Gene Therapy Of Interleukin 12 Expressed In Mesenchymal Stem Cells For Glioma In Rats

Malignant glioma is the most common primary neurologic neoplasm.Current therapy for intracranial glioma centers on surgical resection withradiation and chemotherapy. But tumor recurrence is common. Tumor cellsmigrating away from the primary tumor site often form tumor microsatellites atdistal sites, these microsatellites are responsible for the eventual recurrence ofthe tumor after initial treatment. Now gene therapy paradigms for experimentalbrain tumors models have been conducted, and they were thought torevolutionize the treatment of patients with gliomas. Selection of therapeuticgene and delivery vehicles for gene can influence curative effect of genetherapy.Bone mesenchymal stem cells(BMSCs)represent a subpopulation ofnon-hematopoietic pluripotent cells within the bone marrowmicro-environment due to their ability to differentiate into mesenchymallineages and neuronal lineages. This neurogenic potential of BMSCs wasrevealed under special culture conditions in vitro or following intracranialimplantation. Intracranially implanted BMSCs can migrate away from theinitial injection site towards tumor site. Taken together, these findings implythat NSCs could be replaceable with BMSCs as a therapeutic vehicle for genetherapy against glioma. Moreover, BMSCs also have the advantage of ease ofpropagation in vitro and implantation of autologous BMSCs into patients withmalignant glioma is ethically unproblematic. The properties of BMSCs makethis cell population a very attractive candidate for the cell-based delivery oftherapeutics to tumor sites.Interleukin-12 (IL-12), a heterodimer composed of two subunits, is amultifunctional cytokine produced by antigen-presenting cell. IL-12 is one ofthe most effective immunomodulators for cancer treatment. Recently, IL-12was reported to inhibit angiogenesis. In fact, recombinant IL-12 exhibitssignificant antitumor activity in various animal models. But there were alsoadverse effects, which interrupted the clinical trials. Since intratumoral IL-12gene transfer could minimize the adverse effects of systemic rIL-12administration, the effectiveness against tumors of gene therapy of IL-12,hasbeen evaluated in various animal models. Many studies have shown thatintratumoral IL-12 gene transfer is a feasible and well-tolerated procedure butexerts only mild antitumor effects. These activities have provided a rationalefor the use of IL-12 gene in cancer immunotherapy.In this experimental study, we established a retrovirus expression plasmidcooperated with IL-12 gene;BMSCs were isolated from rat bone marrow andtransinfected with retrovirus expression vector for evaluation of anti-tumoractivities in vitro and in vivo.Isolation and Identification of BMSCsBMSCs were separated from bone marrow by density gradientcentrifugation and selected by plastic adhesion according to references. Atnear-confluence, cells were subcultured after trypsin digestion. To characterizethese cells, immunocytochemistry was performed to identify specific antigens,such as CD44, CD29.Musashi and Nestin expressed on the surface of BMSCswere also detected. The results were CD29 (46%), CD44 (70%),Musashi(12.85%) and Nestin(3.17%). We also induced BMSCs differentiationby incubation with different culture media to identify, ALP positive cells ofosteogenic differentiation of BMSCs were detected by Gomori staining,adipogenic differentiation of BMSCs were detected by oil red O staining,andneural differentiation of BMSCs expressed GFAP and MAP-2. The resultsdemonstrated that these cells have the capacity to differentiate into osteoblast ,adipocyte and neuronal lineages .They were stem cells but not mature cells.Construction of pLEGFP-mIL12 recombinant plasmid and transinfectionof recombinant plasmid into rat BMSCsDNA of mIL-12 obtained from pNGVL-mIL12 plasmid was cloned intopLEGFP-C1 using standard procedures to develop recombinant plasmidpLEGFP-mIL12. pLEGFP-C1 and pLEGFP-mIL12 were transferred intopackage cell line PA317 using lipofectamine 2000 respectively. In order to testtiter of virus production, NIH3T3 cell line was employed for stable productionof retrovirus. BMSCs were transinfected with retrovirus produced by PA317.The EGFP-positive cells (BMSCs/pLEGFP-C1 and BMSCs/pLEGFP-mIL12)can be selected with medium containing G418 and observed by confocal lasermicroscopy. Growth curves of BMSCs, BMSCs/pLEGFP-C1 andBMSCs/pLEGFP-mIL12 were not different. That indicated growth ofretrovirus and mIL-12 gene tranfected BMSCs have no change. Production ofmIL-12 in