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Personalizing the treatment of head and neck cancer in vitro: The 3D-OTC model

ABSTRACT: Background: Treatment of head and neck squamous cell carcinoma (HNSCC) remains challenging and the survival rates of affected patients remain poor. A three-dimensional organotypic co-culture (3D-OTC) model where patient derived tumor tissue is cultured on human-derived fibroblasts (dermal equivalent, DE) was evaluated regarding its comparability to primary tumor tissue and the identification of potential individualized therapeutic targets. Methods: 3D-OTC models were cultured from n=10 HNSCC patients for up to 21 days. The growth pattern at the DE was compared to tumor budding of corresponding resection specimens. Furthermore, we immunohistochemically determined the immune cell infiltrate of primary tumor tissue and corresponding 3D-OTC models. Spatially resolved gene expression analysis (“Xenium in situ”) was performed for separate regions of interest within the 3D-OTC specimens and within primary tumor tissue. Up-regulated and down-regulated genes of the 3D-OTC samples were included in gene set enrichment analysis and up-regulated genes between invasive (invading the DE) and non-invasive tumor cells within the 3D-OTC samples were included in drug sensitivity analyses using publicly available cell line data. Results: The growth pattern observed at the DE was associated with tumor budding in primary tumor tissue. The density of CD3-/CD20-/CD56-positive cells was lower in 3D-OTC samples compared to primary tumor tissue. No such changes were observed for CD68-positive cells and no significant changes in the density of the immune cell infiltrate were detected during the cultivation period. The centroids and dispersion of the gene expression of the 3D-OTC samples did not differ from the corresponding primary tumor tissue. The regions of interest within the 3D-OTC samples showed distinct functional states in gene set enrichment analysis. The comparison of genes up-regulated in invasive tumor parts of the 3D-OTC samples with cell line data revealed potential therapeutic targets. Conclusion: The 3D-OTC model morphologically and transcriptomically resembles primary tumor tissue and its biology while preserving the tumor microenvironment. Furthermore, the 3D-OTC model allows the standardized evaluation of tumor tissue by the definition of transcriptomically separate regions of interest and thus, could help to evaluate the impact of personalized therapeutic interventions on the tumor and its microenvironment in vitro.

ORGANISM(S): Homo sapiens

PROVIDER: GSE324934 | GEO | 2026/05/11

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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Personalizing the treatment of head and neck cancer in vitro: The 3D-OTC model

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