Biology subjects

Gjeta, B.

Publications and source records attributed to Gjeta, B..

7 recordsLinked to original sources

bioRxiv2025

An integrated platform for high-throughput phenospace learning of 3D multilineage organoid systems

Complex multilineage organoid systems lack quantitative phenotyping methods preserving spatial architecture at high throughput. Current approaches compromise biological complexity, spatial resolution, or robust homogeneous multilineage assembly. We establish an integrated experimental-computational platform for high-throughput spatial phenotyping of multilineage organoids through developing a modular tumoroid culture system incorporating pancreatic ductal adenocarcinoma (PDAC) cells and cancer-associated fibroblasts (CAFs) in 384-well format with multiplexed whole-mount imaging. We developed Phenocoder, a machine learning framework combining conditional variational autoencoders with spatial graph analysis to extract multiscale organoid features. Rigorous validation demonstrates robust performance in PDAC tumoroids. The platform identifies pathway modulators that disrupt the fibrotic microenvironment and discovers stroma-dependent vulnerabilities, undetectable in monocultures. Extending to immuno-competent tumoroids, we assess fibrosis modulators in combination with T cell bispecific antibodies, identifying treatments that enhance immune cell proliferation and infiltration inducing cancer cell death, validated in patient-derived explants. This platform establishes a generalizable framework for multilineage organoid phenotyping.

cancer biology↗

bioRxiv2025

Integrated cell atlas and tumoroids chart pancreatic cancer therapeutic targets

Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense, fibroblast-rich stroma that actively shapes the tumor microenvironment. Most PDAC cases arise from conserved genetic transformations initiated by oncogenic KRAS mutations, developing into metastatic disease with high mortality rates. To chart universal PDAC cell states and identify therapeutic inroads, we integrated published single-cell transcriptomes from 200 patient samples, and used the atlas to define prevalent cancer cell and cancer-associated fibroblast (CAF) states, gene expression programs, and ligand-receptor interactions. We established modular tumoroids incorporating patient-derived cancer cells and CAFs that recapitulate aspects of ductal architecture and desmoplastic stroma. Single-cell and spatial transcriptomic profiling confirmed preservation of key cellular states and signaling networks in vitro. We identified Syndecan-1 (SDC1) as a CAF-responsive cancer cell receptor correlating with poor patient survival. Functional SDC1 blockade disrupted cancer growth in tumoroids, highlighting therapeutic relevance. This study provides a framework for dissecting cancer-stroma dynamics and identifying actionable targets using patient-derived tumoroid models.

cancer biology↗

bioRxiv2025

Human Lung Alveolar Model with an Autologous Innate and Adaptive Immune Compartment

Lung-resident immune cells, spanning both innate and adaptive compartments, preserve the integrity of the respiratory barrier, but become pathogenic if dysregulated1. Current in vitro organoid models aim to replicate interactions between the alveolar epithelium and immune cells but have not yet incorporated lung-specific immune cells critical for tissue residency2. Here we address this shortcoming by describing human lung alveolar immuno-organoids (LIO) that contain an autologous tissue-resident lymphoid compartment, primarily composed of tissue-resident memory T cells (TRMs). Additionally, we introduce lung alveolar immuno-organoids with myeloid cells (LIOM), which include both TRMs and a macrophage-rich alveolar myeloid compartment. The resident immune cells formed a stable immune-epithelial system, frequently interacting with the epithelium and promoting a regenerative alveolar transcriptomic profile. To understand how dysregulated inflammation perturbed the respiratory barrier, we simulated T-cell-mediated inflammation in LIOs and LIOMs and used single-cell transcriptomic analyses to uncover the molecular mechanisms driving immune responses. The presence of innate cells induced a shift in T cell identity from cytotoxic to immunosuppressive, reducing epithelial cell killing and inflammation. Based on insights obtained with bulk RNA-seq data from the phase 3 IMpower150 trial, we tested whether LIOM cultures could model clinically-relevant but poorly understood pulmonary side effects caused by immunotherapies such as the checkpoint inhibitor atezolizumab3. We observed a decrease in immunosuppressive T cells and identified gene signatures that matched the transcriptomic profile of patients with drug-induced pneumonitis. Given its effectiveness in capturing outcomes and mechanisms associated with a prevalent pulmonary disease, this system unlocks opportunities for studying a wide range of immune-related pathologies in the lung.

cell biology↗

bioRxiv2023

Human intestinal organoids with an autologoustissue-resident immune compartment

The intimate relationship between the epithelium and the immune system is crucial for maintaining tissue homeostasis, with perturbations in epithelial-immune interactions linked to autoimmune disease and cancer. Whereas stem cell-derived organoids are powerful models of tissue-specific epithelial function, these structures lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident lymphocytes (TRMs), a portion of which integrate within the IIO epithelium and survey the barrier. IIO formation was driven by TRM migration and interaction with epithelial cells, as orchestrated by TRM-enriched transcriptomic programs governing cell motility and epithelial inspection. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients, and found that the system recapitulates clinical outcomes and the underlying cellular mechanisms. Inflammation was associated with the emergence of an activated population of CD8+ T cells, which progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and likely mediated by a Th1-like CD4+ population, which initially displayed a cytokine-producing character and subsequently became cytotoxic itself. A system amenable to direct perturbation and interrogation, IIOs allowed us to identify the Rho pathway as a novel target for mitigating immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and the underlying inter-lineage immune interactions, IIOs can be used to broadly study tissue-resident immune responses in the context of tumorigenesis, infectious and autoimmune diseases.

