| Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor of the central nervous system. The innate nature of GBM include considerable heterogeneity, high aggressiveness and well vascularization. Despite the standard treatment of surgical resection of the tumor followed by radiation and/or chemotherapy, the median survival for GBM patients is only12months over the past three decades. Furthermore, the nonspecific nature of conventional therapy for brain tumors often results in damage to the surrounding normal brain and systemic tissue. As a result, It is clear that novel therapies are needed to improve the prognosis and quality of life of patients with GBM.Immunotoxins (ITs), otherwise known as cytototoxins are a class of novel biologicals that are currently under evaluation for glioma therapy. These recombinant proteins consist of a specific antibody or ligand coupled to a toxin protein. Immunotoxins act by specifically targeting surface antigens overexpressed in cancer. The most commonly employed toxins in the construction of immunotoxins include the bacterial toxins Pseudomonas aeruginosa exotoxin A (PE) and diphtheria toxin (DT) and the plant toxin ricin. A single immunotoxin can kill a cancer cell as compared to105molecules of a chemotherapeutic drug that are needed to kill one cancer cell. Obstacles which limit the effectiveness of radiation and chemotherapy such as regional tumor hypoxia, non-specific cell targeting, and natural or acquired resistance are overcome by immunotoxin therapy. So immunotoxin represent a group of anti-cancer therapeutics different from conventional anti-cancer therapies.The specificity of immunotoxin therapy relies on the presence of a moleculal cells. Recent evidence has shown that the EphA2receptor is overexpressed in GBM compared to normal brain tissues. EphA2is a member of the Eph receptor tyrosine kinase family, whose16members can be further divided into "A" and "B" classes, based on sequence homology and binding affinity to their ligand, the Ephrin. Accumulating evidence has indicated that overexpression of EphA2is associated with the development and progression of a number of human malignancies, including those of breast, prostate, melanoma and ovary as well as in GBM. Thus, the EphA2receptor represents a very attractive target for immunotoxin therapy. For the toxin-based targeted therapy for caner, one of the most important issuesto be considered is how to target only caner cells and to protect the surrounding normal tissues from side effects. The major purpose of immunotoxin is to deliver therapy selectively to cancer cells that frequently overexpress cytokine receptors Although these agents selectively bind and kill cancer cells, clinically their use has been limited by:(a) their failure to penetrate and localize in adequate concentrations in cancer target tissue; and (b) their localization in nontarget organs limiting the tolerated dose and narrowing the therapeutic window. Suitable vehicles for delivering immunotoxins to the site of cancer cells in vivo may overcome these obstacles. Recently, human mesenchymal stem cells (hMSCs) have begun to show promiseas potential vehicles for delivering therapeutic genes for the treatment of brain tumors. hMSCs possess tropism for experimental tumors, including gliomas, following intraarterial or intracranial injections. Moreover, these cells can be obtained from patients without ethical concerns, easily expanded in vitro, and genetically modified with viral vectors for the delivery of antitumor substances in vivo. several preclinical trials of stem cell-based gene therapies have shown that hMSCs can be effective tumor-specific delivery vehicles for transgenes to brain tumors. We have also recently produced a VEGF-PE38immunotoxin in hMSCs in vitro, which confirms that hMSCs can serve as a powerful cell-based delivery vehicle for for immunotoxins.With these studies in mind, we attempt to test the possibility that genetically engineered hMSCs could serve as a carrier for the delivery of immunotoxin genes to tumors. In the present study, we construct an adenoviral vetor encoding EphrinA1-PE38, a recombinant immunotoxin based on the EphrinAl ligand