Background and Objective: Access to high-quality patient-derived brain tumor tissues is instrumental for translational neuro-oncology research. Glioblastoma tumor material resected by ultrasonic aspiration (UA) during surgery offers an abundant source of material; however, it is generally not used for research experiments. We hypothesize that UA-derived tumor tissue represents a source of tissue that accurately reflects the immune infiltrates of glioblastomas. Methods: In this study, we have utilized UA-derived tissue and performed a head-to-head comparison with paired resection tissue from the vital tumor core of the same patient. A combination of 16 fluorochrome-conjugated antibodies was designed to identify tumor-infiltrating T, B, and NK lymphocytes and characterize the TILs by spectral flow cytometry. Furthermore, a 5-plex panel was designed to spatially characterize the T cells, macrophages, and tumor cells on the paired UA and resection tissues. Results: UA-obtained cells exhibited a comparable yield and viability, as well as an abundance of tumor-infiltrating T, B, and NK lymphocytes compared to resection sample-derived cells. Importantly, we observed that there is a high concordance with respect to expression intensities of immune checkpoints by T cells in both types of tissue samples. Conclusions: These findings underscore the feasibility and reliability of utilizing the immune infiltrates from ultrasonic aspiration-acquired glioblastoma tissue.

Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.

The oncolytic adenovirus (OV) Delta24-RGD, also known as DNX-2401, is currently under clinical investigation for GBM. We studied multiple components of the immune system in response to local treatment with this agent using both in vitro and in vivo models and patient-derived materials.