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Cancer Cell-Oriented Migration of Mesenchymal Stem Cells Engineered with an Anticancer Gene (PTEN): an Imaging Demonstration

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Cancer Cell-Oriented Migration of Mesenchymal Stem Cells Engineered with an Anticancer Gene (PTEN): an Imaging Demonstration OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration Zhuo-Shun Yang1,* Background: Mesenchymal stem cells (MSCs) have been considered to hold great potential Xiang-Jun Tang2,* as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. Xing-Rong Guo1 This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) Dan-Dan Zou1 engineering on MSCs’ capacity for cancer cell-oriented migration. Xu-Yong Sun3 Methods: MSCs were engineered with a PTEN-bearing plasmid and the expression was con- Jing-Bo Feng1 firmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic Jie Luo1 system. Long-Jun Dai1,4 Results: The expression of transfected PTEN in MSCs was identified by immunoblotting 4 Garth L Warnock analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented 1Hubei Key Laboratory of Stem migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring Cell Research, Taihe Hospital, system, and a phagocytosis-like action of MSCs was also observed. Hubei University of Medicine, Shiyan, People’s Republic of China; Conclusion: MSCs maintained their capacity for cancer cell-directed migration after they 2Department of Neurosurgery, were engineered with anticancer genes. This study provides the first direct evidence of MSCs’ Taihe Hospital, Hubei University of Medicine, Shiyan, People’s Republic tropism post-anticancer gene engineering. of China; 3Guangxi Key Laboratory Keywords: gene therapy, mesenchymal stem cells, phosphatase and tensin homolog, cancer for Transplant Medicine, 303 Hospital of PLA, Nanning, People’s Republic of China; 4Department of Surgery, Introduction University of British Columbia, Vancouver, BC, Canada Cancer is one of the most common life-threatening diseases, accounting for an esti- mated one in four human deaths.1 Despite improved treatment models, many tumors *These authors contributed equally to this work remain unresponsive to conventional cancer therapies. The major obstacle limiting the effectiveness of conventional therapies for cancer is their tumor specificity. An ideal therapeutic strategy would directly target tumors, in both primary and metastatic sites, and possess the ability to act locally over a sustained period of time. Mesenchymal stem cells (MSCs) were first identified in the stromal compart- 2,3 Correspondence: Long-Jun Dai ment of bone marrow by Friedenstein et al in the 1960s. MSCs have generated Department of Surgery, University of considerable biomedical interest since their multilineage potential was first identified British Columbia, 400-828 West 10th 4 Avenue, Vancouver, BC, V5Z 1L8, Canada in 1999. Owing to their easy acquisition, fast ex vivo expansion, and the feasibility Tel +1 604 875 4111 ext 62501 of autologous transplantation, MSCs have become the first type of stem cells to be Fax +1 604 875 4376 Email [email protected] utilized in clinical applications. Recent findings on specific tumor-oriented migration and incorporation of MSCs demonstrate the great potential for MSCs to be used as an Jie Luo ideal carrier for anticancer gene delivery.5–7 Tumor-directed migration and incorpora- Taihe Hospital, 32 Renmin Road, Shiyan, 442000, People’s Republic of China tion of MSCs have been demonstrated by a number of preclinical studies using both Tel +86 719 880 1880 transwell migration assays (in vitro) and animal tumor models (in vivo). The homing Fax +86 719 880 1218 Email [email protected] capacity of MSCs has been demonstrated with almost all tested human cancer cell submit your manuscript | www.dovepress.com OncoTargets and Therapy 2014:7 441–446 441 Dovepress © 2014 Yang et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further http://dx.doi.org/10.2147/OTT.S59227 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Yang et al Dovepress lines, including lung cancer,8 malignant glioma,9 breast can- (MKFPSQLLLLLLFGIPGM) and an eleven amino cer,10 colon carcinoma,11 pancreatic cancer,12,13 melanoma,14 acid transacting activator of transcription (TAT, YGRK- and ovarian cancer.10 The high frequency of MSC migration KRRQRRR) were inserted at the multiple cloning site fol- and incorporation was observed during in vitro coculture and lowed by human PTEN (403 amino acids). The