Efficacy of antitumor vaccines based on photoinduced GL261 glioma cells using photosensitizers from the group of tetra(aryl)tetracyanoporphyrases with different aryl substituents

Abstract

Glioblastomas are solid tumors in the brain that pose a challenge for traditional treatments like surgery, radiation therapy, and chemotherapy. Complete cures are not guaranteed, and these treatments cause numerous side effects. First-line treatment approval has only been granted to Temozolomide (TMZ), the sole chemotherapy drug available. Using TMZ increases the median overall survival from 15 to 17 months. In general clinical practice, innovative interventions have not demonstrated efficacy due to glioma heterogeneity and their immunosuppressive microenvironment. Immunogenic cell death (ICD) activates an immune response against cancer cells by emitting damage-associated molecular patterns (DAMPs) upon cell death/dying. The DAMPs activate an anti-tumor immune response. Photodynamic therapy (PDT) can induce ICD. Integrating the principles of immunogenic cell death into glioma immunotherapy could elicit a targeted immune response against the diverse tumor. Vaccination with dendritic cells represents a promising approach for immunotherapy. The goals of this research are to evaluate the efficacy of a prophylactic vaccine using immunogenically-photoinduced glioma GL261 cells and a dendritic cell vaccine in an orthotopic in vivo model. The glioma cell line (GL261) is cultured in RPMI medium supplemented with 10% serum, L-glutamine, 1% penicillin, and 1% streptomycin in a CO2 incubator. Photodynamic treatment involves using photosensitizers from the tetra(aryl)tetracyanoporphyrazine group with 9-phenanthrenyl (pz I) or 4-(4-fluorobenzyloxy)phenyl (pz III) as side substituents. The GL261 cell line is incubated in a serum-free solution containing a selected porphyrazine for four hours. Subsequently, the solution is exchanged with complete medium and the cells are activated through photodynamic treatment at a dose of 20 J/cm2. The cells are then further incubated for 24 hours in a CO2 incubator. Immunizing mice includes subcutaneously injecting photoinduced GL261 glioma cell lysates twice with a 7-day interval. One week after the final immunization, viable GL261 glioma cells are injected into the brain using a stereotaxis frame. Measuring the neurological status of the animals occurs for 25 days, and tumor localization and volume are detected on day 23 by MRI. The survival rate of the experimental groups was 100% (pz III) or significantly higher (pz I) than that of the control groups. Additionally, neurological symptoms were either not identified (pz III) or were significantly lower (pz I) compared to the control animal groups experienced. The dendritic cell vaccine was based on photoinduced GL261 glioma cells. The femur and tibia bones of C57BL/6J mice were used and differentiated for 9 days in RPMI medium to isolate bone marrow stem cells. The medium was supplemented with 5% fetal bovine serum, 20 ng/ml GM-CSF, 1% L-glutamine, 1mM sodium pyruvate, 50 µM β-mercaptoethanol, 100 units/ml penicillin, and 100 µg/L streptomycin. The culture medium was renewed on days 3 and 6 to maintain stability. Protein levels are quantified in cell lysates collected. 2 mg of protein is added to a dendritic cell suspension, and after 90 minutes, 0.5 µg/ml lipopolysaccharide is added for 24 hours. Intraperitoneal dendritic cell vaccine immunization on animals is conducted, with injections 7 days apart. One week post-last immunization, viable GL261 cells are intracranially injected via a stereotactic frame. The neurological status of the animals was monitored for 25 days, and an MRI was conducted on day 23 to evaluate the brain tumor. The survival rate of animals in the experimental groups did not significantly differ from those in the control groups. However, the experimental groups exhibited significantly lower neurological symptoms, and the tumor was visualized without any areas of necrosis, with a smaller volume.