supernatant of BMSCs detected by ELISA assay was 6.5ng/106/48h.Antitumor activity of BMSCs and BMSCs/pLEGFP-mIL12 in vitroWe utilized a two-chamber culture system to evaluate the effect ofcoculture with BMSCs on the growth of C6 glioma cells in vitro. Theproliferation of C6 cells was inhibited to a significantly greater extent byco-cultivation with BMSCs (15.2±1.6% inhibition rate) than with NIH3T3cells(4.9±1.2% inhibition rate, P﹤0.01).The cell cycle of C6 cells co-culturedwith BMSCs was also detected by flow cytometry (FCM).The results indicatedthat the distribution of cell cycle changed as followed, percentage of G0 /G1period was evidently increased compared with control(P﹤0.01). These resultsindicate that BMSCs themselves possess a direct antitumor effect against C6glioma cells in vitro.We evaluated the migratory nature of BMSCs towards C6 cells in vitro.BMSCs co-cultivation with C6 cell were found to possess significantly greatermigratory capacity than control (P﹤0.01).In vitro the growth of C6 cells cultured in different supernatant ofBMSCs,BMSCs/pLEGFP-C1 and BMSCs/pLEGFP-mIL12 were compared byCCK-8 assay. The proliferation of C6 cells has no change. These resultsindicate that mIL-12 inhibits tumor growth indirectly.EVC304 cells grown in different supernatant of BMSCs,BMSCs/pLEGFP-C1 and BMSCs/pLEGFP-mIL12 showed no significantdifferences in endothelial cell proliferation rates measured by MTT assay.EVC304 cells were then grown on Matrigel in different supernatants to observetubule formation. Endothelial cells grown in different media all showed intacttubule formation and have no difference. These results indicate that mIL-12have no effect on growth of endothelial cell and endothelial cell tubuleformation directly in vitro. There are other mechanisms of mIL-12 inhibitionangiogenesis.Establishment of animal model and transplantation of BMSCs,BMSCs/pLEGFP-C1 and BMSCs/pLEGFP-mIL121. Animal model of glioma was accomplished according to references.2. Migration and differentiation of BMSCs injected into the contralateralstriatum of rat glioma model BMSCs labeled with Hoechst33342 wereinjected into contralateral hemisphere of glioma-bearing rats. We examinedBMSCs migration , differentiation and viability 14d and 21d aftertransplantation. BMSCs labeled by Hoechst33342 were observed byfluorescent microscopy. BMSCs migrated away from the initial injection sitealong the corpus callosum. Some BMSCs were found in the corpus callosumand blood vessel wall. The results of immunocytochemical staining suggestedthat some BMSCs implanted differentiated and expressed MAP-2(33.93% )and GFAP(18.02%).3.Antitumor activity of BMSCs injected intratumorally Animals wereclassified into 3 groups, saline group (control group), BMSCs/pLEGFP-C1group and BMSCs/pLEGFP-mIL12 group.The survival time of rats in different group were compared. Intratumoralinoculation of BMSCs/pLEGFP-mIL12 significantly prolonged survival inglioma-bearing rats (43.6±5.38d ) compared to controls(19.00±1.15d ).There were five rats outlived beyond 60 days. The mean survival time ofglioma-bearing rats injected with BMSCs/pLEGFP-C1 was 22.30±1.19d,theformer survived longer than the controls.BMSCs/pLEGFP-C1 and BMSCs/pLEGFP-mIL12 were injectedintratumorally. We examined migration,viability and antitumuor activity ofBMSCs 21d after transplantation. EGFP-positive BMSCs were observed byfluorescent microscopy. BMSCs were found at the border zone between tumorand normal parenchyma, and also infiltrated into the tumor bed relativelyuniformly.Tumor volumes of rats in different group were also compared .Significantly smaller tumor volumes were present in brains of rats treated withBMSCs/pLEGFP-mIL2 (compared to controls).The tumor volumes of rats indifferent groups were 71.00±15.56mm3(saline group);68.33±11.72 mm3(BMSCs/pLEGFP-C1 group);32.67±12.42 mm3(BMSCs/pLEGFP-mIL12group).Immunohistochemistry results showed BMSCs/pLEGFP-mIL12 therapyalso resulted in increased intratumoral CD4+ and CD8+T-cell infiltration,which was significantly higher than the immune cell infiltration seen in C6tumors inoculated with Saline(P<0.05) .Our results showed BMSCs as a therapeutic vehicle for mIL-12 genetherapy against glioma were injected intratumorally , IL-12-secreting BMSCscan survive ,augmente the antitumor effect and prolonged the survival oftumor-bearing rats compared to treatment with saline. IL-12-secreting BMSCstherapy was associated with enhanced T-cell infiltration in tumor beds. Thus,gene therapy employing BMSCs as a vehicle would be promising as a newtherapeutic approach for refractory brain tumor.