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Gene expression dynamics of branching morphogenesis in pancreatic cancer organoids

ABSTRACT: The transcriptional dynamics occurring at the self-organizing glandular epithelial during branching morphogenesis are pivotal for deciphering various processes ranging from normal tissue growth to cancer formation. For the past decade matrigel© has been widely used as extracellular matrix for the propagation of organoids, here we report that only in the presence of a higher than 70% collagen matrix symmetry breaking can be observed. Comparing the transcriptomic profile of collagen and matrigel grown-organoids we identified that matrigel organoids are more basal-like, have a higher EMT signature (GSEA) compared to the more classical collagen grown organoids. In this study we used single primary murine pancreatic ductal adenocarcinoma cells (PDAC) cells embedded in floating collagen gels and adapted the media supplementation, thus allowing organoids to self-organize into highly complex branched structures, replicating the in vivo tumour architecture. During organoid development we identified four distinct morphogenetic phases, each characterized by a unique pattern of cell proliferation, invasion, matrix deformation and protein expression. In our search for novel molecular drivers of branching morphogenesis we compared the transcriptome of two distinct phases, an early development (day 7) vs a late mature time point (day 13). We compared these two distinct developmental time points in using PCA, GSEA and respective heat maps of major processes illustrating the overexpression of genes associated with: proliferation, ECM interaction, signalling by Rho GTPases and EMT in day-7 organoids and genes associated with ion channel transport at day-13 organoids. An important aspect raised during our investigation was the effect of different genotypes (Ptf1aCre/+; KrasG12D/+, Pdx1Cre/+; KrasG12D/+; TP53fl/fl) on the branch formation of organoids. To this end we generated organoids derived from KC and KPC tumours, which interestingly gave rise to strikingly similar branching organoids. Although the morphology was not significantly altered the transcriptional profiling showed higher proliferation (Myc targets, E2F targets), EMT score for the KPC organoids. We propose that the spatiotemporal synchronized processes of cell proliferation, matrix remodeling, contraction and ion channel flux are key-events of the different morphogenic phases and lead to the formation of these complex structures. Importantly, these dynamic processes are accompanied by strong transcriptional profile changes, with the organoids undergoing de-differentiation during branch elongation and later on activating an epithelial gene expression program upon maturation. Taken together, these results illustrate the ability of tumour cells to self-organize in multicellular complex structures and provide with a novel system to study branching morphogenesis and tumour biology in vitro.

ORGANISM(S): Mus musculus

PROVIDER: GSE200308 | GEO | 2022/08/02

REPOSITORIES: GEO

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Project description:The transcriptional dynamics occurring at the self-organizing glandular epithelial during branching morphogenesis are pivotal for deciphering various processes ranging from normal tissue growth to cancer formation. For the past decade matrigel© has been widely used as extracellular matrix for the propagation of organoids, here we report that only in the presence of a higher than 70% collagen matrix symmetry breaking can be observed. Comparing the transcriptomic profile of collagen and matrigel grown-organoids we identified that matrigel organoids are more basal-like, have a higher EMT signature (GSEA) compared to the more classical collagen grown organoids. In this study we used single primary murine pancreatic ductal adenocarcinoma cells (PDAC) cells embedded in floating collagen gels and adapted the media supplementation, thus allowing organoids to self-organize into highly complex branched structures, replicating the in vivo tumour architecture. During organoid development we identified four distinct morphogenetic phases, each characterized by a unique pattern of cell proliferation, invasion, matrix deformation and protein expression. In our search for novel molecular drivers of branching morphogenesis we compared the transcriptome of two distinct phases, an early development (day 7) vs a late mature time point (day 13). We compared these two distinct developmental time points in using PCA, GSEA and respective heat maps of major processes illustrating the overexpression of genes associated with: proliferation, ECM interaction, signalling by Rho GTPases and EMT in day-7 organoids and genes associated with ion channel transport at day-13 organoids. An important aspect raised during our investigation was the effect of different genotypes (Ptf1aCre/+; KrasG12D/+, Pdx1Cre/+; KrasG12D/+; TP53fl/fl) on the branch formation of organoids. To this end we generated organoids derived from KC and KPC tumours, which interestingly gave rise to strikingly similar branching organoids. Although the morphology was not significantly altered the transcriptional profiling showed higher proliferation (Myc targets, E2F targets), EMT score for the KPC organoids. We propose that the spatiotemporal synchronized processes of cell proliferation, matrix remodeling, contraction and ion channel flux are key-events of the different morphogenic phases and lead to the formation of these complex structures. Importantly, these dynamic processes are accompanied by strong transcriptional profile changes, with the organoids undergoing de-differentiation during branch elongation and later on activating an epithelial gene expression program upon maturation. Taken together, these results illustrate the ability of tumour cells to self-organize in multicellular complex structures and provide with a novel system to study branching morphogenesis and tumour biology in vitro. In the updated submission we include new data sets from our recent publication. More specifically we focus on how we can capture inter- and intratumoral heterogeneity using our previously published organoid system (Randriamanantsoa, Papargyriou et al. 2022) of pancreatic cancer and how these morphologically diverse organoids respond to targeted therapy. We aim to understand the following four biological questions: First, we aim to address intertumoral heterogeneity by culturing classical and basal-like PDAC cells in 2D culture conditions and 3D floating collagen gels. Indeed, in both cases we observe a clear separation between mesenchymal and epithelial organoids and describe pivotal pathways (f.e. TGF pathway) that are differentially regulated between the two culture conditions (2D vs 3D). In detail, we culture 3 epithelial (ID: 8442, 9591, 53631) and 3 mesenchymal (ID: 8028, 9091, 16992) murine pancreatic cancer lines in established 2D conditions and in 3D floating collagen gels. Next, we focus on understanding the transcriptional background of distinct organoid morphologies derived from the same parental cell lines of either epithelial or mesenchymal origin. In more detail we transcriptionally analyze specific epithelial (line ID 9591) as a bulk population and their subclones: TEBBO, cystic branched, thick-branched and tree-like and mesenchymal pancreatic cancer line (line ID 16992) as a bulk population and their subclones: branched mesenchymal, firework and star-like. After performing a 102-drug screening on these clones we identified potent target therapy drugs acting in specific phenotypes. To validate the effects on the transcriptional level we treated all the epithelial organoid phenotypes with the combination of AZD5153 (BET/BRD4 inhibitor)+Poziotinib (pan-HER inhibitor) and the mesenchymal phenotypes with monotreatments of the following drugs: Birinapant (SMAC antagonist) Poziotinib (pan-HER inhibitor), Saracatinib (Src inhibitor) and RO5126766 (RAF/MEK inhibitor). Furthermore, to translate our findings in a human setting we developed a new media composition which enables the formation of branching organoids in pancreatic cancer patient-derived organoids (PDOs). We examined the effect of our newly developed media, the Basal Branching PDO Media on the transcriptome of 3 PDOs (PDO line ID: B211, B250, B320) compared to previously established media conditions such as the Full PDO Media.

