repair pathways. Functionally, CLF-based combinations selectively targeted transformed fallopian tube secretory epithelial cells while sparing non-transformed counterparts, demonstrated broad activity in patient-derived ovarian cancer ascites spheroids, and significantly inhibited tumor growth and prolonged survival in vivo. Collectively, our findings identify nuclear CTSL as a mechanistic driver of replication stress that remodels the replication machinery, impairs DNA repair, and creates a therapeutically exploitable vulnerability in ovarian cancer. We propose that nuclear CTSL promotes a transition from replication competence to replication catastrophe, thereby establishing a conceptual framework for biomarker-guided therapeutic strategies targeting CTSL-dependent replication stress.

cancer biology↗

bioRxiv2026

Integrative single cell analysis of CD8+ T-cells across early and advanced oral cancers reveals signatures of anti-tumour activity

Tumour-targeting CD8 T cells drive responses to every major form of cancer immunotherapy. Identifying them, however, remains an unsolved problem in solid tumours. The antigens they recognize are rarely defined and almost never shared between patients. We profiled 51,459 CD8+ T cells by paired single-cell RNA and T-cell receptor sequencing across 28 samples from 17 HPV-negative oral cancers spanning primary tumours, draining lymph nodes, metastases, and pembrolizumab-treated recurrences. We found that clonotypes that were expanded and shared across anatomical sites and timepoints were enriched within tumours and progressively selected over disease evolution and checkpoint blockade. Designating these shared-expanded clones as putative tumour-targeting cells, we trained a machine learning classifier that identifies them from transcriptome data alone. This 108-feature random forest signature recapitulated programmes of tumour reactivity and generalized to an integrated atlas of 89,318 CD8+ T cells from independent cohorts, showing progressive enrichment from normal to malignant tissue, and localized to tumour-proximal niches in spatial transcriptomics. By demonstrating that clonal behaviour across space and time encodes tumour reactivity in the transcriptome, this work establishes a generalizable framework for mapping tumour-engaged immunity without knowledge of the underlying antigen.

cancer biology↗

bioRxiv2026

Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit

Rhabdomyosarcoma (RMS) is a multi-subtype, high-risk pediatric sarcoma with a low mutational burden. The mutations found in RMS often alter genes involved in transcriptional control. Approaches to target dysregulated RMS transcription have remained elusive. Here, we develop a novel approach to target RMS transcription comprising simultaneous targeting of two distinctly acting transcriptional co-activators. We discover a common identity-controlling pan-RMS core regulatory circuit (CRC) composed of oncogenic and lineage-specific myogenic master transcription factors (mTFs). Using a super-enhancer-based reporter screen, we identify the EP300/CBP inhibitor A485 as a potent inhibitor of the pan-RMS CRC, though with efficacy-limiting toxicities. To enhance efficacy, we identify the mTF-binding co-activator EYA2 as a co-factor of this pan-RMS CRC and exploit a new second-generation EYA1/2 inhibitor, LG1-34, to disrupt its function. Combined co-activator inhibition inactivates the CRC and synergistically reduces RMS growth. This strategy dually targets CRC-associated co-activators to cooperatively suppress the RMS transcriptome and enforce cell death.

cancer biology↗

bioRxiv2026

A patient-centric therapeutic paradigm uncouples prostate cancer suppression from systemic metabolic collapse

The clinical benefits of cancer therapies are often compromised by the tolerable adverse effects that impair systemic organismal health and may evolve into latent life threats. Here, we identified profound abiraterone-induced but androgen-independent metabolic perturbations in prostate cancer patients and developed Lifehug-9892 to balance tumor therapy with systemic metabolic homeostasis. By integrating population cohorts with high-resolution metabolomics, we demonstrate that abiraterone induces profound systemic lipidomic dysregulation, characterized by the massive, pathological accumulation of desmosterol. Abiraterone inhibits but stabilizes DHCR24, leading to a metabolic trap in patients showing elevated levels of both desmosterol and cholesterol. Desmosterol accumulation is highly lipotoxic, potently triggering endothelial cell senescence and necrosis, macrophage foam cell formation, murine atherosclerosis, and hepatic senescence. To mechanistically uncouple and therapeutically rescue this systemic metabolic collapse, Lifehug-9892 was rationally designed to selectively retain on-target CYP17A1 inhibition while completely sparing DHCR24 function. Lifehug-9892 maintains potent tumor-suppressive activity while fully preserving the desmosterol-cholesterol metabolic axis and preventing systemic cardiovascular and hepatic damage. Our study uncovers a critical mechanistic link between drug-induced metabolic dysregulation and organismal health in cancer patients, providing a biochemical framework for developing patient-centric targeted therapies that preserve host homeostasis.

cancer biology↗

bioRxiv2026

Ex vivo glioblastoma migration phenotypes define clinical recurrence and tumor heterogeneity

Glioblastoma's pronounced migratory capacity underlies its diffuse invasion, presenting a formidable barrier to successful treatment. Ex vivo characterization of glioblastoma cells isolated from freshly resected clinical samples under physiologically relevant conditions revealed two distinct migratory phenotypes, Fast Migrating (FM) and Slow Migrating (SM). These phenotypes reflect distinct mechanosensitivity profiles and are associated with pharmacological responses that support the motor clutch model of cell migration. Analysis of genes associated with these phenotypes revealed a transcriptomic signature that closely associated with in vitro cell migration, histological invasion in patient specimens, and clinical survival. Single-nucleus RNA sequencing revealed that FM and SM cells coexist within a single glioblastoma, with FM cells enriched at the periphery and SM cells localized to the tumor core. Collectively, our study demonstrates the utility of ex vivo glioblastoma characterization, allowing decoding of tumor heterogeneity and clinical prognostication as well as providing a framework for deconvoluting the complex cancer phenotype.

cancer biology↗