- Open Access
PhiC31/PiggyBac modified stromal stem cells: effect of interferon γ and/or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on murine melanoma
© Bahrambeigi et al.; licensee BioMed Central Ltd. 2014
Received: 8 August 2014
Accepted: 14 November 2014
Published: 26 November 2014
TRAIL and IFNγ are promising anti-cancer cytokines and it has been shown that IFNγ may sensitize cancer cells to TRAIL. Adipose derived mesenchymal stem cells (ADSCs) are attractive vehicles for delivering anti-cancer agents. In this study, we evaluated the therapeutic potential of PhiC31 (φC31) recombinase and/or piggyBac transposase (pBt) modified ADSCs expressing either TRAIL, IFNγ, or co-expressing TRAIL/IFNγ in mouse models of melanoma.
The expression and bioactivity of mouse IFNγ and TRAIL in φC31 and pBt modified cells were confirmed. We examined the effects of modified ADSCs on signal intensity of red fluorescence protein expressed by melanoma cells in subcutaneous tumors or established lung metastases and on survival (6 mice per group). We also conducted a flow cytometric analysis of systemic CD4+CD25+FOXP3+ T regulatory cells (Tregs) and histological analysis of melanoma tumors. Data were analyzed by Student t test, ANOVA, and log-rank tests. All statistical tests were two-sided.
We demonstrated non-viral DNA-integrating vectors can be used for stable transgene expression. IFNγ inhibited melanoma cell growth in vitro probably via IFNγ-induced JAK/STAT1 signaling pathway activation. Murine TRAIL induced apoptosis in the human cell lines CAOV-4 and Ej-138, while MCF7 and B16F10 cells appeared to be insensitive to TRAIL. Treatment of melanoma cells with IFNγ did not influence their response to TRAIL. In contrast, results from in vivo studies showed that IFNγ-expressing ADSCs, engrafted into tumor stroma, inhibited tumor growth and angiogenesis, prevented systemic increase of Tregs, increased PD-L1 expression and CD8+ infiltration (but not interleukin-2+ cells), and prolonged the survival of mice (68 days, 95% confidence interval [CI] =52 to 86 days compared to 36 days, 95% CI =29 to 39 days for control, P < .001).
For the first time, we employed DNA integrating vectors for safe and stable modification of MSCs. Our data indicate potential of non-virally modified IFNγ-expressing ADSCs for treatment of melanoma through direct effects of IFNγ. This study may have a significant role in the management of cancer in the future.
Mesenchymal stem cells (MSCs) are emerging as promising tools for combined cancer gene/cell therapies since they have the unique ability of targeting tumor cells. Several recent studies have successfully used viral-based gene transfer approaches to modify MSCs. However, immunogenicity, risk of insertional mutagenesis, and accidental production of self-replicating viruses are of concern and remain a problem for viral systems. Non-viral gene delivery methods represent a simpler and safer alternative, as long-term expression of the therapeutic genes can be achieved though their stable integration into the host genome using DNA-based gene transfer vectors. Commonly used non-viral integrating vectors permanently integrate DNA into the host genome via either a recombinase or transposase. PhiC31 (φC31) recombinase and piggyBac transposase (pBt) are two representatives of DNA-based gene transfer vectors that are under intensive development[4, 5]. The site-specific recombinase of bacteriophage φC31 integrates the complete plasmid construct carrying an attB sequence into pseudo attP site in the mammalian genome. Compared to φC31, pBt insert only the transposon cassette including the transgenes situated inside of terminal repeat elements (TREs). We used the φC31 and pBt systems to achieve long term gene expression of therapeutic agents in murine adipose derived MSCs (ADSCs).
