2016 Fiscal Year Research-status Report
Investigation of the therapeutic interaction of mesenchymal stem cells with glioblastoma stem-like cells and its biological mechanism
| Project/Area Number |
16K19996
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| Research Institution | Kanazawa University |
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Principal Investigator |
Pyko Ilya 金沢大学, がん進展制御研究所, 博士研究員 (00731853)
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| Project Period (FY) |
2016-04-01 – 2018-03-31
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| Keywords | glioblastoma / temozolomide / mesenchymal stem cells / GSK 3β |
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| Outline of Annual Research Achievements |
Studies in vitro and in animal model were carried out complementary to optimize mesenchymal stem cells (MSCs) transplantation for benefits to treatment of glioblastoma (GBM) by enhancing anti-tumor effect in combination with GSK3β inhibitors and temozolomide (TMZ). For in vitro study, we examined the interaction between patient-derived GBM stem-like cells (SCs) and human adipose tissue-derived MSCs in the presence and absence of GSK3β inhibition and TMZ. I found that the interaction of MSCs with GBM-SCs is enhanced by a combined treatment by GSK3β inhibitor and that MSCs co-cultured with GBM-SCs participate in regulation of GBM stemness phenotype similar to that observed under GSK3β inhibition. For animal model study, at present we examine effects of intracranial transplantation of MSCs, neural-transdifferentiated MSCs and primary mouse neural stem cells (NSCs) against GBM in mice bearing mouse GBM cell lines or human GBM-SCs. Our experiments showed that transplantation of MSCs and GSK3β inhibition synergizes for treatment of experimental GBM. This observation encouraged us to investigate biological mechanisms by which MSCs regulate GBM stemness phenotype under the control by GSK3β starting from the screening for changes in stem cell markers’expression in GBM-SCs under GSK3β inhibition.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The following steps have been performed for the study. (1) preparation of 30 GBM-SC cultures from patients with primary GBM; among which 25 with high O6-methylguanine DNA methyltransferase (MGMT) expression and resistant to temozolomide were used in experiments;
(2) 7 primary cultures of bone marrow MSCs and a primary NSC culture were isolated from mice;
(3) 10 primary MSC cultures were isolated from human adipose tissue samples with confirmed phenotype (CD14, CD29, CD31, CD34, CD44, CD45, CD90, CD105);
(4) I have investigated the effects of TMZ, GSK3β inhibition and co-culture of primary GBM-SCs and MSCs on GBM stemness phenotype by proliferation assay, time-lapse microscopy and by evaluation of stem cell markers’ gene expression by QRT-PCR;
(5) I have investigated the effects of GSK3β inhibition and intra-tumor transplantation of MSCs on neurological state and survival of mice with GBM models generated by using human GBM-SCs or mouse GBM cell lines that adequately represent human GBM.
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| Strategy for Future Research Activity |
Studies in vitro and in animal model will be carried out complementary to optimize mesenchymal stem cells (MSCs) transplantation for benefits to treatment of glioblastoma (GBM) by enhancing anti-tumor effect in combination with GSK3β inhibitors and temozolomide (TMZ).
For in vitro study, we isolate GBM-SC culture from patients’ GBM tumors, MSCs from human adipose tissues and mice bone marrow, and primary NSC cultures from mice. We examine effects of MSCs, NSCs, GSK3β inhibition, TMZ and various combination of them against GBM-SCs. To address our working hypothesis of interaction between GBM-SCs and MSCs via GSK3β-mediated signaling, we investigate (a) c-Myc and O6-methylguanine DNA methyltransferase (MGMT) expression and MGMT promoter methylation; and (b) c-Myc and DNA (cytosine-5)-methyltransferase 3A binding to MGMT promoter according to our previous study (Carcinoenesis 2013).
For animal model study, we examine effects and underlying mechanism of intracranial transplantation of MSCs, neural-transdifferentiated MSCs and primary mouse NSCs against GBM in mice bearing mouse GBM cell lines or human GBM-SCs. We observe survival as well as consequence of neurological state and cognitive functions in mice following transplantation of the respective cells.
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