Project description:Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. The goals and objectives of this study were 1.) to characterize and validate the molecular identity of human primary epithelial and stromal/mesenchymal breast cells maintained long-term in novel ex vivo culture conditions in serum free medium. 2.) To analyze changes in gene expression profiles of normal human primary epithelial and stromal/mesenchymal breast cells upon long-term ex vivo co-culture when compared to corresponding monocultures 3.) To study the dynamic reciprocity between normal human primary epithelial and stromal/mesenchymal breast cells. 4.) To identify critical molecular pathways and biomarkers controlling epithelial and/or stromal cell growth and quiescence. Human primary epithelial progenitor cells and mesenchymal stem cells bearing fluorescent tags were either co-cultured in novel ex vivo culture conditions on ECM coated meshes in serum free medium (M5) or cultured as monocultures in the same conditions for 30 days. The cultures were then dissociated and epithelial and stromal/mesenchymal cells from either co-cultures or monocultures separated by FACS. Gene expression profiling of epithelial or stromal/mesenchymal cells was performed. Clean gene expression profiles from three different epithelial and stromal/mesenchymal cell extracts either grown in co-cultures or monocultures were successfully obtained.
Project description:The aim of this study is to compare the transcriptomic profiles of various ex vivo models of pancreatic cancer. First, primary tumours and their corresponding xenografts in nude mice were analysed by RNA-seq. Monocultures of pancreatic cancer cells derived from the xenografts were then prepared as 2D monolayers, Matrigel-embedded organoids, spheres in suspension and 3D cultures in self-assembling peptide amphiphile (PA) hydrogels. Seven-day cultures were then analysed by RNA-seq. The results suggest that all ex vivo monocultures retain patient-specific transcriptional profiles, especially cancer stem cell signatures, while being deficient in the expression of stromal components such as the core matrisome. Correlations with the primary tumours were generally higher in PA hydrogels than organoids. Biased gene expression signatures were identified in certain models. This is the first study to explore the transcriptomic signatures of four different ex vivo models matched to their primary tumours of origin.
Project description:Bacterial-fungal monocultures and co-cultures in which Penicillium solitum #12 are grown with one of 4 bacterial growth partners (Glutamicibacter arilaitensis JB182, Brevibacterium linens JB5, Pseudomonas psychrophila JB418, Escherichia coli K12) grown on 10% cheese curd agar (CCA) and extracted using acetonitrile. Extracts were analyzed via LC-MS(/MS) and processed with MZmine2 for analysis with MetaboAnalyst 5.0 and GNPS feature based molecular networking. Dataset 3 contains monocultures and pairwise co-cultures in which full plate (100 mm) extractions were performed. Dataset 4 contains monocultures in which plug (30 mm) extractions were performed. Dataset 5 contains monocultures and pairwise co-cultures in that were grown regularly or on nitrocellulose membranes using plate count agar with milk and salt (PCAMS).
Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.
Project description:Analysis of LNCaP cell molecular differences in monocultures and in co-cultures in the presence of R1881. Human osteoblast molecular signatures were also identified from the tissue engineered bone monocultures. LNCaP cells molecular profile was altered by co-culturing with human osteoblasts compared to LNCaP monocultures with or without R1881 stimulations. These results provide insights into the behavioral change of LNCaP cells in a bone-like microenvironment.
Project description:These 12 arrays are the basis for Figure 1 of the "An interferon-response induced by tumor-stroma interaction in a subset of human breast cancers" manuscript. Figure 1: Effect of heterotypic interaction between breast cancer cell line MDA-MB231 and CCL-171 fibroblast. Biologically independent replicates of the mono-cultured fibroblast CCL-171, the breast cancer cell line MDA-MB231 and the mixed co-culture of CCL-171 and MDA-MB231 were grown for 48h at low serum conditions and characterized by DNA microarray hybridization. Hierarchical clustering of a total of 4333 elements that display a greater than 3-fold variance in expression in more than 3 different experimental samples. Data from individual elements or genes are represented as single rows, and different experiments are shown as columns. Red and green denote expression levels of the samples. The intensity of the color reflects the magnitude of the deviation from baseline. Unsupervised hierarchical clustering of the experiments grouped the biological replicates together. Gene expression varied considerably between fibroblast and MDA-MB231 as expected for cells of mesenchymal or epithelial origin respectively. The co-culture profile showed mainly intermediate expression levels. However, the vertical black bar marks a cluster of genes induced in all co-cultures compared to both mono-cultures indicating that they are induced by heterotypic interaction Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:To study the differences in gene expression between PDAC organoids grown either as monocultures or co-cultures with panctreatic stellate cells in normoxia and hypoxia.
Project description:Analysis of LNCaP cell molecular differences in monocultures and in co-cultures in the presence of R1881. Human osteoblast molecular signatures were also identified from the tissue engineered bone monocultures. LNCaP cells molecular profile was altered by co-culturing with human osteoblasts compared to LNCaP monocultures with or without R1881 stimulations. These results provide insights into the behavioral change of LNCaP cells in a bone-like microenvironment. In this study, LNCaP cells cultured in the hydrogel were prepared and co-cultured with or without human osteoblasts (in the form oftissue engineered bone). Similarly, tissue engineered bone monocultures were also prepared 4-6 weeks earlier before co-culturing with the LNCaP cells. These cultures were maintained up to 24 days in RPMI growth media (+10% FBS) before they were androgen-starved for 48 hours. Cells were either treated with 1nM R1881 or continued to be androgen-deprived (without R1881 with 0.008% ethanol) for another 48 hours prior to cell harvest for gene expression analysis. Biological triplicates were prepared for each condition.
Project description:Objective: Cartilage formation is stimulated in mixtures of chondrocytes and human adipose-derived mesenchymal stem cells (MSCs) both in vitro and in vivo. During co-culture, human MSCs perish through an unknown process. The goal of this study is to elucidate the mechanism by which adipose tissue-derived MSC cell death occurs in the presence of chondrocytes. Methods: Human primary chondrocytes were co-cultured with human MSCs derived from three donors. The cells were cultured in mono-culture or co-culture (20% chondrocytes and 80% MSCs) in pellets (200.000 cell/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or co-culture. Western blot and immune fluorescence assays were performed to quantify the level of Caspase-3, LC3B and P62. Results: RNA sequencing revealed significant up-regulation of >90 genes in the three co-cultures when compared to monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Western blotting shows a clear up-regulation of the autophagic machinery with no substantial activation of the apoptotic pathway. Conclusion: In co-cultures of human MSCs with primary chondrocytes, MSCs perish through autophagy. We propose that this sacrificial cell death may perhaps contribute to the trophic effects of MSCs on cartilage formation.