Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype

Abstract

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems

“…Evidence of this was also provided in a recent characterization of the used 3D models by Braun et al. describing core region with necrotic and damaged cells, similar to tumors from GBM patients ( 11 ). In addition to the necrotic areas of these complex models, it was observed that an outer layer with proliferating cells circumscribed the organoid.…”

“…Since in 3D organoids, cells in the core of the cell aggregate may have reduced access to essential nutrients, hypoxia and nutrient deficiency might trigger stress responses (74). Evidence of this was also provided in a recent characterization of the used 3D models by Braun et al describing core region with necrotic and damaged cells, similar to tumors from GBM patients (11). In addition to the necrotic areas of these complex models, it was observed that an outer layer with proliferating cells circumscribed the organoid.…”

“…showed that 3D GBM models based on the same tumor specimens exhibited significantly altered mRNA expression patterns compared to corresponding 2D models. Single-cell sequencing also revealed changes in immunoregulatory signaling pathways, such as IL-4 and IL-13 ( 11 ). Interestingly, our smallRNA-Seq supports this finding, as the miRNA cargo in 3D cultures are associated with the IL-4 and IL-13 cytokine signaling pathway.…”

“…Conventional cultures (2D) were maintained in standard cell culture flasks and passaged when 80% confluency was reached. Tumor organoid models (3D) were generated by mixing 20000 GBM cells in 80 µl Matrigel™ largely following a previously published protocol ( 11 , 17 ). 3D cultures were further kept on an orbital shaker to prevent attachment, multiple 3D spheroids were kept in 5 ml petri dish.…”

“…One promising approach to ameliorate some aspects of in vitro (2D) culturing are 3D tumor organoid models. They are shown to maintain and regenerate a heterogeneous tumor cell population and thereby better represent the complex microenvironment found in GBM ( 10 , 11 ).…”