The cytokine type II interferon (IFNγ) can be used as a therapeutic agent as it exerts a variety of different anti-tumor effects, including inhibition of cancer cell proliferation, repression of tumor angiogenesis, and the induction of tumor cell apoptosis[7, 8]. IFNγ also stimulates the host immune response and enhances tumor cell apoptosis via tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). TRAIL in its role as a death ligand binds to the surface death receptors (DR; DR1 and DR2) and induces apoptosis in a variety of neoplastic cells while sparing most normal cells. Cancer cells have variable levels of sensitivity to TRAIL-mediated apoptosis and studies have shown that IFNγ pre-treatment can sensitize some of the resistant cancer lines to TRAIL[11–15]. Besides, IFNγ/TRAIL combination immunotherapy has been shown to synergistically induce tumor cell death. However, to yield significant anti-tumor activity, multiple high-dose systemic administration of these cytokines is necessary which is associated with adverse side effects[10, 17]. To overcome this limitation, several studies used cytokine-expressing MSCs to mitigate cancer progress in tumor models including melanoma[18–20]. Therefore, in this study we aimed to investigate antitumor activity of φC31/pBt modified murine ADSCs expressing IFNγ and TRAIL individually, or co-expressing Trail/IFNγ in vitro and in mouse subcutaneous or lung metastasis models of melanoma.
Characterization of murine ADSCs
Stable modification of ADSCs and melanoma B16f10 Cells by DNA integrating vectors
Effect of IFNγ on viability and apoptosis of melanoma cells and the possible involvement of JAK/STAT1 dependent signaling pathway
Bioactivity of TRAIL and In Vitro effect of IFNγ on sensitization of melanoma cells
Plasma levels of IFNγ after injection of IFNγ-producing ADSCs to mice
ELISA was performed to evaluate the effect of stem cell therapy on systemic levels of IFNγ at three time points (days 10, 16, and 21). The systemic levels of IFNγ in all of the experimental groups were below the detection level of our ELISA assay, indicating very low plasma levels.
In Vivo effect of modified ADSCs on growth of subcutaneous melanoma and melanoma pulmonary tumors, and survival analysis of mice with melanoma metastases
Murine ADSC homing
The effect of cell therapy on infiltration of CD4+, CD8+ ,FOXP3+ and IL2+ Cells
The effect of cell therapy on population of systemic CD4+CD25+FOXP3+ T regulatory cells (Tregs)
Population analysis of peripheral blood mononuclear cell (PBMC) isolated by Ficoll density gradient revealed two separate populations of PBMCs; lymphocytes and monocytes (Figure 9C, left panel). We initially analyzed PBMCs and lymphocytes for CD4 (Figure 9C, right panel) and subsequently CD4+ lymphocytes for CD25+ FOXP3+ by FACS analysis (Figure 9D). We noted that the mean percentage of CD4+ cells significantly decreased in ADSC-injected groups in a statistically meaningful manner (ANOVA, P = .021 for EGFP-ADSCs and P =0.002 for IFNγ-ADCS compared to normal mice, Figure 9E). Moreover, our results indicated that the population of Tregs in mice treated with IFNγ-ADCS (P =0.109) and IFNγ/TRAIL-ADSCs (P =0.797) were comparable to the population of Tregs in normal mice. In contrast, the population of Tregs were increased significantly in a statistical manner in mice with established B16F10 lung metastases, which were injected with PBS only, EGFP-ADSCs or TRAIL-ADSCs (ANOVA, P < .001, Figure 9E).