and a truncated form of Pseudomonas exotoxin A, which could specifically target EphA2receptor-overexpressing tumor cells. We also confirm the production of EphrinAl-PE38in hMSCs via adenoviral transduction and the potent killing activity of these EphrinAl-PE38-carrying hMSCs in EphA2-overexprssing GBM cells in vitro. Utilizing the tumor tropism of hMSCs, the administration of hMSCs that were transiently transduced with these virus would result in a significant in vivo anticancer effect against U251glioma. Our study will explore a new strategy for the site-specific treatment of glioma. This study includes four chapters:Chapter I. Construction and package of recombinant adenoviral vectors expressing EphrinAl-PE38Objective:To construct and package the EphrinAl-PE38expressing recombinant adenoviral vector, and then to assess, purify, and determine the virus titre for establishing the foundation of subsequent research.Method:The extracellular domain of EphrinA1and PE38genes were amplified by polymerase chain reaction (PCR). The PCR products and the shuttle plasmid pAdTrack-CMV were digested by Restriction enzymes respectively. Subsequently, the resulting three DNA fragments were religated using T4DNA ligase. The new plasmids were identified by restriction enzyme digestion and DNA sequencing. pAdTrack-EphrinAl-PE38was then cut by Pmel and the fragment of interest cloned by homologous recombination in the competent BJ5183cells, which were prepared by transforming pAdEasy-1bacteria using the way of Calcium chloride. Then the adenovirus was packaged in293cells after linearization by Pac I. The virus was verified by PCR and virus titer was determined by infection of293cells with serially diluted vector stocks, followed by observation of GFP-positive cells. Results:Restriction endonuclease digestion and sequence analysis revealed that the fusion immunotoxin gene fragments EphrinAl-PE38were cloned into the pAdtrack-CMV vector. Next, the virus was packaged in293cells and titers were determined from the plaque-forming units (PFU) assay and GFP-expressing units assay in293cells. Typically, viral titers of approximately109pfu/ml were obtained.Conclusion:We have successfully constructed the combinant adenovirus vector pAd-EphrinAl-PE38and the virus was packaged in293cells. The result provide the basis for search of the targeted cytotoxic activity to glima cellsChapter Ⅱ. Establishment of EphrinAl-PE38-secreting human bone marrow-derived mesenchymal stem cellsObjective:To establish a simple protocol to isolation, culture and identification of hMSCs and investigate the basic characterises of hMSCs. To observe the infection efficiency, the influence on cell proliferation and the fusion gene expression of hMSCs after Ad-EphrinAl-PE38infection.Method:Human adult bone marrow stroma stem cells of were isolated by gradient density centrifugation and cultured in conditioned medium. Morphological observations were performed with phase-contrast microscope and cell phenotype and generation cycle were detected by flow cytometry. hMSCs were infected with Ad-EphrinA1-PE38at various multiplicity of infection (MOI), the infection efficiency were perfomed by microscope fluorescence; the proliferation of hMSCs was determined by CCK-8, After adeno virus infection, EphrinAl-PE38transgene expression were confirmed by RT-PCR and ELISA transcription and translation level respectively.Result:We obtained hMSCs successfully by using gradient density centrifugation. The typical morphology of hMSCs was with long spindle shape, little volume and large caryoplasmic ratio. In addition, most of them grew adherently with obvious syntropy and arrayed in whirlpool. The positive ratio of cell phenotype was counted as following respectively:CD29(97.77%), CD34(1.4%), CD44(92.73%), CD45(0.07%), CD105(90.94%), HLA-DR(0.14%). The ratio of cells in S+G2+M stage was18.03%by analysis of generation cycle, which demonstrated that hMSCs maitained a strong proliferative ability. Green fluorescence can be observed24h and became strongest at7d after Ad-EphrinA1-PE38infection. The infection rate at day7varied with the MOI:100(24%),400(43%),800(88%)1200(90%), however, cell pathology phenomenon