immunology↗

bioRxiv2022

Multimodal spatiotemporal phenotyping of human organoid development

Organoids generated from human pluripotent stem cells (PSCs) provide experimental systems to study development and disease. However, we lack quantitative spatiotemporal descriptions of organoid development that incorporate measurements across different molecular modalities. Here we focus on the retina and use a single-cell multimodal approach to reconstruct human retinal organoid development. We establish an experimental and computational pipeline to generate multiplexed spatial protein maps over a retinal organoid time course and primary adult human retina, registering protein expression features at the population, cellular, and subcellular levels. We develop an analytical toolkit to segment nuclei, identify local and global tissue units, infer morphology trajectories, and analyze cell neighborhoods from multiplexed imaging data. We use this toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components, and global patterning in each organoid and primary tissue. In addition, we generate a single-cell transcriptome and chromatin accessibility time course dataset and infer a gene regulatory network underlying organoid development. We then integrate genomic data with spatially segmented nuclei into a multi-modal atlas enabling virtual exploration of retinal organoid development. We visualize molecular, cellular, and regulatory dynamics during organoid lamination, and identify regulons associated with neuronal differentiation and maintenance. We use the integrated atlas to explore retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death. Finally, we show that mosaic CRISPR/Cas genetic perturbations in retinal organoids provide insight into cell fate regulation. Altogether, our work is a major advance toward a virtual human retinal organoid, and provides new directions for how to approach disorders of the visual system. More broadly, our approaches can be adapted to many organoid systems.

systems biology↗

bioRxiv2022

Reconstructing cell interactions and state trajectories in pancreatic cancer stromal tumoroids

Interlineage communication within a cancer microenvironment can augment cancer cell behaviour and impact response to therapy. Patient-derived cancer organoids provide an opportunity to explore cancer cell biology, however it is a major challenge to generate a complex cancer microenvironment in vitro. Here, we established a stromal tumoroid culture system modeling pancreatic ductal adenocarcinoma (PDAC) that reconstitutes multilineage interactions between cancer, endothelial, and fibroblast cells and recapitulates several aspects of primary tumors. Whole-mount immunohistochemistry on cleared tumoroids reveals organized vessel, desmoplastic fibroblast, and glandular cancer cell phenotypes that emerge over time. Time-course scRNA-seq measurements show that tumoroid formation activates fibroblasts, altering the extracellular matrix (ECM) composition and inducing cancer cell signal-response signatures and metabolic state change. Comparison between tumoroids with normal or cancer associated fibroblasts (CAFs) reveals different ECM compositions, as well as differential effects on cancer cell behaviors and metabolism. We identify Syndecan 1 (SDC1) and Peroxisome proliferator-activated receptor gamma (PPARG) as receptor and metabolic nodes involved in cancer cell response to CAF signals, and blocking SDC1 disrupts cancer cell growth within the tumoroid. Tumoroids from multiple PDAC patients revealed co-existence of subpopulations associated with classical and basal phenotypes, and CAF-induced migration behaviors emerged in certain patient tumoroids. Comparisons between patient tumoroids revealed a multigene migration signature that develops over time reflecting a stress response mechanism that correlates with worse clinical outcome. Altogether, stromal tumoroids can be used to explore dynamic and reciprocal interactions between cancer, CAF and endothelial cell states, and our data provides new inroads into the discovery of personalized pancreatic cancer therapies.

cancer biology↗

bioRxiv2019

Aboave-Weaire’s law in epithelia results from an angle constraint in contiguous polygonal lattices

It has long been noted that the cell arrangements in epithelia, regardless of their origin, exhibit some striking regularities: first, the average number of cell neighbours at the apical side is (close to) six. Second, the average apical cell area is linearly related to the number of neighbours, such that cells with larger apical area have on average more neighbours, a relation termed Lewis law. Third, Aboav-Weaires (AW) law relates the number of neighbours that a cell has to that of its direct neighbours. While the first rule can be explained with topological constraints in contiguous polygonal lattices, and the second rule (Lewis law) with the minimisation of the lateral contact surface energy, the driving forces behind the AW law have remained elusive. We now show that also the AW law emerges to minimise the lateral contact surface energy in polygonal lattices by driving cells to the most regular polygonal shape, but while Lewis law regulates the side lengths, the AW law controls the angles. We conclude that global apical epithelial organization is the result of energy minimisation under topological constraints.

biophysics↗