In the present study, we demonstrated that φC31 and/or pBt mediated non-viral gene transfer provides prolonged and high levels of transgene expression in ADSCs. Both the recombinase and the transposase systems worked well for modification of ADSCs. However, genomic modifications mediated by φC31 may result in chromosomal rearrangements, whereas pBt is deactivated during the excision of the transposon from the helper-independent pmhy GENIE-3 construct. We observed robust levels of IFNγ and TRAIL expression in GM-ADSCs conferring upon them the ability to be efficiently used for cancer therapy. We also demonstrated that exogenously administered ADSCs survive and proliferate in the tumor environment. When co-injected with melanoma cells, ADSCs mostly stayed along with melanoma cells. We explored the effect of TRAIL-ADSCs and we showed that murine TRAIL, with a homology of 65% to human TRAIL is active on human CAOV-4 and Ej-138 cell lines. However, certain tumor cells such as melanoma B16F10 and human MCF-7 breast cancer cell lines used in this study are not amenable to TRAIL-mediated apoptosis. IFNγ may sensitize cancer cells to TRAIL-induced apoptosis by upregulating Caspase-8 through a Stat1/IRF1 dependent pathway[11–16]. Still, we did not observe sensitization of melanoma cells to TRAIL in the presence of IFNγ. Therefore, melanoma resistance is probably based upon lower expression of functional death receptors (DR1 and DR2). Actually, a recent study suggests the role of NFκB-mediated inflammatory signals through the death receptor DR5 which may promote malignant behaviors of melanoma cells. Interestingly, the effect of IFNγ on melanoma B16F10 cells was remarkable. IFNγ has been clinically applied to treat a variety of malignancies. Phase I trials of IFNγ gene transfer into cancer cells in patients with metastatic melanoma have been performed, albeit with low therapeutic efficacy[25–27]. Furthermore, it has been shown that human IFNγ produced by genetically modified MSCs was able to inhibit proliferation and induce apoptosis of human leukemia K562 cells in vitro. We demonstrated that IFNγ secreted by GM-ADSCs is able to directly affect melanoma cells in vitro, probably through activation of JAk1/Stat1 pathway. Moreover, co-injection of IFNγ-ADSCs with melanoma cells reduced the growth of subcutaneous melanoma tumors. More importantly, intravenously injected IFNγ-ADSCs significantly reduced growth of pre-established melanoma lung metastases. In addition, systemic delivery of IFNγ-ADSCs does not elevate IFNγ amounts in blood circulation, suggesting local expression of IFNγ. We also explored the anti-tumor effects of IFNγ through the activation of the immune response. We noted a significantly increased infiltration of CD8+ cells, while the CD4+ and IL2+ cell count in the tumor environment of IFNγ-ADSC treated mice remained constant. Additionally, we confirmed IFNγ-induced overexpression of PD-L1 in melanoma tumor cells[31, 32]. These observations suggest a possible role of PD-L1 in impairing T cell function because of; 1) the absence of infiltrated T helper 1 cells (no statistically significant difference in CD4+ and IL2+ cells) which is critical for the development of cell-mediated immune response, and 2) the accumulation of exhausted and inflamed CD8+ cells which failed to produce IL2. The latter could explain why melanoma metastases avoided complete eradication in spite of significant infiltration of CD8+ cells. Actually, CD8+ T cells and IFNγ may induce the expression of indoleamine-2,3-dioxygenase (IDO) and PD-L1 in the melanoma tumor microenvironment, and inhibit the activation of additional T cells trafficking. Therefore, we hypothesize that the observed anti-tumor effect was primarily due to apoptosis in the melanoma tumor and tumor endothelial cells. A similar observation has been reported by Kakuta et al., where they examined effects of IFNγ receptor-deficiency and NK cell depletion on the melanoma tumor growth and suggested that IFNγ prevents the murine melanoma metastases by directly inhibiting cell growth when the tumor mass is small and in an earlier developmental stage. It is also noteworthy that the population of infiltrated FOXP3+ cells in IFNγ-ADSC treated mice did not differ to those in mice of other treatment groups, indicating that the higher systemic Tregs (in control and EGFP-ADSC injected mice) was possibly due to immunosuppression mediated by tumor progression. Therefore, intravenous injection of IFNγ-ADSC indirectly maintained normal levels of systemic Treg through inhibition of melanoma growth. However, systemic injection of GM-ADSCs showed immunosuppressive properties probably associated with PD-L1 expression by ADSCs. The selective blockage of PD-L1 may be a potential strategy to improve the therapeutic value of IFNγ-producing ADSCs for melanoma treatment.
