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Gene expression profiling of PDX-derived lung squamous carcinoma cells (TUM622) grown in 2D vs. 3D culture

ABSTRACT: Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding tumor microenvironment (TME). Investigation of tumor cell-TME interactions could be greatly facilitated by models that mimic human disease on both the genotypic and phenotypic levels. Three dimensional (3D) cultures represent an important means to study the impact of tumor cell-TME interactions on specific aspects of neoplastic phenotypes. Here, we developed a novel 3D culture system (termed TUM622) derived from a patient-derived xenograft (PDX) of a lung squamous carcinoma (LUSC) that was grown in extracellular matrix (Damelin et al, Cancer Research; 71(12) June 15, 2011). Single TUM622 cells established acinar-like structures with proper apical-basal polarity, remained non-motile and demonstrated heterogeneous expression of stem-like and differentiation markers similar to the original PDX model and patient tumor, but nonetheless exhibited hyperplasia. Transcriptional profiling and gene set enrichment analysis (GSEA) revealed many similarities to the established acinar models, as well as modulation of the Wnt signaling pathway and stem cell signatures. These results demonstrate that the tumor microenvironment significantly influences the plasticity of NSCLC tumor cells. In order to gain a better understanding of the cell intrinsic mechanisms regulating TUM622 acinar morphogenesis in 3D, we performed transcriptional profiling of TUM622 cells collected from 2D and 3D cultures.We identified genes that vary more than 1.5-fold with reproducible changes among the biological replicates (p < 0.05) and found a total of 9227 genes differentially expressed, indicating that changing culture conditions from 2D to 3D exerts a major influence on gene expression in TUM622 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122538 | GEO | 2018/12/19

REPOSITORIES: GEO

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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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Gene expression profiling of PDX-derived lung squamous carcinoma cells (TUM622) grown in 2D vs. 3D culture

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