Even though a patient has a good immune system, tumors are shielded from it, because tumors grow by suppressing the host’s immune-response by various mechanisms. They are keeping their local microenvironment immune suppressed by producing immune suppressive cytokines like IL-10 and TGF-b, express immune checkpoint ligands like Programmed Death Ligand 1 (PDL1), and harbor immune suppressive cells like Tregs and MDSCs. To overcome these barriers a stronger anti-tumour immune-response is essential. We evaluated a whole cell vaccine with extracorporeal Rutherrin®-PDT treated cancer cells (RuVaCareTM) to break the suppressive barrier in the RG2-glioblastoma model. Rutherrin®-PDT induced strong immunogenic cell death (ICD) in glioblastoma cells in-vitro. RuVaCareTM supernatants showed significantly higher level of extracellular ATP, which is known to induce recruitment of antigen presenting cells (APCs) and their activation by eliciting an effective anti-tumour immune-response. Extracellular calreticulin (CRT) is one of the hallmarks of ICD; its expression went up in more than 85% cells undergoing Rutherrin®-PDT mediated cell death. There was a close to 10 times increase in expression of HSP 70 in RuVaCareTM. Immunostimulatory cytokines IFNa, IL-1b and GMCSF expression is high in the RuVaCareTM. In-vivo efficacy of the RuVaCare™ was evaluated in orthotopic RG2 rat glioblastoma model. There was a significant increase (~43% with 2-time vaccine and 87% in the 6-time vaccine) in survival in the RuVaCare™ vaccinated groups compared to unvaccinated controls. Increased intratumoral CD8+T-cell numbers are shown to be correlated with increased survival in glioblastoma rats, with RuVaCare™ there was a significant increase in the number of CD8+T-cells.
KEYWORDS: Ruthenium, Animal model studies, Photodynamic therapy, In vivo imaging, Brain, Brain cancer, Photons, In vitro testing, Tissues, Magnetic resonance imaging
Glioblastoma is a highly aggressive and common brain cancer in adults with a grave prognosis, and aggressive radio and chemotherapy provide only a 15 months median survival. We evaluated the tolerability, and efficacy of the Ruthenium-based photosensitizer TLD-1433 in the formulation with apo-Transferrin (Rutheriin®) in the RG2, rat glioblastoma model. The specific tumour uptake ratio, PDT threshold, of the RG2 rat glioblastoma models and normal brain in vivo were determined as well as the survival post-PDT and the extent of CD8+T cell infiltration post-PDT. Results were compared with those obtained by 5-ALA-induced PpIX mediated PDT in the same animal model. As both photosensitizers have different photophysical properties, the number of absorbed photons required to achieve an equal cell kill is compared during in-vitro and in vivo studies. A significantly lower absorbed energy was enough to achieve LD50 with Rutherrin® versus -PpIX mediated PDT. Rutherrin® provides higher selective uptake ratio (SUR>20) in RG2 tumours compared to normal brain, whereas the SUR for ALA-induced PpIX was 10.6 in the same tumour model. To evaluate the short-term tissue response in vivo enhanced T2-weighted MR images provided the spatial extent of edema, which is twice post PpIX-PDT versus Rutherrin®-PDT suggesting reduced non-specific damage typically associated with a secondary wave of neuronal damage. A significant survival increase was observed in Rutherrin® treated rats bearing RG2 versus PpIX-PDT for the selected treatment conditions, associated with an increased CD8+T cell infiltration in the tumours. Rutherrin®-PDT was well tolerated providing safe and effective treatment of RG2 -induced glioblastoma.
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