We were able to show that systematically transplanted ADSCs engrafted into tumor cells, however the fact that stem cells also migrate to many other organs makes it difficult to trace them. In fact, there is no clear consensus amongst researchers where else such treated stem cells end up after injection. It is well known that systemically infused MSCs typically become trapped within the lungs as the first micro capillary network they encounter (pulmonary "first-pass" effect). Within 24h MSCs migrate to other organs, in particular the liver and also the spleen[41, 42]. Consecutively, MSCs appear at injured tissue sites and tumors as well as bone marrow, liver and spleen[43, 44]. This redistribution attributes to the observed reduction of MSCs present in the lungs, as does cell death. Kidd et al. showed that in addition to the specific tropism of MSCs in tumor sites, some MSCs remained in the lungs, as well as some disseminating into the liver. An alternate study showed that few days after tail vein stem cell injections, cells dissipated from the animals and were undetectable within one week after injection.
Studies have attempted to address the eventual fate of the MSCs within tumor microenvironments. MSC may differentiate towards tumor-associated fibroblast (TAF) phenotypes. In addition, MSCs may acquire endothelial-like characteristics, but their involvement in vasculogenesis is complex. Comşa et al. indicated that MSCs, negative for CD31, have a clear tendency to form capillary-like structures in the presence of tumor-derived VEGF. The same proved true in our hands where ADSCs co-injected with melanoma cells organized into capillary-like structures, whereas, they did not differentiate into three mesodermal lineages in the melanoma tumor environment.
In order to translate stem cell-based anticancer strategies into clinical therapy, it is essential to identify and minimize treatment-associated risks. Only with improvements in safety, quality, and efficiency of stem cell/gene therapy for inoperable or malignant tumors, clinical scenarios can be envisaged. For the first time we employed DNA integrating vectors including PhiC31and PiggyBac transposase systems for safe and stable modification of MSCs. We modified ADSCs to produce IFNγ and TRAIL. Then evaluated antitumor effects of cytokine-producing GM-ADSCs in murine models of melanoma. The present study is the first in vivo attempt to use ADSCs as a vehicle for IFNγ-mediated immunotherapy and demonstrates the potential of non-virally modified IFNγ-ADSCs for melanoma cancer therapy. This may have a significant role in the management of cancer in the future.
Materials and methods
Isolation of ADSCs and culture
ADSCs were isolated from inguinal fat pads of 4–5 week-old male C57BL6 mice (N = 8; 21.5 ± 0.7 g body weight). Isolated cells were grown in culture medium (CM) consisting of Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Carlsbad, CA), 10% Fetal bovine serum (FBS; Invitrogen Carlsbad, CA), 1 g/L glucose, 1% L-glutamine, and 1% penicillin-streptomycin (Invitrogen Carlsbad, CA). For details please see Additional file1.
ADSCs cultured at passage 6 were used for adipogenic, osteogenic, and chondrogenic differentiation. As a first step, cells were cultured for 3 weeks in differentiation medium and consecutively stained with Oil red O, Alizarin red S, Von Kossa S, Alcian blue, and Toluidine blue. For a detailed description of the experiments please see Additional file1.
Immunotyping of ADSCs
Immunotyping of surface markers was performed in passage 6 with BD Calibur™ (Becton Dickinson San Jose, CA) using monoclonal antibodies against mouse CD11b, CD24, CD34, CD45, CD73, CD90.1, CD105, CD133, CD146, CD309 and CXCR4 (all form eBioscience, San Diego, CA). A total of 5 × 105 cells from passage 6 were incubated with each antibody (Ab) in PBS with 3% bovine serum albumin (BSA; Sigma, St. Louis, MO) for 40 min at 4°C. The Cells were then washed with PBS and fixed with BD fixation reagent (BD Biosciences, San Jose, CA sciences). Analysis of the FACS data was carried out with a FlowJo software version 10 (Treestar, OR).
