Biology subjects

Vlckova, P.

Publications and source records attributed to Vlckova, P..

7 recordsLinked to original sources

bioRxiv2026

Epithelial Stem Cell Fate Determines Chemoradiotherapy Response in Rectal Cancer

Rectal cancers are often treated with neoadjuvant chemoradiotherapy (CRT), yet 85% of patients do not achieve a pathological complete response. To identify the molecular determinants of CRT response, we profiled the single-cell signalling, DNA-damage, cell-cycle, apoptotic, and cell-fate responses of 2,769 patient-derived organoid cultures treated with CRT, cancer-associated fibroblasts (CAFs), and signal-rewiring agents. We find that CRT response is determined by stem cell-fate. CRT triggers comparable DNA-damage in isogenic proliferative (proCSC) and revival (revCSC) colonic stem cells, but proCSC retain damage and die whereas revCSC resolve damage and persist. Both CRT and CAFs drive proCSC to a common treatment-resistant revCSC fate and high revCSC predicts worse survival in patients. Pharmacologically constraining stem-cell plasticity increases CRT sensitivity, and Spatial Perturbation of ARrayed Tumour Assembloids (SPARTA) confirms YAP/TEAD inhibition improves chemotherapy responses in human stromal-tumour models. These results suggest that cancer cell-fate, not genotoxic damage itself, ultimately governs response to standard-of-care chemoradiotherapy. HIGHLIGHTSO_LIRectal cancer stem cell-fate determines chemoradiotherapy-induced apoptosis C_LIO_LIproCSCs retain DNA-damage and die, whereas revCSCs repair damage and persist C_LIO_LICAFs and chemoradiotherapy converge on a common chemo-radioresistant revCSC state C_LIO_LISPARTA reveals TEAD inhibition blocks DNA-repair persisters in stromal assembloids C_LI

cancer biology↗

bioRxiv2026

Stromal Prostaglandin is a Dominant Spatial Regulator of Cell-fate Plasticity in Colorectal Cancer

Colorectal cancer (CRC) tumours with high stromal content have a worse outcome, but the mechanisms governing this are unclear. Using high-throughput single-cell perturbation analysis of CRC patient-derived organoids (PDOs) and cancer-associated fibroblasts (CAFs) we find that epithelial cells with high stromal-communication potential are marked by the transcriptional co-repressor DACH1. To define the causal regulators of stromal-epithelial signalling, we developed a novel CRISPR screening platform to perturb the CAF secretome and measure epithelial stem cell responses at single-cell resolution. Intercellular CRISPR screening and full factorial ligand analysis revealed that stromal Prostaglandin E2 (PGE2) is a dominant regulator of CRC cell-fate plasticity. Stromal PGE2 converts DACH1+ epithelia from a chemosensitive proliferative colonic stem cell (proCSC) fate into a chemorefractory and prometastatic revival colonic stem cell (revCSC) fate. PGE2-driven epithelial transdetermination is rapid and reversible, providing an acute mechanism for stromal-driven plasticity in CRC tumours. Genetic and pharmacological inhibition of stromal COX2 inhibits epithelial plasticity, trapping CRC epithelia in an anti-metastatic and chemosensitive proCSC fate. PTGS2+ CAFs support a spatially resolved revCSC to proCSC plasticity gradient in human CRC tumours marked by increasing DACH1 expression. These results reveal that stromal prostaglandin is a dominant spatial regulator of poor-prognosis cell-fates and may explain the benefit of anti-COX therapies in both preventing and treating CRC.

cancer biology↗

bioRxiv2023

Chemotherapeutic regulation of the ROS/MondoA-dependent TXNIP/GDF15 axis; and derivation of a new organoid metric as a predictive biomarker

Chemotherapy, the standard of care treatment for cancer patients with advanced disease, has been increasingly recognised to activate host immune responses to produce durable outcomes. Here, in colorectal adenocarcinoma (CRC) we identify chemotherapy-induced Thioredoxin Interacting Protein (TXNIP), a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15). GDF15 is a negative prognostic factor in CRC and promotes the differentiation of regulatory T cells (Tregs), through CD48 ligation. Intriguingly, multiple models including patient-derived tumor organoids demonstrate that loss of TXNIP/GDF15 axis functionality is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker. These findings illustrate a potentially common pathway where chemotherapy-induced epithelial stress drives local immune remodelling for patient benefit, with disruption of this pathway seen in refractory or advanced cases.

cancer biology↗

bioRxiv2023

A Single-cell Perturbation Landscape of Colonic Stem Cell Polarisation

Cancer cells are regulated by oncogenic mutations and microenvironmental signals, yet these processes are often studied separately. To functionally map how cell-intrinsic and cell-extrinsic cues co-regulate cell-fate in colorectal cancer (CRC), we performed a systematic single-cell analysis of 1,071 colonic organoid cultures regulated by 1) CRC oncogenic mutations, 2) microenvironmental fibroblasts and macrophages, 3) stromal ligands, and 4) signalling inhibitors. Multiplexed single-cell analysis revealed a stepwise epithelial differentiation landscape dictated by combinations of oncogenes and stromal ligands, spanning from fibroblast-induced Clusterin (CLU)+ revival colonic stem cells (revCSC) to oncogene-driven LRIG1+ hyper-proliferative CSC (proCSC). The transition from revCSC to proCSC is regulated by decreasing WNT3A and TGF-{beta}-driven YAP signalling and increasing KRASG12D or stromal EGF/Epiregulin-activated MAPK/PI3K flux. We find APC-loss and KRASG12D collaboratively limit access to revCSC and disrupt stromal-epithelial communication - trapping epithelia in the proCSC fate. These results reveal that oncogenic mutations dominate homeostatic differentiation by obstructing cell-extrinsic regulation of cell-fate plasticity. HighlightsO_LI1,071-condition single-cell transition map of colonic stem cell polarisation regulated by oncogenic and mircoenvironmental cues. C_LIO_LIFibroblasts polarise WT colonic epithelia towards Clu+ revCSC via TGF-{beta}1 and YAP signalling. C_LIO_LIAPC-loss and KRASG12D drive a Birc5+, Lrig1+, and Ephb2+ proCSC fate via MAPK and PI3K. C_LIO_LIOncogenic mutations disrupt stromal regulation of epithelial plasticity, trapping cells in the proCSC fate. C_LI

