In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma

Abstract

Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of cancer-related death in children. While it is clear that surgery (if possible), and radiotherapy are beneficial for treatment, the role of chemotherapy for these tumors is still unclear. Therefore, we performed an in vitro drug screen on primary glioma cells, including three DIPG cultures, to determine drug sensitivity of these tumours, without the possible confounding effect of insufficient drug delivery. This screen revealed a high in vitro cytotoxicity for melphalan, doxorubicine, mitoxantrone, and BCNU, and for the novel, targeted agents vandetanib and bortezomib in pHGG and DIPG cells. We subsequently determined the expression of the drug efflux transporters P-gp, BCRP1, and MRP1 in glioma cultures and their corresponding tumor tissues. Results indicate the presence of P-gp, MRP1 and BCRP1 in the tumor vasculature, and expression of MRP1 in the glioma cells themselves. Our results show that pediatric glioma and DIPG tumors per se are not resistant to chemotherapy. Treatment failure observed in clinical trials, may rather be contributed to the presence of drug efflux transporters that constitute a first line of drug resistance located at the blood-brain barrier or other resistance mechanism. As such, we suggest that alternative ways of drug delivery may offer new possibilities for the treatment of pediatric high-grade glioma patients, and DIPG in particular.

Figure 1. a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.

Figure 2. Western blot for detection of P-gp, MRP1, and BCRP1 in pHGG cultures.

MW represents approximate molecular weight of these proteins, as indicated at the right. The pHGG lanes were loaded with 20 µg of protein, the lanes with positive controls were loaded with 5 µg of protein.

Figure 3. Immunohistochemical staining of ABC-transporters in pHGG sections.

Expression of P-gp (A) and BCRP1 (C) is located to the endothelial cells of the tumor vasculature. Whereas MRP1 (B) expression is visualized mainly in the cytoplasm of tumor cells as well as in the vasculature.