Plasmids pCAG-DsReds, pCMVInt, pDRBB2, pBEB, pISRE-TA-Luc (Clontech, Mountain View, CA), pBLB, pmhy GENIE-3, coding sequences (CDS) of murine full-length IFNγ, TRAIL and the sequence of SV40 Promoter were used in this study. The plasmid pBEB (carrying the attB, the enhanced green fluorescent protein [EGFP] reporter gene) and the vector pmhy GENIE-3 (containing EGFP gene, a hyperactive self inactivating piggyBac sequence and piggyBac transposon) were used as a control plasmids for integrase and transposase systems. We prepared the vector pDsRed-attb-zeo (carrying eRFP, attB, eukaryotic zeocin resistance gene) using the plasmid pCAG-DsReds (expressing the Red fluorescent protein [eRFP] reporter gene), the plasmid pDRBB2 (carrying attB and antibiotic resistance gene for zeocin under the prokaryotic T7 promoter) and a synthesized sequence of T7 promoter. The vector pBTB (carrying attB and antibiotic resistance gene for G418 and a murine full-length TRAIL) was prepared using the vector pBLB (carrying the attB and firefly luciferase [FL] under a chicken beta actin and rabbit beta globin intron [CAG] promoter) and synthesized murine TRAIL coding sequence (CDS; protein ID = NP_033451.1). The plasmid pISRE-TA-Luc-attB was prepared using the vector pISRE-TA-Luc (carrying FL gene located downstream of ISRE enhancer element and a minimal TA promoter; Clontech, Palo Alto CA) and pBLB. All integrase-related constructs were co-transfected with the plasmid pCMVInt (producing φC31 integrase) to achieve stable cell modification. Plasmids pDsRed-attb-zeo, pmhy GENIE-3 and the coding sequence of the full-length murine IFNγ (protein ID = NP_032363.1) were used for construction of pmhy GENIE-3-IFNγ. CDS of murine full-length IFNγ, TRAIL and the sequence of SV40 Promoter with pertinent restriction sites were synthesized by Generay Biotech (Co., Ltd). All vectors were purified by EndoFree® plasmid maxi kit (Qiagen, Valencia, CA) before transfection. For additional information on vector construction please see Additional file1.
Murine ADSCs transfection, selection, and transgene expression
Nucleofection was perfomed by nucleofector device 2b (Amaxa Biosystems) using the Human MSC Nucleofector kit (Amaxa Biosystems) with program X-001 (mouse T cells). About 2 μg of DNA plasmid and 5 × 105 ADSCs were used for each nucleofection experiment and done in triplicate for each plasmid construct. The vector pMAX was utilized for evaluation of nucleofection efficiency. ADSCs modified with pBEB/pCMVInt, pBIB/pCMVInt, pmhy GENIE-3, and pmhy GENIE-3-IFNγ are referred to as EGFP/Int-ADSC, TRAIL-ADSC, EGFP-ADSC and IFNγ-ADSC respectively. TRAIL-ADSCs were modified with pmhy GENIE-3-IFNγ to generate ADSCs co-expressing TRAIL and IFNγ (referred as IFNγ/TRAIL-ADSC). After each nucleofection, 500 μl of CM was added to each nucleofector cuvette and cells were seeded into 6 well plates at 37°C in 5% CO2. Forty eight hours after each nucleofection, selection with related resistant antibiotic was initiated. ADSCs co-nucleofected with the vectors pBEB/pCMVInt (EGFP/Int-ADSC) and pBTB/pCMVInt (TRAIL-ADSC) were exposed to 1000 μg/mL of G418 sulfate (Roche, Indianapolis, IA) for two weeks and maintained in CM with 800 μg/mL of G418 sulfate. ADSCs nucleofected with the plasmids pmhyGENIE-3 (GFP-ADSC) and pmhy GENIE-3-IFNγ (IFNγ-ADSC) were selected with 200 μg/mL of hygromycin (Roche, Indianapolis, IA) for about 10 days and maintained under selection pressure with 100 μg/mL of hygromycin, giving rise to EGFP-ADSCs and IFNγ-ADSCs. After about 2 weeks 15 × 104 of TRAIL-ADSCs were