Lactate receptor GPR81 drives breast cancer growth and invasiveness through regulation of ECM properties and Notch ligand DLL4
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE203441 | GEO | 2022/05/23
REPOSITORIES: GEO
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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).
2021-10-01 | E-MTAB-10862 | biostudies-arrayexpress
Project description:Tumor microenvironments present significant barriers to anti-tumor agents. Molecules involved in multicellular tumor microenvironments, however, are difficult to study ex vivo. Here, we generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers. Gene ontology analysis revealed that upregulated genes were primarily related to immune response, wound response, lymphocyte stimulation and response to cytokine stimulation, whereas downregulated genes were primarily associated with apoptosis. Among the 142 genes, 27 are located in the membrane and related to biologic processes of cellular movement, cell-to-cell signaling, cellular growth and proliferation and morphology. Western blot analysis validated elevation of MMP2, BAFF/BLyS/TNFSF13B, RANTES/CCL5 and TNFAIP6/TSG-6 protein expression in spheroids as compared to monolayers. Thus, we have reported the first large scale comparison of the transcriptional profiles using an ex vivo matrix-free spheroid model to identify genes specific to the three-dimensional biological structure of tumors. The method described here can be used for gene expression profiling of tumors other than mesothelioma. We generated a matrix-free tumor spheroid model using the NCI-H226 mesothelioma cell line and compared the gene expression profiles of spheroids and monolayers using microarray analysis. Microarray analysis experiments were performed in triplicates. A total of six samples (three for monolayers and three for spheroids) were analyzed.
2012-06-20 | E-GEOD-37629 | biostudies-arrayexpress
Project description:Cancer progression and wound healing share some characteristics. Most colorectal cancers are differentiated adenocarcinomas that maintain three-dimensional structures to some extent. Hence, disruption of the architecture can provoke remodeling similar to the remodeling of normal intestinal epithelium. We used our recently developed three-dimensional culture system to investigate the response of cancer cell spheroids to mechanical disruption. Specifically, we developed a protocol for homogenous disruption of the spheroids that maintained the cell-cell contacts. After disruption, 9 spheroids from 9 patient samples reformed within a few hours, and 2 showed accelerated spheroid growth. Stemness increased after spheroid disruption, as assessed by marker expression, spheroid forming capacity, radiation sensitivity, and tumorigenesis. The spheroid-forming capacity increased in 6 of 11 spheroids. The disruption signature, as determined by gene expression profiling, supported the incidence of remodeling and predicted the prognosis of the colorectal cancer patients. WNT and HER3 signaling was increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increases in growth and stemness after disruption. Thus, disorganized architecture in patient tumors might reflect the processes of disruption and subsequent remodeling and represent a cause rather than simply a consequence of malignancy progression. Gene expression in cancer tissue-originated spheroids (CTOSs) was measured at pre, 6 hr, 24 hr, and 4 days after mechanical disruption. One experiment was performed at each time point.
2015-12-10 | E-GEOD-75867 | biostudies-arrayexpress
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.
2014-01-03 | E-GEOD-53759 | biostudies-arrayexpress
Project description:Immortal spheroids were generated from fetal mouse intestine using the culture system developed to culture organoids from adult intestinal epithelium. Spheroids are made of a monostratified polarized epithelium displaying a poorly differentiated intestinal phenotype. The proportion of spheroids generated from intestinal explants progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Spheroid cells show indefinite self-renewing properties but exhibit a transcriptome strikingly different from that of adult intestinal stem cells reminiscent of incompletely caudalized progenitors. The receptor Lgr4, but not Lgr5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the E14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, thus qualifying them as a new type of intestinal stem-like cells. Two-channel microarray experiments were performed from spheroid/organoid pairs isolated each from a given embryo. Following initial seeding of small intestine from a given embryo/mouse (at E16, E18 or P0), spheroids and organoids were selectively picked up for each animal and replated for 3 passages to reach sample homogeneity. Hybridization was performed on the 4 independent pairs with dye-swap.
2014-01-21 | E-GEOD-49803 | biostudies-arrayexpress
Project description:Summary: Melanoma spheroids grown under neural crest cell conditions are highly plastic migratory/invasive tumor cells endowed with immunomodulator function Background: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi subpopulations of cancer cells some of which may possess stem cell-like properties supporting the notion of plasticity. Although useful for certain tumors, the use of the sphere-formation assay to identify stem cell-like or tumor initiating cells subpopulations in human melanoma has been recently challenged. Our study reveals that this assay predicts a functional phenotype associated with aggressive behavior of tumor cells. Methodology/Principal Findings: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic (SLM8) or advanced primary (Mela1) tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages, and showed enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not extensive self-renewal or enhanced tumorigenicity when compared to their adherent counterparts. To determine whether melanoma spheroids in our model could predict a molecular or functional phenotype, we performed gene expression profiling experiments using Affymetrix microarrays. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that these spheroids are endowed with enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Thus, our findings reveal novel immune-modulator function of melanoma spheroid cells and suggest specific roles for these spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroid cells reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and could therefore, be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability. 12 Total samples were analyzed: SLM8 adherent (SLMA) and spheroids (SLMS) cells, and Mela1 adherent (MelaA) and spheroid (MelaS) cells, all performed in triplicates. Paired statistical analyses were performed using Student's paired t-test on the gene signal intensities (gene level) and results were considered statistically significant at p-values <=0.05 and fold-change >=1.5.
