An advanced patient-derived organoid co-culture system for investigating immune interactions and therapeutic strategies in glioblastoma

Glioblastoma (GBM) is a highly aggressive, incurable primary brain tumor with dismal prognosis. The GBM tumor microenvironment (TME) is composed of infiltrating brain-derived and blood-derived immune cells. However, GBM is characterized as a cold tumor with a strong immunosuppressive microenvironment and is largely unresponsive to current immunotherapies. Understanding the functional crosstalk between tumor cells and immune components across distinct microenvironmental niches has been limited due to the lack of GBM preclinical models that reconstitute the complex TME.
To this aim, we utilized bulk and single-cell RNA-sequencing, flow cytometry and functional assays to interrogate the immunological status of GBM cells and the phenotypic states constituting the immune compartment in GBM patient tumors and patient-derived models. While the majority of GBM cells were Major Histocompatibility Complex class-II (MHC-II) negative, MHC-I expression was retained in various tumor niches and could be further increased upon 24-hour exposure to IFN-y in all GBM tumor cell cultures. Tumor cells had low basal expression of PDL-1, which was elevated following IFN-y exposure but not hypoxia. The immune cell composition of the TME included monocyte and microglia-derived tumor-associated macrophages as well as T cells, presenting T regulatory and exhausted phenotypes. CellChat-based ligand-receptor interaction analysis revealed multiple crosstalk axes between GBM tumor cells, heterogeneous tumor-associated macrophages and T cells.
To functionally investigate the dynamic immune ecosystem of GBM tumors ex vivo, we further established co-cultures from GBM patient-derived organoid (PDO) with T cells isolated from GBM patients or healthy donor blood as 3D organoids. We showed that such co-cultures recapitulate the functional crosstalk observed in patient tumors, where GBM tumor cells directly influence the phenotypes of T cells. As a consequence of direct crosstalk, we observed elevated expression of exhaustion markers LAG-3 and TIM-3, an increased Treg frequency amongst T cell infiltrates within GBM organoids compared to non-infiltrated T cells.
Finally, we utilized this co-culture system to test the efficacy of a Class II Transactivator (CIITA)-armed adenovirus in GBM cells. We showed that CIITA expression led to effective GBM killing by T cells in an MHC-II independent manner.
Overall, this study highlights the value of ex vivo GBM PDOs as a functional platform for investigating the dynamics of the GBM microenvironment and monitoring tumor-immune cell interactions at basal levels or in response to immunotherapies.