cancer biology↗

bioRxiv2022

OPS-γδ: allogeneic opsonin-secreting γδT cell immunotherapy for solid tumours mediates direct and bystander immunity

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous {beta}T cells. These approaches are limited by tonic signalling of synthetic constructs and costs associated with manufacture of bespoke patient products. {gamma}{delta}T cells are an emerging alternative chassis for cellular therapy, possessing innate anti-tumour activity, potent antibody-dependent cytotoxicity (ADCC) and minimal alloreactivity. We present an immunotherapeutic platform technology built around the V{gamma}9V{delta}2 {gamma}{delta}T cell chassis, harnessing specific characteristics of this cell type and offering an allo-compatible means of delivering cellular therapy that recruits bystander immunity. We engineered {gamma}{delta}T cells to secrete synthetic opsonins and stabilized IL15 (stIL15). Using GD2 as a model antigen we show how opsonin-secreting V{gamma}9V{delta}2 (OPS-{gamma}{delta}) have enhanced cytotoxicity and also confer this benefit on lymphoid and myeloid bystander cells. Reflecting the secreted nature of the engineered efficacy modules, the entire product rather than just the gene-modified fraction exhibited enhanced activation and cytotoxic profiles, superior persistence and proliferative capacity even upon repeated tumour challenge. Secretion of stIL15 abrogated the need for exogenous cytokine supplementation during expansion and further mediated functional licensing of bystander NK cells. Compared to unmodified {gamma}{delta}T cells, stIL15-OPS-{gamma}{delta} cells exhibited superior in-vivo control of subcutaneous tumour and persistence in the blood. stIL15-OPS-{gamma}{delta} cells were further efficacious in 3D patient-derived osteosarcoma models, where efficacy could be boosted with the addition of immunomodulatory aminobisphosphonate drug, zoledronic acid. Together the data identify stIL15-OPS-{gamma}{delta} cells as a novel allogeneic platform combining direct cytolysis with bystander activation to effect solid tumour control. One Sentence SummaryArmoured, opsonin-secreting OPS-{gamma}{delta} cell immunotherapy is built on the innate strengths of the V{gamma}9V{delta}2 cell chassis for allogeneic solid tumour targeting.

immunology↗

bioRxiv2022

Cancer-Associated Fibroblasts Regulate Patient-Derived Organoid Drug Responses

Patient-derived organoids (PDOs) can model personalized therapy responses, however current screening technologies cannot reveal drug response mechanisms or study how tumor microenvironment cells alter therapeutic performance. To address this, we developed a highly-multiplexed mass cytometry platform to measure post translational modification (PTM) signaling in >2,500 colorectal cancer (CRC) PDOs and cancer-associated fibroblasts (CAFs) in response to clinical therapies at single-cell resolution. To compare patient- and microenvironment-specific drug responses in thousands of single-cell datasets, we developed Trellis -- a highly-scalable, hierarchical tree-based treatment effect analysis method. Trellis single-cell screening revealed that on-target cell-cycle blockage and DNA-damage drug effects are common, even in chemorefractory PDOs. However, drug-induced apoptosis is patient-specific. We found drug-induced apoptosis does not correlate with genotype or clinical staging but does align with cell-intrinsic PTM signaling in PDOs. CAFs protect chemosensitive PDOs by shifting cancer cells into a slow-cycling cell-state and CAF chemoprotection can be reversed by inhibiting YAP. HighlightsO_LI>2,500 single-cell PTM signaling, DNA-damage, cell-cycle, and apoptosis responses from drug-treated PDOs and CAFs. C_LIO_LITrellis: hierarchical tree-based treatment effect method for single-cell screening analysis. C_LIO_LIPDOs have patient-specific drug responses that align with cell-intrinsic PTM signaling states. C_LIO_LICAFs chemoprotect PDOs by altering PDO cell-state via YAP signaling. C_LI

cancer biology↗

bioRxiv2019

Single-Cell Signalling Analysis of Heterocellular Organoids

Organoids are powerful biomimetic tissue models. Despite their widespread adoption, methods to analyse cell-type specific post-translational modification (PTM) signalling networks in organoids are absent. Here we report multivariate single-cell analysis of cell-type specific signalling networks in organoids and organoid co-cultures. Simultaneous measurement of 28 PTMs in >1 million single small intestinal organoid cells by mass cytometry reveals cell-type and cell-state specific signalling networks in stem, Paneth, enteroendocrine, tuft, goblet cells, and enterocytes. Integrating single-cell PTM analysis with Thiol-reactive Organoid Barcoding in situ (TOBis) enables high-throughput comparison of signalling networks between organoid cultures. Multivariate cell-type specific PTM analysis of colorectal cancer tumour microenvironment organoids reveals that shApc, KrasG12D, and Trp53R172H cell-autonomously mimic signalling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type specific signalling analysis of healthy and cancerous organoids.

cell biology↗