Gene therapy of ovarian cancer using IL-21-secreting human umbilical cord mesenchymal stem cells in nude mice
- Yunxia Zhang†1, 2,
- Jing Wang†1,
- Mulan Ren†1Email author,
- Miao Li2,
- Dengyu Chen2,
- Junsong Chen2,
- Fangfang Shi3,
- Xiaoying Wang2 and
- Jun Dou1, 2Email author
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 4 December 2013
Accepted: 16 January 2014
Published: 20 January 2014
The human umbilical cord mesenchymal stem cells (hUCMSCs) have the ability to migrate into tumors and therefore have been considered as an alternative source of mesenchymal progenitors for the therapy of malignant diseases. The present study was aimed to investigate effect of hUCMSCs as vehicles for a constant source of transgenic interleukin-21 (IL-21) on ovarian cancer in vivo.
The hUCMSCs were engineered to express IL-21 via lentiviral vector- designated ‘hUCMSCs-LV-IL-21’, and then were transplanted into SKOV3 ovarian cancer xenograft-bearing nude mice. The therapeutic efficacy and mechanisms of this procedure on ovarian cancer was evaluated.
The isolated hUCMSCs were induced to differentiate efficiently into osteoblast and adipocyte lineages in vitro. The expressed IL-21 in the supernatant from hUCMSCs-LV-IL-21 obviously stimulated splenocyte’s proliferation. The hUCMSCs-LV-IL-21 significantly reduced SKOV3 ovarian cancer burden in mice indicated by tumor sizes compared with control mice. The expressed IL-21 not only regulated the levels of IFN-γ and TNF-α in the mouse serum but also increased the expression of NKG2D and MIC A molecules in the tumor tissues. The down regulation of β-catenin and cyclin-D1 in the tumor tissues may refer to the inhibition of SKOV3 ovarian cancer growth in mice. In addition, hUCMSCs did not form gross or histological teratomas up to 60 days posttransplantation in murine lung, liver, stomach and spleen.
These results clearly indicate a safety and usability of hUCMSCs-LV- IL-21 in ovarian cancer gene therapy, suggesting the strategy may be a promising new method for clinical treatment of ovarian cancer.
KeywordsUmbilical cord mesenchymal stem cells Interleukin-21 Ovarian cancer Gene therapy
More than forty years ago, the study showed that cells isolated from the stroma of bone marrow were capable of osteogenesis, suggesting the characteristics of mesenchymal stem cells (MSCs). Subsequent studies have demonstrated that MSCs exist not only in the bone marrow, but also in cord blood, cancellous bone, adipose tissue, synovium and umbilical cord[2–7]. The human umbilical cord contains an inexhaustible, noncontroversial source of stem cells. Human umbilical cord mesenchymal stem cells (hUCMSCs) grow as adherent cells with mesenchymal morphology, have self-renewing, express cell surface markers displayed by MSCs, and may be differentiated into bone, cartilage, adipose, muscle, and neural cells, etc. The properties promotes their potential utility in several intractable human disease such as neurodegenerative disease, cancer, heart disease, etc. Since hUCMSCs have the ability to migrate into tumors, they could be used as vector for targeted cancer therapy[9, 10].
There is increasing evidence that ovarian cancer is the first leading cause of death among gynecologic malignancies. The high mortality rate of ovarian cancer is usually attributed to late diagnosis of this tumor due to a lack of early symptoms. Even though the treatment of optimal cytoreductive surgery followed by systemic chemotherapy is initially effective in 80% of patients, recurrent cancer is inevitable in the vast majority of cases. Furthermore, recurred tumors became unresponsive to the addition of chemoradiation following an initial response[11, 12]. The second-and third-line (and beyond) therapies are aimed at palliative care to prolong the time of tumor progression and to improve the patient’s life quality. Therefore, the novel therapeutic strategies are desperately needed for treatment of this malignant tumor.
