(H-283) Synergistic Eradication of Glioblastoma in Rodent Models by DNA-Damage through the Combined Use of Non-Ionizing Pulsed Electric Fields and Standard-of-Care Chemotherapy
Assistant Professor University of Massachusetts Amherst, Massachusetts, United States
Introduction:: Standard of care treatment of unresectable glioblastoma with combination of radiation and chemotherapy presents risk of damage to healthy brain despite careful planning and fractionation of radiation. Despite considerable advances in radiation dose planning and physical treatment delivery technology, it remains a difficult task to prevent exposure of healthy tissue peripheral to the tumor volume, as well as those situated at the entry and exit points of radiation beams used in particle therapy. Bleomycin (BLM) can cause double strand DNA breaks similar to radiation (RT), but the use of BLM is limited by its poor diffusion into cells and significant side effects during systemic administration at therapeutic dose. Non-ionizing pulsed electric fields (PEF) can temporarily increase trans-membrane transport of bleomycin, which results in multifold increase of drug delivery to cells even at nanomolar concentration of the drug. This combinatorial treatment approach is termed electrochemotherapy (ECT). The aim of this study was to determine whether ECT can be used as an alternative to RT for enhancing chemotherapy. We investigated whether this approach could reproduce the effect of radiation and synergizes with drugs acting on DNA repair pathways (temozolomide, TMZ and olparaib, OLA).
Materials and Methods:: Two brain cancer cell lines (T98G & U87), with low and high sensitivity to standard of care drugs Temozolomide and Olaparib, were used for this study. In vitro testing of treatment groups of PEF (voltage and pulse number) +/- BLM (dose escalation) and following cohorts 1) Sham, (2) PEF (pulsed electric field of 1400V/cm), (3) Free drug (BLM), (4) ECT (BLM+PEF), (5) ECT+TMZ, was screened in 3D tumor models. The 3D models were constructed by seeding glioblastoma cells in collagen scaffolds for viability (flow cytometry), proliferation (CCK-8). DNA damage was assessed with gH2AX activation via flow cytometry and confocal microscopy. Congenic assays, ECT radiation to gamma irradiation were compared at 2Gy, 5Gy, and 10Gy.
Results, Conclusions, and Discussions:: Results:
γH2A is known to be activated in the presence of DNA damage, a common radiation indicator. Cells were not sensitive to Bleomycin without electroporation (Figure 1A). No significant activation of γH2A was observed between cells treated with BLM (free drug), but ECT (BLM+PEF) showed significant upregulation of γH2A. There was no significant loss of cell viability post ECT Figure 1B-C. After 72 hours, synergy of combination ECT with TMZ under dose escalation conditions was evaluated. Electrochemotherapy with adjuvant TMZ increased yH2A activation when compared to TMZ and sham controls Figure 1D. Compared to treatment with Bleomycin and/or Temozolomide, electroporation in the presence of combination of both drugs significantly reduced total cell counts in the cancer cell line at both time point (p = < 0.005) Figure 1E. Electroporation in combination with Bleomycin plus Temozolomide following 72 hours incubation had the greatest cytotoxic effect with a cell viability reduction of 24% (p = < 0.005) Figure 1E. Effects of synergy of electrochemotherapy with Olaparib (chemotherapeutic agent) was also studied in both T98G and U87 cell lines (data not shown).
Discussion:
The study investigated the effectiveness of electrochemotherapy (ECT) in combination with chemotherapeutic drugs that act on DNA repair pathways in reducing total cell counts in glioblastoma cell lines. The results showed that electroporating cells at 1400V/cm, 8 pulses, 100µs increased uptake of impermeable BLM. ECT induced significant upregulation of γH2A, associated with DNA damage pathways, whereas BLM free drug treatment alone did no elicit such activation. Additionally, the combination of ECT and TMZ at all ratios led to increases γH2A activation compared to TMZ or sham controls. Moreover, electroporation in the presence of both BLM and TMZ significantly reduced cell proliferation in the glioblastoma cell lines and had the greatest cytotoxic effect with a cell viability of 24% after 72 hours of treatment. These findings suggest that the combination of ECT with TMZ may be an effective treatment for unresectable glioblastoma.
Conclusion:
Within this in vitro model, electrochemotherapy with Bleomycin can assist treatment of Temozolomide resistant glioblastoma. These results provide compelling evidence to further explore this combination therapy.