Project description:MicroRNAs (miRNAs) regulate developmental events such as branching morphogenesis, epithelial to mesenchymal transition (EMT) and its reverse process mesenchymal to epithelial transition (MET). In this study, we performed small RNA sequencing of a breast epithelial progenitor cell line (D492), and its mesenchymal derivative (D492M) cultured in three-dimensional reconstituted basement membrane (3D-rBM) matrix. Large differences in miRNA expression were seen between D492 and D492M. Among the most downregulated miRNAs in D492M was miR-203a, a miRNA that plays an important role in epithelial differentiation. When analyzed over time (21 days) in 3D culture, increased expression of miR-203a was seen in D492, concomitant with increased complexity of the branching structures. When miR-203a was overexpressed in D492M (D492MmiR-203a), a partial reversion towards epithelial phenotype was seen. Gene expression analysis of D492M and D492MmiR-203a revealed that peroxidasin (PXDN), a collagen IV cross-linker was one of the most significantly downregulated genes in D492MmiR-203a. Bioinformatic analysis revealed that PXDN has three putative binding sites for miR-203a in its 3-prime UTR. When a miR-203a mimic was transfected into D492M, reduced PXDN expression was seen. Conversely, inhibition of miR-203a in D492MmiR-203a with a miR-203a inhibitor increased PXDN expression, demonstrating that miR-203a acts as a repressor of PXDN. To verify the binding of miR-203a to the 3’-UTR of PXDN we set up a luciferase reporter assay that confirmed the binding. Collectively, we have demonstrated that miR-203a expression temporally correlates with branching morphogenesis in 3D-rBM and is suppressed in the mesenchymal state. Overexpression of miR-203a in D492M induces a partial MET and reduces the expression of PXDN. Furthermore, we demonstrate that miR-203a is a novel repressor of PXDN. MiR-203-PXDN axis may be an important regulator in branching morphogenesis, EMT and basement membrane remodeling. \

Project description:Full title: Comprehensive Characterization of Three-Dimensional Models for Prostate Cancer Growth and Invasion in Laminin-rich Extracellular Matrix Prostate Cancer (PrCa) cells undergo acinar morphogenesis and spheroid formation in three-dimensional (3D) culture, supported by laminin-rich extracellular matrix (lrECM, Matrigel). We developed miniaturized 3D model systems that facilitate investigation of morphogenesis and invasion of normal and PrCa cell lines in lrECM. Primary and non-transformed cell lines formed round structures with strong cell-cell contacts and epithelial polarization, lumen and a complete basal lamina (BL). In contrast, most PrCa cell lines formed either defective, “mass” spheroids with incomplete BL, or invasive “stellate” structures. The bioinformatic analyses of genome-wide mRNA expression data revealed massive alteration of key functional and signaling pathways in 3D cultures, with lipid and steroid metabolism, epigenetic reprogramming, and differentiation-related transcription factors induced across all cell lines by lrECM. In invasive cells, AKT, PI3Kinase, mTOR, and hedgehog signaling pathways were most highly activated, validated by small molecule inhibitors compounds specifically targeting key regulatory molecules. Compounds against AKT and PI3kinase pathways were significantly more effective in invasive cells, compared to mass or round/normal phenotype spheroids, and monolayer culture. A severe morphologic conversion was observed in PC-3 and PC-3M cells, transforming initially round, normal-appearing epithelial spheroids into rapidly invading cell masses. Markers for EMT (epithelial-mesenchymal transition) were highly expressed already in early stage, round spheroids prior to invasive conversion, and were not further increased in invasive cells. This indicates that PrCa cells can display extraordinary plasticity. EMT may be involved in providing a metastable genotype that allows morphological transformation, but is not be required for invasive processes themselves. Total RNA was obtained from non-transformed prostate epithelial cells and prostate cancer cells cultured in monolayer and three-dimensional laminin-rich extracellular matrix (growth factor-reduced Matrigel).

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Gene expression dynamics of branching morphogenesis in pancreatic cancer organoids

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