In our previous studies, interleukin-21 (IL-21) was involved in T and NK cell activation and induced a strong cell-mediated immune responses in the tumor vaccine approaches[14, 15]. In fact, IL-21 has been extensively applied to significantly augment antitumor immunity in multiple murine tumor models and clinical trials, such as metastatic lymphoma, melanoma[17, 18], ovarian cancer, etc. Previously, we constructed recombinant pIRES2-IL-21-EGFP and transfected it into CD34+ human umbilical cord blood stem cells (UCBSCs) for treatment of ovarian cancer. The animal experiment results indicated that transferring CD34+ UCBSC- IL-21 into A2780 ovarian cancer xenograft-bearing nude mice augmented the therapeutic effect on ovarian cancer, however, IL-21 expression was gradually decreased in the mouse tumor sites 21 days posttransplantation. In order to last the expression of IL-21 in tumor-bearing mice, we used lentiviral vector as delivery of IL-21 in this study. Accumulating evidence indicates that lentiviral vector has evolved as a benchmark tool for stable gene transfer into cells with a high replicative potential and provides a rich resource for numerous applications in experimental platforms and therapeutic settings.
Since hUCMSCs have attracted much attention due to their availability, low immunogenicity, as well as strong tropism for tumors in contrast to hMSCs from other sources[8–10], we particularly focused on the investigation using hUCMSCs to express IL-21 via lentiviral vector to assess the therapeutic effect of hUCMSCs- LV-IL-21 on SKOV3 ovarian cancer xenograft-bearing mouse model in the current study. Here, we show that hUCMSCs themselves do not form tumors, and that tumor growth in ovarian cancer bearing mice is significantly inhibited following systemic administration of IL-21-expressing hUCMSCs.
Cell lines, mice, and primers
Human ovarian cancer cell line SKOV-3 and human embryonic kidney (HEK) 293T cells were purchased from the Cellular Institute in Shanghai, China. SKOV-3 cells were maintained in complete media consisting of RPMI 1640, 2 mmol/L-glutamine, 100 u/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS, Gibco BRL, USA). HEK293T cells were cultured in DMEM medium plus 10% FBS, 2 mmol/LL-glutamine, 100 u/ml penicillin, and 100 μg/ml streptomycin. All cells were cultured at 37°C in a humidified 5% CO2 atmosphere.
Balb/c nude mice of 4–5 weeks of age were acquired from the Animal Center of Yang Zhou University of China and were raised under sterile conditions in air-filtered containers at the Experimental Animal Center, Medical School of Southeast University. All the experiments were performed in compliance with the guidelines of the Animal Research Ethics Board of Southeast University, China.
The used primer sequences are as follows:
The PCR primer sequence for IL-21 gene was 5′-GGCGCTAGCATGGATCG CCTCCTGATTAGACTT-3′ (sense), and 5′-TGCGCGGCGTCGGTGGAGAGAT GCTGAT-3′ (antisense). The PCR primer for natural killer glucoprotein 2 domain (NKG2D) gene was 5′-CTCCATTTTTTTTCTGCTGCTTC-3′ (sense), and 5′-AAG GCTGGCATTTTGAGACATAC-3′ (antisense). The PCR primer for MICA gene was 5′-AAGACCAAGACACACTATCACGC-3′ (sense), and 5′-GGTGTCGTGGC TCAAAGATAC-3′ (antisense). The PCR primer for actin gene was 5′-GTCCACCG CAAATGCTTCTA-3′(sense), and 5′-TGCTGTCACCTTCACCGTTC-3′ (antisense). All the primers were synthesized by Company of Gene and Technology of China in Shanghai.
Isolation and culture of hUCMSCs
All human umbilical cord blood samples were obtained from the Department of Gynecology & Obstetrics of Zhongda Hospital, Southeast University (Nanjing, Jiangsu, China) and were used in accordance with the ethical guidelines and accepted human studies protocols at Southeast University School of Medicine. The length of the umbilical cords in pregnancy (more than 37 weeks) was about 15 cm. hUCMSCs were harvested as described previously[21, 22]. To ensure a homogeneous population of stem cells, passage-4 cells were characterized by flow cytometry (FCM) to analyze specific surface antigens of MSC lineage including CD29, CD34, CD45, and CD90. Approximately 106 cells per vial were used for each antigen detection. Nonspecific binding was first blocked with a staining buffer containing 2% FBS for 15 min, and cells were then incubated with single label antigen for 20 min on ice. Mouse isotype antigen served as a negative control. FCM analysis was conducted using a FAC scan flow cytometer (BD Biosciences). Passage-4 hUCMSCs were used to test the ability of osteoblastic and adipogenic differentiation as described in a previous reports[23–25].