fusion protein in vivo xenograft tumors, respectively; these findings were was designated as TAT-PTEN-red fuorescent protein (RFP) found to be consistent, and were independent of tumor type, with the predicted molecular weight of 74 kDa. immunocompetence, and delivery route of MSCs. In order to utilize MSCs as the therapeutic vehicles for Plasmid transfection of MSCs cancer treatment, it is critical to maintain the tumor-tropic PTEN-bearing expression plasmid was transfected into MSCs capacity of these cells. However, whether such a homing with an electroporation method. MSCs were collected and property is altered by the engineering process of adding washed with Opti-MEM (Life Technologies, Carlsbad, CA, anticancer genes to the MSCs remains largely unknown. In USA) three times, then resuspended with Opti-MEM at a our recent pancreatic cancer study, the cancer cell-oriented density of 1×107cells/mL. MSCs suspension (90 µL) was migration of MSCs was demonstrated using a real-time cell placed into each cuvette and 10 µg plasmid (1 µg/µL) was analyzer system (xCELLigence; Roche Diagnostics, India- added. The electroporation was performed according to the napolis, IN, USA).13 In the present study, imaging techniques manufacturer’s instruction (NEPA21; Nepa Gene Co, Ltd., were utilized to directly verify the cancer cell-oriented migra- Chiba, Japan). After a series of trials on this particular cell tion of MSCs which were engineered with an anticancer gene, type, the most optimized transfection conditions were defined phosphatase and tensin homolog (PTEN), which functions as a voltage of 150 V and a 5 millisecond pulse length. The as the central negative regulator of the PI3K-AKT-mTOR PTEN-engineered MSCs were designated as MSCPTEN. Two (phosphoinositide 3-kinase – protein kinase B – mammalian days after transfection, the culture media were collected as target of rapamycin) pathway in controlling apoptosis. conditioned media for coculture studies. Materials and methods Immunoblotting and enzyme-linked Cells and culture conditions immunosorbent assay (ELISA) analysis MSCs were isolated from human pancreas and expanded Immunoblotting analysis was used to detect the cellular expres- ex vivo as previously described.13,15 Based on the mini- sion of PTEN in the MSCs. The MSCs transfected with PTEN mal criteria for defining human MSCs established by the or mock infected were harvested in lysis solution. Whole-cell International Society of Cellular Therapy, theses MSCs were lysates (50 µg) were separated through 12% denaturing sodium verified by both membrane biomarker determination and dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and functional differentiation. They fulfilled the characteristics transferred to a nitrocellulose membrane. The membrane was of human MSCs, exhibiting positive expression of cluster incubated overnight with mouse anti-PTEN antibody (1:1000; of differentiation (CD) 44+, CD73+, CD95+, and CD105+, R&D Systems Inc., Minneapolis, MN, USA). A separate and negative expression of CD34–. The results of adipogenic membrane was prepared and incubated overnight with mouse and osteogenic differentiation also met the standards. MSCs anti-red fluorescent protein (RFP) antibody (1:1000; Abcam were cultured in Minimum Essential Medium (MEM) with PLC, Cambridge, England). This was followed by a 1 hour 10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin- incubation with goat-anti-mouse immunoglobulin (Ig)G con- streptomycin solution (all from Life Technologies, Carlsbad, jugated to horseradish peroxidase (HRP) (1:2500). The blots were developed using enhanced chemiluminescence detection CA, USA) and incubated at 37°C in a humidified, 5% CO2 atmosphere. A human glioma cell line (DBTRG) was pur- (Amersham Bioscience, Baie D’Urfe, QC, Canada). chased from American Type Culture Collection (ATCC, The soluble PTEN in the culture supernatants was mea- Manassas, VA, USA) and used as target cells in the present sured using ELISA as per the manufacturer’s instruction study. DBTRG cells were maintained as suggested by ATCC (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Con- and their culture condition was kept consistent with MSCs. ditioned media collected from the corresponding cultures were equally concentrated using 10,000 molecular weight cut Construction of PTEN-bearing off (MWCO) (cat # 42406; EMD Millipore, Billerica, MA, expression vector USA) and protein concentrations were determined using
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