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Effect of 2D and 3D cell culture system on trophoblast extracellular vesicles physio-chemical characteristics and potency

ABSTRACT: In this project, trophoblast cell analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension cultures (3D) and how the cell growth environment affects the physical and biochemical properties of EVs produced by them was studied. Importantly, proteomic cargo profile of EVs derived from 2D and 3D cultures was compared. There was substantial differences in the proteomic cargo profile and potency of EVs derived from two culture systems. Findings of this study will help in selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Placenta, Epithelial Cell, Cell Culture

SUBMITTER: SUBHASHINI MUHANDIRAM MUHANDIRAM RALALAGE

LAB HEAD: Alireza Fazeli

PROVIDER: PXD048789 | Pride | 2024-05-08

REPOSITORIES: Pride

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Project description:Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterise extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems.Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterisation was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analysed by high-resolution mass spectrometry.Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR- 323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response.Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

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Effect of 2D and 3D cell culture system on trophoblast extracellular vesicles physio-chemical characteristics and potency

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