occurred in MOI1200group. Infection with the adenovirus at MOI not exceeding800did not alter the morphology of hMSCs compared to untransduced cells. In RT-PCR analysis,, a700bp band of EphrinAl-PE38expression could be seen in Ad-EphrinA1-PE38transduced hMSCs, but not in untransduced hMSCs. Subsequently, to further verify whether EphrinA1-PE38secreted from the transduced hMSCs, we performed an ELISA assay on the media collected from the transduced hMSCs to determine the concentration of secreted EphrinA1-PE38at different time points after transduction. The result showed that a high level of EphrinA1-PE38could be detected at day6, and this high level of EphrinA1-PE38persisted for approximately12days and then began to decline until day33.Conclusion:we have successfully established a protocol to isolate and culture hMSCs. hMSCs have the similar morphologies and phenotype with typical MSCs, and also have very strong proliferation ability. hMSCs could be efficiently infected at suitable MOI without causing any unfavorable effects on cell proliferation. EphrinA1-PE38could be expressed and secreted from transduced hMSCs into the culture media over certain period.Chapter Ⅲ. The selective killing effect of EphrinA1-PE38secreted from transduced hMSCs on GBM cells in vitroObjective:To investigate the cytotoxicity of EphrinA1-PE38secreted from transduced hMSCs against GBM cells that overexpress EphA2receptor, and also the potential mechanism involved in cell cytotoxicity was explored.Method:To develop an EphA2-targeted therapy against human glioma, we evaluated the expression of the EphA2receptor in four different human glioma cell lines (U251, A172, SHG-44and U87) and parallel control cell line (U-251[EphrinA1](+), T47D and hMSCs) by Western blot analysis. To test the specific killing effect of EphrinAl-PE38on GBM cells that overexpress the EphA2receptor, proliferation assays were performed, cells were plated at1×104cells/well in96-well plates and cultured with an increasing amount of the supernatants(12.5ul,25μl,50μl and100μl) harvested from hMSCs-EphrinAl-PE38cells. The supematants from untransduced hMSCs were used as a control. The experiment was terminated at different time points (12,24,48, and72hours), and the cell proliferation was assessed using a CCK-8kit. For blocking study, the supematants containing EphrinAl-PE38were pre-incubated with an anti-EphrinAl antibody for1hour at37℃before addition to the U251cells. After further incubation for72hours, the cell proliferation was measure as described above. The data from the proliferation assays are reported as a percentage of the untreated control cells. For apoptosis assay, Apoptotic cells induced by the immunotoxin were confirmed by TUNEL and Annexin V-PI double staining.Result:The result of Western blot showed that four different human glioma cell lines (U251, A172, SHG-44and U87) expressed detectable but variable levels of EphA2protein as an immunoreactive band at-130kDa. In marked contrast, the expression of EphA2was barely detected in U-251[EphrinAl](+), T47D and hMSCs. The control β-actin staining on the same blots demonstrated an equal amount of protein was loaded in each lane. To investigate the cytotoxicity of EphrinAl-PE38secreted from transduced hMSCs against target cells, we examined the tumor cell proliferation in the presence of the supematants from transduced or untransduced hMSCs. In EphA2-overexpressing GBM cells, a dose-dependent killing effect by the supematants from Ad-EphrinAl-PE38transduced hMSCs was observed (Fig.4), whereas untransduced hMSCs supematants did not induce any cell death. To test the specificity of EphrinAl-PE38on GBM cells that overexpress the EphA2receptor, several cell lines that have that express low or undetectable levels of EphA2naturally or by down-regulation were evaluated. As negative control cells, T47D and hMSCs were used because these cells hardly express the EphA2receptor. In addition, EphA2-expression on U251[EphrinA1](+) cells was markedly down-regulated by transfection of EphrinAl. These cells responded weakly or not at all to treatment with the supernatants containing EphrinAl-PE38. Next, a blocking experiment was performed as