Forsøksdyr: Inhibition of gap junctions via drug repurposing of meclofenamate sensitizes primary glioblastoma cells to temozolomide-mediated cell death

Godkjenningsdato 16.11.2020

Godkjenningsperiode 16.11.2020-01.05.2021

Gap junctions have recently been shown to interconnect glioblastoma cells to a multicellular syncytial network, thereby enabling intercellular long-distance communication, mechanisms of therapy resistance as well as brain microinvasion. Against this backdrop, gap junctions have become an attractive target for novel therapeutic approaches. However, given the lack of clinically-approved gap junction inhibitors, a clinical implementation of gap junction-targeted therapies has been considered far from feasible, yet.

In a recent project we have shown that meclofenamate (MFA) – a clinically-approved nonsteroidal anti-inflammatory drug – inhibits gap-junction-mediated intercellular cytosolic traffic in preformed in-vitro glioblastoma cellular networks. Our preliminary data further suggest MFA-mediated inhition of intercellular communication via gap junctions to profoundly sensitize glioblastoma cells to standard chemotherapeutic agent temozolomide-induced cell death. In respect of MFA as a FDA-approved drug, these data – for the first time - harbor the potential of bridging the idea of a gap-junction-targeted therapeutic approach into an instant clinical implementation.

In vivo validation is required to determine the translational relevance. A holistic interrogation of how treatment effects are modified by the complex in vivo microenvironment of GBM characterized by cellular heterogeneity (cancer vs. normal cells), oxygenation/hypoxia, blood flow and intracranial pressure will be necessary. Thus the proposed in vivo study aims to confirm our encouraging in vitro findings and establish the modality for future patient trials.

We will utilize n=50 non- obese diabetic immune-deficient mice (Nod-SCID) based on statistical power analysis for sample size required to distinguish significant effects between treatment groups and to standardize the xenograft take rate. We have extensive experience with intracranial implantations. A neurosurgeon and laboratory assistant will undertake the surgical procedures, ensuring rapid, safe and reproducible intracranial tumor implantations to minimize animal discomfort. We will utilize a stereotaxic-frame and aseptic technique for accurate co-ordinates, reproducibility and limited infection. Treatment with drugs and/or immune-cells will commence after confirmed MRI visible tumor to ensure valid results and limit repeated experiments. Longitudinal non-invasive MRI will be used to monitor tumor growth to ensure statistical robustness. Upon neurological sequelae, tumors will be harvested and analyzed ex vivo by various cellular and molecular methods that will be correlated with survival outcomes. Minimal distress for the animals is expected, as we utilize local and post-op analgesia. Gaseous anesthesia during surgical procedures will reduce mortality due to hypothermia and hypervolemia, as animals recover more rapidly. Humane endpoints include severe neurological sequelae and/or loss of 20% body-weight. Animals will be sacrificed by CO2 inhalation and decapitation, previously deemed satisfactory.