INVESTIGATION OF THE IMPACT OF RADIATION AT DIFFERENT FRACTIONATION DOSES ON MOLECULAR BARRIER PERMEABILITY IN AN IN-VITRO BLOOD BRAIN BARRIER MODEL

Project no. 23021 · Status: Kapanmış (Yayın bekleniyor) · 2021

Summary

Objective: Currently, brain cancer and its metastases are among the leading causes of death. Despite many treatment approaches such as surgery, chemotherapy and radiotherapy, no significant success has been achieved in prolonging the survival of these patients. The yield of drugs used in the biological treatment of cancer is very low because the blood-brain barrier (BBB) prevents the therapeutic agents from reaching the tumor. Our aim is to evaluate the changes in the permeability of the BBB with different fraction doses of radiotherapy and to prepare the ground for treatment strategies that may benefit from this impaired function. Materials and Methods: The in vitro model of BBB was established by seeding cells in a 24-well plate with a porous membrane-separated insert. Human umbilical vein endothelial cells (HUVEC) and human astrocyte cells (NHA) were used for seeding. The accuracy of the model was verified by transendothelial electrical resistance (TEER) measurements. A device developed by the Department of Physics was used for TEER measurement. Radiation was delivered in single fractions of 2, 4, 6, 8, 16 and 25 Gy. After ionising radiation, the cells were incubated with the TEER device for 72 hours, with measurements taken every two hours. In addition, glioblastoma (GBM) cell lines were seeded at the bottom of the 24-well plate where the insert was placed to assess drug dose efficiency, which can vary according to membrane permeability. Two different drugs were applied to the irradiated BBB model. Determination of membrane permeability by ELISA, MTT proliferation assay and cell wound healing assay were used. Results: There was a significant decrease in TEER values in all dose groups after irradiation compared to the control group (p<0.001). No significant difference was found when fraction dose were evaluated (p=0.639). The most significant dose-independent decrease occurred between hours 2 and 6. It was noted that the change in the rate of resistance decline increased in direct proportion to dose. Temozolomide (TMZ), used to evaluate the proliferation of GBM cell lines seeded on the bottom after irradiation, was effective on GBM cell proliferation and the lowest dose for effective permeability was 4 Gy. In the cell wound healing experiment performed as an indicator of repopulation and repair capacity, it was observed that while bridging started in the early hours after irradiation in the TMZ-treated groups at low fraction doses, there was no wound closure at the end of 72 hours at high fraction doses. When the results of ELISA measurements made after the application of trastuzumab (TRAS), which is 750 times larger than TMZ, were evaluated, TRAS levels below 500 ng/ml were found at doses of 2, 4 and 6 Gy. TRAS levels of 1500 ng/ml were measured at doses of 8, 16 and 25 Gy. Conclusion: Our study demonstrates that different doses of ionising radiation combined with chemotherapy may increase tumour access to therapeutic agents and evaluates the time course of this relationship. We conclude that permeability through the BBB is related to molecular weight, which increases proportionally with increasing dose fraction.

View details