Effect of Trp53 mutations on mouse oviductal epithelial cell spheroids
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE227681 | GEO | 2023/06/13
REPOSITORIES: GEO
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Project description:Spheroids are 3D multi-cell aggregates formed in non-addherent culture conditions. In ovarian cancer (OC), they serve as a vehicle for cancer cell dissemination in the peritoneal cavity. We investigated genes and networks upregulated in three dimensional (3D) versus two-dimensional (2D) culture conditions by Affymetrix gene expression profiling and identified ALDH1A1, a cancer stem cell marker as being upregulated in OC spheroids. Network analysis confirmed ALDH1A1 upregulation in spheroids in direct connection with elements of the beta-catenin pathway. A parallel increase in the expression levels of beta-catenin and ALDH1A1 was demonstrated in spheroids vs. monolayers an in successive spheroid generations by using OC cell liness and primary OC cells. The percentage of Aldefluor positive cells was significantly higher in spheroids vs. monolayers in IGROV1, A2780, SKOV3, and primary OC cells. B-catenin knock-down decreased ALDH1A1 expression and chromatin immunoprecipitation demonstrated that beta-catenin directly binds to the ALDH1A1 promoter. Both siRNA mediated beta-catenin knock-down and a novel ALDH1A1 small molecule enzymatic inhibitor described here for the first time, decreased the number of OC spheroids (p<0.001) and cell viability. These data strongly support the role of beta-catenin regulated ALDH1A1 in the maintenance of OC spheroids and of a stem cell phenotype and propose new ALDH1A1 inhibitors targeting this cell population. Different gene profiles were observed in ovarian cancer spheroids versus ovarian cancer monolayers. Nine samples were analyzed in triplicate. Each group is a reference.
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Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).
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Project description:Cell-based approaches utilizing autologous human renal cells require their isolation, expansion in vitro and reintroduction back into the host for renal tissue regeneration. Nevertheless, human kidney epithelial cells (hKEpC) lose their phenotype, dedifferentiate and assume the appearance of fibroblasts after relatively few passages in culture. We hypothesized that growth conditions may influence hKEpC phenotype and function. hKEpC retrieved from human nephrectomy tissue samples showed the ability to reproducibly form kidney-spheres when grown in suspension culture developed in non-adherent conditions. Genetic labeling and time-lapse microscopy indicated, at least in part, the aggregation of hKEpC into 3D-spheroids rather than formation of pure clonally expanded spheres. Characterization of hKEpC spheroids by real-time PCR and FACS analysis showed up-regulation of some renal developmental and 'stemness' markers compared to monolayer and mostly an EpCAM+CD24+CD133+CD44+ spheroid cell phenotype. Oligonucleotide microarrays which were used to identify global transcriptional changes accompanying spheroid formation, showed predominantly up-regulation of cell matrix/cell contact molecules and cellular biogenesis processes and down-regulation of cell cycle, growth and locomotion. Accordingly, hKEpC spheroids proliferated slowly as indicated by low Ki-67 staining, but when grafted in low cell numbers onto the chorioallantoic membrane (CAM) of the chick embryo, they exclusively reconstituted various renal tubular epithelia. Moreover, efficient generation of kidney spheroids was observed after long-term monolayer culture resulting in re-establishment of tubulogenic capacity upon CAM grafting. Thus, generation of a specialized quiescent niche in hKEpC spheroids may provide a functional benefit for kidney-derived cells in vivo. 6 samples, 3 adherent and 3 spheroids samples
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