Construction of recombinant lentiviral vector containing IL-21 gene
To generate the IL-21 expression lentivirus vector, we amplified IL-21 by PCR from template plasmid pRSC-IL-21 using the designed primers as aforesaid. The lentivirus IL-21 and the lentiviral without IL-21 gene were produced from the transient transfection of the HEK293T cells with pHAGE-CMV-IL-21-IZsGreen, psPAX2, and pMD2.G plasmid DNAs (kindly provided by Professor Lu Chun, Department of Microbiology and Immunology, Nanjing Medical University, China) plus Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. The IZsGreen expressed by HEK 293T cells was detected using fluorescence microscopy.
Transduction and analysis of IL-21 expression
hUCMSCs were plated in 100 mm plates at 6 × 106 cells per plate. After one day, the cells were 70% confluent. The cells were incubated sequentially with the 48 hour and 72 hour lentivirus-IL-21 and lentivirus supernatants respectively for 12 h. Following the last transduction, the cells were washed and incubated with fresh growth medium to allow puromycin-resistance expression. Two days later, puromycin selection was performed by incubating the cells in growth media supplemented with 1 μg/mL puromycin (Clontech Laboratories Inc.) for five days. After selection, hUCMSCs containing lentivirus-IL-21 (named hUCMSCs-LV-IL-21) and hUCMSCs lentivirus without IL-21 (named hUCMSCs-LV-Vec) were selected respectively. Then IL-21 expression was analyzed by western blotting. To identify the bioactivity of UCMSCs-LV-IL-21, the splenocytes from the normal nude mice were incubated with the supernatant from the cultured hUCMSCs-LV-IL-21 for 72 h, then the splenocyte proliferative activity was detected by FCM as described in a previous report.
Evaluation of gene therapy of ovarian cancer efficacy
Balb/c nude mice of 4–5 weeks of age were randomly assigned to PBS control group, hUCMSCs group, hUCMSCs-LV-Vec group, and hUCMSCs-LV-IL-21. In establishing an ovarian cancer xenograft-bearing nude mice model, 5 × 106 SKOV3 cells were subcutaneously injected into a mouse’s right flank. About 7 days after the injection, tumors were visible to the naked eye. Then, 100 μl PBS, 1 × 106 hUCMSCs or hUCMSCs-LV-Vec or hUCMSCs-LV-IL-21 were injected into the mouse’s tumor sites, and tumor growth was monitored each day. The tumor volumes were evaluated every 3 days by measuring two perpendicular diameters of the tumors using calipers. Three mice per group were used and the experiment was repeated two times. All mice were sacrificed at 35 days after injection of different hUCMSCs. In addition, 1 × 106 hUCMSCs were injected into 5 nude mice to observe if the cells could form tumors by 60 days of observation.
ELISA for IL-21, IFN-γ and TNF-α
Mouse serum cytokines were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (eBioscience Company, USA). Briefly, samples were diluted 1:5 with PBS, and each cytokine was captured by the specific primary monoclonal antibody and detected by biotin labeled secondary antibody. The plates were read at 450 nm using a microplate reader (Bio-Rad Labs, Hercules, CA). The samples and standards were run in triplicate, and the sensitivity of the assays was 0.1 U/ml for interferon-γ (IFN-γ), IL-21, and tumor necrosis factor-α (TNF-α), respectively[29, 30].
