Immortalized mesenchymal stem cells: a safe cell source for cellular or cell membrane-based treatment of glioma

BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumour tropism, anti-tumour, and immunoregulatory properties. The limited passage and self-differentiation abilities of MSCs in vitro hinder preclinical studies of mesenchymal stem cells, and makes the MSC-based treatment of tumours lack a stable, uniform, and homogeneous source of cells. In this study, we focused on the safety of immortalised mesenchymal stem cells (im-MSCs) and, for the first time, studied the feasibility of im-MSCs as candidates for the treatment of glioma.METHODS: The im-MSCs were constructed by the lentiviral transfection of genes, and the proliferative capacity of immortalised MSCs and the proliferative phenotype of MSCs and MSCs co-cultured with glioma cells (U87) were measured using CCK-8 and EdU assays. After long-term culture, karyotype analysis of im-MSCs was conducted. In addition, the tumourigenicity of engineered MSCs was evaluated using soft agar cloning assays. Moreover, the engineered cells were injected into the brain of female BALB/c nude mice. Finally, the cell membranes of im-MSCs were labelled with DIO or DIR to detect their ability to be taken by glioma cells and target in situ gliomas using an IVIS system.RESULTS: Im-MSCs expressed CD73, CD90, CD105, CD29, and CD44 but did not express CD45, CD34, CD14, CD11b, or CD31. Engineered MSCs maintained the ability to differentiate into mesenchymal lineages in vitro. Im-MSCs showed stronger proliferative capacity than unengineered MSCs without colony formation in soft agar, no tumourigenicity in the brain, and normal chromosomes. MSCs or im-MSCs co-cultured with U87 cells showed enhanced proliferation ability, but did not show malignant characteristics in soft agar cloning experiments. Immortalised cells continued to express homing molecules. The cell membranes of im-MSCs were taken up by glioma cells and targeted in situ gliomas in vivo.CONCLUSIONS: Im-MSCs are promising candidates for cellular anti-glioma therapy. Im-MSCs provide a safe, adequate, quality-controlled, and continuous source of cells or cell membranes for the treatment of glioma.

Thymidylate synthase gene expression in solid tumors predicts for response to pemetrexed in vitro

13058 Background: Pemetrexed (P) is a novel antifolate which targets thymidilate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The aim of the present study was to identify gene expression thresholds for these enzymes in human tumor specimens in order to separate P-sensitive from P-resistant patients. Methods: Soft-agar cloning assays were performed on freshly biopsied tumor cells exposed one hour to clinically achievable concentrations of P. In parallel, RNA was isolated, transcribed to cDNA and subsequently used for multiplex real-time PCR. Gene expression data were normalized against beta-actin transcripts followed by correlation against cloning assay results. Iterative calculations (fourfold analysis) were done for each enzyme separately to find the best cutoff for prediction of sensitivity to P. Results: Sensitive and resistant tumor samples were statistically significant different in gene expression of TS, DHFR, and GARFT (p < 0.003). 81% of all tumors with a TS copy number < 144 (related to 104 copies β-actin) were sensitive to P in vitro. (specificity = 0.69; chi2 = 14.14). Statistical tests demonstrated that gene expression of TS, DHFR, and GARFT are dependent variables and that TS transcription is the leading variable. The combination of TS, DHFR, and GARFT expression data was not superior to TS alone. Conclusions: TS expression is the most meaningful predictor for sensitivity (≤ 144 copies) or resistance (> 144 copies) to Pemetrexed in fresh tumor tissue. This observation forms a rationale for clinical trials using TS expression as predictor for clinical response. No significant financial relationships to disclose.