nucleofected with pmhy GENIE-3-IFNγ and selected with hygromycin to create TRAIL/IFNγ co-expressing ADSCs (TRAIL/IFNγ-ADSCs). These cells were maintained in a CM containing G418 (800 μg/mL) and hygromycin (100 μg/mL). Surface and intracellular staining of TRAIL-ADSCs and EGFP/Int-ADSC (control) were done with anti-TRAIL Ab (eBioscience, San Diego, CA). Expression of IFNγ was confirmed by Western blot. Protein extraction was performed by lysing EGFP-ADSCs and IFNγ-ADSCs with Cell lysis buffer (Sigma, St. Louis, MO) supplemented with a protease inhibitor cocktail (100 μg/mL; Sigma, St. Louis, MO). Proteins were separated in 12% SDS PAGE and transferred to nitrocellulose membrane (Amersham Biosciences/GE Healthcare, Pittsburgh, PA). The membrane was blocked in buffer containing PBS, 10% powdered nonfat milk (Sigma, St. Louis, MO), and 0.05% Tween-20 (Sigma, St. Louis, MO). Blotting was performed with a monoclonal rabbit anti-mouse IFNγ Ab at dilution of 1:1000. After twice washing the membrane with blocking buffer, blotting continued using a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody at 1:1000 dilution (Abcam, Cambridge, MA). Interferon-γ protein band was detected after adding the enhanced chemiluminescence reagent (Amersham Biosciences/GE Healthcare, Little Chalfont) by an imaging system (Bruker Inc, Ettlingen, Germany) using UV-Epi-Illumination source with 30 seconds of exposure time.
Cancer cell culture, transfection, and selection
The mouse melanoma cell line B16F10, human breast cancer cell line MCF-7, human bladder carcinoma derived cell line Ej-138, and human ovarian carcinoma cell line CAOV-4 were purchased from the Pasteur institute of Iran. Cancer cells were maintained in CM at 37°C in a 5% CO2 atmosphere. In this study, melanoma cells which were modified with pBEB/pCMVInt and pDsRed-attb-zeo/pCMVInt vectors are referred to GFP-melanoma and RFP-melanoma cells respectively. Lipofection was used for modification of melanoma cells. To create transgenic melanoma B16F10, cells at a density of 2 × 105 per well in triplicate for each group were seeded in 6-well plates. After 24 h, cells were co-transfected with the vector pBEB/pCMVInt, pBLB/pCMVInt, pISRE-TA-LUC-attb/pCMVInt, and pDsRed-attb-zeo/pCMVInt using lipofectamine 2000™ transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. EGFP-melanoma, FL-melanoma and ISRE/FL-melanoma were selected, and maintained with 1000 μg/mL of G418. RFP-melanoma was exposed to 400 μg/mL zeocin (Invitrogen, Carlsbad, CA) and after about two weeks maintained in CM with 100 μg/mL of zeocin.
Bioactivity of IFNγ and TRAIL produced by ADSCs
To study the effect of IFNγ-ADSC and TRAIL-ADSC on melanoma B16F10 cells we evaluated proliferation and apoptosis by MTT (Sigma, St. Louis, MO) and Annexin V apoptosis (Roche, Indianapolis, IA) assays. MTT and Annexin V apoptosis assays were performed as explained in Additional file1.
Monitoring the induction of the STAT1/2 components of Jak/STAT-mediated signal transduction pathways
FL-melanoma and ISRE/FL-melanoma cells were seeded into 12 well plates and incubated at 37°C in 5% CO2. Supernatants from either EGFP-ADSCs or IFNγ-ADSCs were added to these cells once they reached cell densities of 105 and 3 × 105 respectively. Two days later, a volume of ONE-Glo™ luciferase assay reagent (Promega Corp. Madison, WI) equal to that of the CM was added to each well and samples were mixed thoroughly. Luminescence imaging was performed in an imaging system (Bruker Inc, Ettlingen, Germany) using a UV-Epi-Illumination source with a 15 min exposure time.