RNA isolation and quantitative RT-PCR
Total RNA was obtained using the Qiagen RNeasy kit (Qiagen, CA). Isolated RNA samples were dissolved in RNase-free water, and RNA quantity was measured using NanoDrop (Thermo Fisher Scientific). cDNA was synthesised from 10 μg of total RNA at a volume of 100 μl using ImProm RT-II™ (Promega) according to the manufacturer’s instructions. cDNA samples were diluted with sterile deionised water to a total volume of 100 μl, and 2 μl was added to a PCR reaction. Quantitative RT-PCR (qRT-PCR) was performed on an ABI step one plus real-time system (Applied Biosystems, USA). We analyzed the relative expression levels of NKG2D and MIC A genes using the primers described above.
The protocol was based on a previously published report. Briefly, 1 × 106 cells were collected and lyzed in a protein extraction buffer (Novagen, WI) according to the manufacturer’s protocol. Western blotting was performed after 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins (15 μg/lane) were electrotransferred onto a nitrocellulose membrane. The rabbit antibody specific to mouse IL-21 or human β-catenin or human cyclin-D1 was added to the membrane for 1 h, the membrane was washed for 5 min with antibody wash solution three times, and the subsequent steps were performed according to the Western-Breeze Kit’s protocol (Invitrogen). Immunoreactive bands were detected by the Odyssey scanning instrument (LICOR Odyssey, USA).
Histopathology of tissues and tumors
Mice were sacrificed 35 days (gene therapy of ovarian cancer experiment) after the SKOV3 cell challenge or 60 days (tumor formation experiment) after the injection of hUCMSCs. The tissues of the spleens, livers, stomachs, and lungs as well as the tumor tissues were removed from the nude mice and fixed in 10% formalin, and then embedded in paraffin. Serial thin tissues and tumor sections (4 μm) were cut and mounted on SuperFrost Plus glass slides, fixed in methanol, and stained in hematoxylin and eosin (H&E).
μm formalin-fixed, paraffin embedded slides were incubated with the rabbit anti human β-catenin antibody or rabbit anti human cyclin-D1 antibody (Sigma) after over night incubation at 4°C. The samples were then labeled with horseradish peroxidase conjugated streptavidin (Invitrogen) and the chromogenic reaction developed using liquid DAB substrate pack (Biogenex, San Ramon, CA) according to the manufacturer’s instructions. β-catenin and cyclin-D1 stained cells from random and non-overlapping fields were counted under a light microscope, magnification of 200× .
Statistical comparisons were performed using the Student’s t-test method or single factor analysis of variance to test for any statistically significant differences in the results between the experiment group and the control group. P < 0.05 was considered statistically significant. Analyses were performed with the Graph Pad Prism 5.0 statistical software package (Graph Pad Company, USA).
Culture and identification of hUCMSCs
Transduction and analysis of IL-21 expression
Efficacy of hUCMSCs-LV-IL-21 therapy for ovarian cancer in nude mice and the related mechanism investigation
Down-regulation of β-catenin and cyclin-D1 in tumor tissues
Histological analysis of the spleen, liver, lung, stomach and tumor tissues in the nude mice
In the study, we first constructed the recombinant lentiviral vector for lasting the expression of IL-21, and then the hUCMSCs transducted by IL-21 gene packaged lentivirus had been injected into mouse ovarian cancer model to assess the effect of hUCMSCs-LV-IL-21 on ovarian cancer in vivo.
Our experimental results showed that hUCMSCs-LV-IL-21 induced powerful tumoricidal activities against human ovarian tumor in the xenograft-bearing Balb/c nude mouse model after the injection of SKOV3 cells. This effectiveness was reflected in the tumors showing slower growth and the smaller sizes (Figure 3), in the tumor cell necrosis, and in the observation that the vascular bleeding was more obvious in the tumor tissues of mice treated with hUCMSCs-LV-IL-21 group than other groups (Figure 5H).
The IL-21 secreted by the hUCMSCs-LV-IL-21 may stimulate the innate immune response to SKOV3 ovarian cells as well as induce hUCMSCs-LV-IL-21 to differentiate more immune cells, enhance the immune activity, and play a key role in killing tumor cells or drive tumor cell apoptosis. Although hUCMSCs can also elicit antitumor immune responses in contrast to the control group, this antitumor efficacy was especially true of the mice transplanted with hUCMSCs-LV-IL-21 that secrete IL-21 for long time.