2011-03-28 | E-GEOD-26980 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE14818: Gene expression analysis of glioblastoma spheroid cultures I GSE14819: Array CGH analysis of glioblastoma serum grown adherent cultures GSE14820: Array CGH analysis of glioblastoma cell lines GSE14821: Array CGH analysis of single spheroids from glioblastoma spheroid cultures GSE14822: Array CGH analysis of glioblastoma spheroid cultures GSE14823: Array CGH analysis of glioblastomas GSE16666: Gene expression analysis of glioblastoma spheroid cultures II Refer to individual Series
2010-02-12 | E-GEOD-14824 | biostudies-arrayexpress
Project description:Intro: Spheroid culture especially on MCF-7 cell has been used as in vitro CSC model for anti-CSC drug discovery. The role of miRNAs in the regulation of mRNA specifically looking at the self-renewal capacity and the drug resistance of the spheroid-enriched CSCs models has not been evaluated. Therefore, a comprehensive profiling of the miRNAs will provide an insight into the regulatory mechanisms associated with breast cancer and CSCs. Methods: Tumour spheroid of MCF-7 was generated and characterised for the biological features and CSCs characteristic comparing to the monolayer parental MCF-7 cells. Differential expression of miRNA between the spheroid and parental cells was evaluated using next generation sequencing (NGS). The differentially expressed miRNAs were then subjected to target genes predictions, followed pathway analyses to better understand the mechanism associated with spheroid-enriched CSCs. Results: Spheroids generated from MCF-7 cell line under serum-free condition were found to be enriched with CSCs characteristics. miRNA-NGS analysis revealed 119 differentially expressed miRNAs between the spheroids and parental, with 24 up-regulated and 94 down-regulated. The gene ontology (GO) analysis showed that the predicted genes of the differentially expressed miRNAs were enriched in various biological processes. Pathway analysis revealed that the differentially expressed miRNAs and their target genes were associated with a variety of KEGG pathways, which could explain the self-renewal capability, and the higher drug resistance in spheroids when compared to parental.
2016-05-10 | GSE68246 | GEO
Project description:cancer spheroid has been widely used as in vitro model of cancer stem cells (CSCs), yet little is known about their phenotypic characteristics and microRNAs (miRNAs) expression profiles. The objectives of this research were to evaluate the phenotypic characteristics of MDA-MB-231 spheroid-enriched cells for their CSCs properties and also to determine their miRNAs expression profile. Similar to our previously published MCF-7 spheroid, MDA-MB-231 spheroid also showed typical CSCs characteristics namely self-renewability, expression of putative CSCs-related surface markers and enhancement of drug resistance. From the miRNA profile, miR-15b, miR-34a, miR-148a, miR-628 and miR-196b were shown to be involved in CSCs-associated signalling pathways in both models of spheroids which highlights the involvement of these miRNAs in maintaining the CSCs features. In addition, unique clusters of miRNAs namely miR-205, miR-181a and miR-204 were found in basal-like spheroid whereas miR-125, miR-760, miR-30c and miR-136 were identified in luminal-like spheroid. Our results highlight the roles of miRNAs as well as providing novel perspectives of the relevant pathways underlying spheroid-enriched CSCs in breast cancer. Keywords: Breast
2016-11-17 | GSE75396 | GEO
Project description:Cancer progression and wound healing share some characteristics. Most colorectal cancers are differentiated adenocarcinomas that maintain three-dimensional structures to some extent. Hence, disruption of the architecture can provoke remodeling similar to the remodeling of normal intestinal epithelium. We used our recently developed three-dimensional culture system to investigate the response of cancer cell spheroids to mechanical disruption. Specifically, we developed a protocol for homogenous disruption of the spheroids that maintained the cell-cell contacts. After disruption, 9 spheroids from 9 patient samples reformed within a few hours, and 2 showed accelerated spheroid growth. Stemness increased after spheroid disruption, as assessed by marker expression, spheroid forming capacity, radiation sensitivity, and tumorigenesis. The spheroid-forming capacity increased in 6 of 11 spheroids. The disruption signature, as determined by gene expression profiling, supported the incidence of remodeling and predicted the prognosis of the colorectal cancer patients. WNT and HER3 signaling was increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increases in growth and stemness after disruption. Thus, disorganized architecture in patient tumors might reflect the processes of disruption and subsequent remodeling and represent a cause rather than simply a consequence of malignancy progression.
2015-12-10 | GSE75867 | GEO