In vivo studies and optical imaging
Eight-week-old male C57BL/6 mice (30.1 ± 0.6 g body weight) were purchased from the Pasteur institute of Iran. Handling of the animals was performed according to the guidelines of the Institutional Animal Care and Ethics Committee of Isfahan University. For the subcutaneous models of melanoma, 2 × 106 RFP-melanoma cells were delivered either alone or concurrently with 7.5 × 105 ADSCs of each group described above, in 200 μl of PBS into the right flank of the mice. The day of melanoma injection was defined as day 0. At day 21, in vivo red fluorescence optical imaging of subcutaneous RFP-melanoma bearing mice was performed. Subsequently, all mice were euthanized via pentobarbital overdose. In lung metastatic models, 7.5 × 105 RFP-melanoma cells were delivered into the lateral tail vein of twelve mice per group. Ten days later, the mice received 7.5 × 105 of GM-ADSC in a volume of 200 μl of PBS solution (control group received only 200 μl of PBS) by tail vein injection. Six mice from each group were sacrificed with pentobarbital overdose at day-28. Subsequently, their lungs were dissected and ex vivo red fluorescence optical imaging of lung samples was performed. The six remaining mice from each group were kept alive for long-term survival analysis. For imaging, animals were anesthetized with 2% isoflurane and images were captured using a multi-wavelength source with a 2 min exposure time in the In-Vivo F Pro small-animal imaging system (Bruker Inc, Ettlingen, Germany).
Serum levels of IFNγ
Frozen serum collected from blood samples at days 10, 16, and 21 was used in triplicate to quantify IFNγ protein levels by enzyme-linked immunosorbent assay (ELISA) using the mouse IFNγ Elipair kit (Abcam, Cambridge, MA) in accordance with the manufacturer’s protocol. Samples were analyzed at an absorbance of 450 nm with minimal sensitivity of 15 pg/mL for IFNγ detection.
Analysis of systemic CD4+ and CD4+CD25+FOXP3+ regulatory T cells (Treg)
Peripheral blood was taken from sub-ocular regions of normal C57BL/6 mice (control samples) and lung metastatic melanoma bearing mice at day 28 post melanoma cell injection. Blood was overlaid onto 3 mL of Ficoll-Paque and after centrifuging at 1800 rpm for 15 minutes, peripheral blood mononuclear cells (PBMCs) at the interface were collected. PBMCs were washed twice in PBS and then Treg cells were analyzed using a mouse Treg detection kit (Miltenyi Biotec, Auburn, CA). PBMCs were stained with FITC-conjugated monoclonal anti-mouse CD4 Ab and APC-conjugated monoclonal anti-mouse CD25 Ab for 30 minutes at 4°C in the dark. Subsequently, the PBMCs were incubated with a PE-conjugated monoclonal anti-mouse Foxp3 Ab, following the staining protocol provided by Miltenyi Biotec. FACS data analysis was performed with BD CellQuest Pro or FlowJo software.
Tissue sections were used for differentiation staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and immunohistochemistry (IHC) analysis. IHC was performed for EGFP, Ki67, murine IFNγ, murine IL2, programmed cell death 1 ligand 1 (PD-L1; B7-H1), CD31, CD4+, CD8+ and FOXP3+. For a detailed description of the experiments please see Additional file1.
Statistical analysis was performed using SPSS version 19 (SPSS Inc, Chicago, IL). The statistical differences between the groups were assessed by Student t test, ANOVA, and log-rank tests. Survival was defined as the date of melanoma cell injection to the date of death. P values less than .05 were considered statistically significant. All statistical tests were two-sided. Data are presented as mean values with 95% confidence intervals (CIs).
This research was supported by grant 189048 from Isfahan University of Medical Sciences. The sponsor had no role in the design of the study; the collection, analysis, and interpretation of the data; the writing of the article and the decision to submit the article for publication. We would like to thank Dr Michele Pamela Calos at Stanford University for generously providing the plasmids pBEB, pBLB, pCMVInt, and pDrBB2 and Dr Constance Cepko at Harvard Medical School for kind gift of the plasmid pCAG-DsReds. We thank Dr Yousef Gheisari and Dr Mohammad Ali Daneshmand for skillful technical assistance with immunohistochemistry.
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