The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patient-derived glioma stem-like cells (GSC). Combination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots. PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10 μM. TMZ monotherapy at 50 μM and 100 μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100 μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes. PARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.

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.

Despite remarkable advancements in (neuro)-oncology, glioblastoma (GBM) remains the most aggressive primary brain tumor with a dismal overall survival of 14-17 months. A critical factor contributing to its poor prognosis is the high local recurrence rate, observed in 80-90 % of patients. This is largely due to the tumor's highly infiltrative growth pattern, which renders complete surgical resection impossible. Although surgery remains a cornerstone of initial treatment, current surgical techniques lack the precision to reliably detect and remove infiltrative margins. Recent state-of-the-art trials have proposed more aggressive surgical strategies aiming to reduce residual disease and mitigating local recurrence. However, implementing such extensive resections requires intraoperative imaging tools with enhanced, real-time sensitivity-capabilities that current technologies lack. Here, we report the preclinical validation of a near-infrared (NIR), fatty acid-based probe formulated in micelles (mFA-ICG) for image-guided surgery of GBM. This includes a comprehensive characterization of its pharmacokinetics, biodistribution, and toxicity profile. Notably, the micellar formulation demonstrated high stability, extended half-life, deep tissue penetration, improved targeting, enhanced fluorescence signal (compared to clinical standards), and a favorable safety profile in relevant models. We further validated the efficacy of mFA-ICG in orthotopic patient-derived, transgenic and organotypic patient' tissue slice models of GBM where our data confirm both in- and ex vivo specificity of NIR signal. Collectively, these findings support the safety and translational potential of mFA-ICG and suggest it may significantly improve delineation at the infiltrative edge. These advantages position mFA-ICG as a promising candidate for a trial in GBM patients.

INTRODUCTION: Overexpression of STAT3 has been demonstrated in high-grade gliomas. STAT3 is situated at a point of convergence of several signaling pathways implicated in gliomagenesis. However, it is unknown whether STAT3 is capable of inducing tumor formation or progression. We studied STAT3 in a murine glioma model to determine its impact on tumor formation and grade. METHODS: Using the RCAS/Ntv-a system, we expressed constitutively active STAT3 (CA) in mice via somatic cell transfer in the forebrains of mice independently and with PDGFB (known to induce glioma). Mice were euthanized 12 weeks after RCAS vector injection, and the brains were examined for tumor formation. Analysis of proliferation (phosphohistone H3) and activated STAT3 expression (phosphorylated STAT3) was performed. RESULTS: Gliomas formed in 21/29 mice (72%) co-injected with RCAS-STAT3CA + RCAS-PDGFB but only in 11/26 mice (42%) injected with RCAS-PDGFB alone (chi-squared, p = 0.02). High-grade gliomas (HGGs) with classic phenotypic features including necrosis were observed in 14 mice (48%) injected with RCAS-STAT3CA + RCAS-PDGFB but in only five mice (19%) injected with RCAS-PDGFB alone (chi-squared, p = 0.02). No tumors were detected in mice injected with STAT3CA alone. Immunohistochemical analysis revealed pSTAT3 expression localizing to areas adjacent to foci of necrosis in HGGs. The portion of pSTAT3-positive cells in PDGFB + STAT3CA-induced HGGs was significantly higher than that in the PDGFB-induced HGGs (pairwise comparison, p = 0.0002). The mitotic index in HGGs induced by PDGFB + STAT3CA was significantly higher in PDGFB + STAT3CA-induced tumors than in either high- or low-grade PDGFB-induced tumors (pairwise comparison, p = 0.006 and p = 0.004, respectively). CONCLUSION: STAT3 enhances tumor formation in a PDGFB-dependent model of glioma. STAT3 expression also promotes progression to HGG and promotes tumor cell proliferation. Expression of pSTAT3 correlates with areas of necrosis, a cardinal feature of HGGs. These results confirm the relevance of STAT3 signaling in glioma biology.