To understand the mechanisms of antitumor efficacy induced by hUCMSCs- LV-IL-21, we examined the serum cytokines of IL-21, IFN-γ, and TNF-α, which have an important functions in antitumor immunity and their levels indirectly represent the capacity of rejection of tumor in bearing-tumor nude mice. The secreted IL-21 by hUCMSCs-LV-IL-21 can induce secondary cytokine production. The secreted IFN-γ and TNF-α by the NK cells or the macrophage, particularly the IFN-γ, can react to the NK cells, increase its cytotoxic activity, and play a key role in killing tumor cells or induce tumor cell apoptosis. The presented data showed that the IL-21, IFN-γ and TNF-α were respectively enhanced in the mice treated with hUCMSCs-LV-IL-21 group compared with other groups. Although it has been shown that serum levels of cytokines delivered by engineered stem cells have no obvious role in contrast to cytokine given systemically, the local production of cytokine appears to be the key for tumor growth attenuation. The stem cell-based gene therapy can also avoid potential systemic adverse effect of cytokines. We speculate that hUCMSCs-LV-IL-21 elevated IFN-γ and TNF-α related to the secretion of IL-21, and that the elevated IFN-γ and TNF-α might markedly enhance the NK cytotoxicity that was supported by increase of expression of NKG2D and MIC A in the tumor tissues of the mice treated with hUCMSCs-LV-IL-21group. The enhanced NK cytotoxicity may be closely associated with the tumor cell necrosis, apoptosis, and the vascular bleeding in the tumor tissues (Figure 5H).
The canonical Wnt signaling pathway includes stabilization of cytosolic β-catenin, nuclear translocation, and gene regulation. Genes regulated by Wnt signaling are involved in metabolism, proliferation, cell cycle, and apoptosis. Therefore, the proliferation of tumor cells can be influenced by regulating canonical Wnt signaling pathway[34, 37]. It has been suggested that hUCMSCs could inhibit tumor growth through the regulation of this pathway. We supposed that canonical Wnt signaling pathway might be involved in the inhibitory effects on ovarian cancer cells mediated by hUCMSCs-LV-IL-21. To test the hypothesis, we examined the molecular expression of β-catenin in tumor tissues. The results of western blotting and immunohistochemistry showed that the expression of β-catenin was down regulated in tumor tissues of mice treated with hUCMSCs, hUCMSCs-LV-Vec and hUCMSCs-LV-IL-21, respectively, especially in the hUCMSCs-LV-IL-21 group. To further evaluate whether known β-catenin target molecule was also reduced, we analyzed the expression of cyclin-D1. Consistent with the observed reduction in β-catenin, the cyclin-D1 expression was also down-regulated in same samples. We guess that the hUCMSCs-LV-IL-21 reduced β-catenin expression and might decrease β-catenin translocation to the nucleus to decrease the expression of cyclin-D1 that may reduce the transcription of its specific target gene, resulting in inhibition of tumor cell growth.
In conclusion, our data show that hUCMSCs display very similar characteristics to bone marrow-MSCs and could represent a valuable alternative for cell-based therapies. Transplanting hUCMSCs-LV-IL-21 into SKOV3 ovarian cancer xenograft-bearing nude mice has a definite therapeutic effect on inhibition of ovarian cancer growth. This augmented the therapeutic efficacy may contribute to the secreted IL-21 from hUCMSCs-LV-IL-21 and increase of the expression of NKG2D and MIC A, and assisting in exerting an antitumor effect through augmenting NK cytotoxicity. We speculate that the hUCMSCs-LV-IL-21 play an anti-tumor effect through regulating canonical Wnt signaling pathway. A more detailed understanding of the β-catenin and cyclin-D1 role in Wnt signaling pathway may be helpful in the investigation of the molecular mechanisms presented in hUCMSCs-LV-IL-21, and may improve therapeutic strategies involving hUCMSCs-LV-IL-21-mediated targeting therapy of ovarian cancer.
This work was supported in part by the 973 National Nature Science Foundation of People’s Republic of China (2011CB933500) and part by the National Natural Science Foundation of China (No. 81071769, No. 81202372).
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