The effects of physical microenvironment stimuli on Temozolomide-induced resistant glioblastoma

Abstract

Glioblastoma (GBM) is the primary malignant brain tumor In 2016 the World Health Organization (WHO) define GBM as the grade IV glioma The current standard of care for GBM patients are surgical resection followed by adjuvant radiation therapy and chemotherapy Temozolomide (TMZ) is the oral alkylating agent and GBM chemotherapy standard agent TMZ methylates DNA and interferes with replication that results in cell death Resistance to TMZ is one of the major barrier for GBM therapy Aldehyde dehydrogenase 1 (ALDH1) is a member of the ALDH enzyme family which catalyzes the oxidation of intracellular aldehydes ALDH1 is directly involved in therapy resistance of GBM and is regulated by autophagy during chemotherapy Autophagy helps cancer cells to overcome the stressful conditions during cancer progression Up to data a few papers show that in the development of therapeutic resistant GBM involve the autophagy and epithelial-mesenchymal transition (EMT) processes EMT or so called mesenchymal differentiation (MD) process in gliomas that makes epithelial cells transform to mesenchymal cells The reversibility of MD suggests that environmental signals strongly correlate with aggressive tumor phenotypes Tumor microenvironment plays important role in the development of chemoresistance and in malignant progression The recent studies showed that elevated extracellular matrix stiffness independently foster GBM aggression Thus we hypothesize that when GBM grows in altered microenvironment with different stiffness ALDH1 autophagy and MD maybe altered in parental and TMZ-induced resistant GBM (TIR-GBM) cell lines In this experiment we used U87MG and 1306MG human GBM cell lines to develop TIR-GBM First we found that autophagy and MD markers were enhanced in TIR-GBM cell lines Then we established two different stiffness polyacrylamide (PA) gels to culture parental and TIR-GBM cell lines By using atomic force microscope (AFM) to measure gel stiffness and control glass we demonstrated that the stiffness of hard matrix was 10 5 kPa soft PA gel was 0 1 kPa and glass was 181 8 kPa The soft matrix softness was similar to human brain tissue and the hard substrate stiffness was similar to human skin or muscle tissue We observed that TIR-GBM cell lines cultured on soft substrate morphology of the cells changed from spindle to round shape and have smaller size In hard substrate the cellular area of resistant U87MG became smaller and length of resistant 1306MG became longer Finally we demonstrated that TIR-U87MG showed different responses with microenvironment In parental cell lines autophagy markers Beclin and LC3 showed low expression levels however it was increased in resistant cell lines When TIR-U87MG grew on soft gel LC3 positive cells were increased however numbers of positive cells in TIR-1306MG were reduced There was no significant alternation in autophagy markers when TIR cell lines grew on hard gel When U87MG with TMZ-resistance was grew on a soft matrix the fluorescence intensity of ALDH1 autophagy mesenchymal markers-Fibronectin and Vimentin were enhanced but it not found in hard gels On the contrary epithelium marker β-catenin fluorescence intensity was reduced However Fibronectin and Vimentin were increased The results indicated when environment changed to soft gel certain cellular phenotypes are altered such as ALDH1 autophagy Fibronectin and Vimentin intensity were decreasd in TIR-1306MG It also found in hard gels β-catenin was significant increase after TIR-1306MG cultured on soft matrix but it not found in hard gels These results also clearly demonstrated that when U87MG compared with 1306MG showed different responses to the cultured microenvironments In summary when GBM grow on soft or hard environmental in chemoresistant it indeed resulted in changes in ALDH1 expression and may either promote or diminished autophagy and MD processes

Date of Award	2019
Original language	English
Supervisor	Chun-I Sze (Supervisor)