another measurement of the specificity of EphrinAl-PE38. When utilizing an EphrinAl antibody incubated with EphrinAl-PE38for1h at37℃prior to adding the immunotoxin to the U251cells, we found that the sensitivity of the U251to EphrinAl-PE38was blocked in a dose-dependent manner. To assess apoptosis induction by EphrinAl-PE38in U251cells, apoptotic cells in U251cells were identified by the TUNEL assay after incubation with the EphrinAl-PE38supernatants for48h. A significant increase in nuclear morphology and the presence of apoptotic bodies was observed in U251cells cultured in the presence of supernatants containing EphrinAl-PE38compared to the corresponding control supernatants from untransduced hMSCs.Conclusion:Our findings show that EphrinAl-PE38from transduced hMSCs selectively kills GBM cells that overexpress the EphA2receptor. The cytotoxic effect of EphrinAl-PE38on GBM cells was mediated through the EphrinAl ligand.Chapter IV. Effect of hMSCs transduced with Ad-EphrinAl-PE38on growth of established U251flank tumorsObjective:To investigate the delivery and therapeutic efficacy of EphrinAl-PE38released by hMSCs and aim to obtain reliable evidence that genetically modified hMSCs may function as a novel therapeutic vehicle for targeted delivery of immunotoxin to malignant gliomas.Method:For initiation of xenografts, U251cells (5x106) that were suspended in100μl of PBS were inoculated subcutaneously into the flanks of4-6-week-old female BALB/c nude mice. One week after implantation, the mice were randomized into three groups. In each group (n=10), either hMSCs or hMSCs-EphrinAl-PE38(1×106cells in10μl of PBS) were administered into the tumor mass. Control animals received PBS alone. Serial measurements of subcutaneous tumor implants were made with calipers, and tumor sizes were calculated as tumor volume=(width)4×length. For confirmation of in vivo immunotoxin secretion, subcutaneous tumor tissue was harvested from U251glioma-bearing mice at1,4,7,9and14day after treatment with intratumoral injection of hMSCs-EphrinAl-PE38,Ⅲuntransduced hMSCs and PBS respectively (n=2for each group). The EphrinAl-PE38protein concentration was determined from tissue homogenate (100μl) using an EphrinAl ELISA kit. To detect hMSCs-EphrinAl-PE38cells in tumor, frozen sections of tumor tissues were prepared and examined using a fluorescence microscope. Cell apoptosis and proliferation were also examined in the primary tumor tissues obtained from the advanced cancer model by immunofluorescence. This was done by staining for cleaved-caspase3and Ki-67in tumor sections derived from hMSCs-EphrinAl-PE38, hMSCs and PBS control groups.Result:To test the ability of hMSCs-EphrinA1-PE38to inhibit tumor growth in vivo, U251flank xenografts model was established and received different treatment. We found that the tumor volume was decreased in animals treated with hMSCs-EphrinAl-PE38compared to those treated with untransduced hMSCs. This decrease was significant on day12,15,18,22,26and30. No significant difference was observed in tumor size between animals treated with untransduced hMSCs and PBS. to determine whether the in vivo antitumor activity of hMSCs transduced with Ad-EphrinA1-PE38was associated with the levels of EphrinAl-PE38expressed in the tumor mass. The results showed that EphrinAl-PE38expressed in tumor tissues was detectable on day2, peaked on day5, and then began to decrease. In contrast, tumors from the untransduced hMSCs-treated and PBS-treated control group contained an undetectable level of EphrinA1-PE38throughout the experimental period. Fluorescence microscopy confirmed GFP-labeled hMSCs-EphrinAl-PE38cells within the tumor bed. For apoptosis analysis, similar to in vitro results, hMSCs-EphrinA1-PE38induced marked apoptosis in the primary tumor compared to untransduced hMSCs and PBS treatment.Conclusion:The intratumoral injection of engineered hMSCs-EphrinAl-PE38was effective at inhibiting tumor growth in a malignant glioma tumor model. These results indicate that gene therapy utilizing EphrinAl-PE38-secreting hMSCs may provide a novel approach for the local treatment of malignant gliomas. |
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