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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 2  |  Page : 362-366

The effects of gene therapy with granulocyte-macrophage colony-stimulating factor in the regression of tumor masses in fibrosarcoma mouse model


1 Drug Applied Research Center, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
2 Immunology Research Center, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
3 Department of Pharmaceutical and Food Control, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

Date of Web Publication23-Jun-2017

Correspondence Address:
Behzad Baradaran
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz
Iran
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Source of Support: Drug applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, Conflict of Interest: None


DOI: 10.4103/0973-1482.159083

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 > Abstract 

Introduction: Cytokine gene therapy is one of the cancer treatment strategies. Recently, granulocyte-monocyte colony-stimulating factor (GM-CSF), as an important cytokine in activating dendritic cells and boosting the anti-tumor immune responses, has been utilized as an immunotherapeutic agent in cancer gene therapy. The purpose of the present investigation was to study the GM-CSF gene therapy effects in regression of tumor masses in fibrosarcoma mouse model.
Materials and Methods: To investigate the therapeutic efficacy of GM-CSF, WEHI-164 tumor cells were transfected with murine GM-CSF plasmid. For cytokine production by transfected cells, enzyme-linked immunosorbent assay test was used. Fibrosarcoma mouse model established with transfected cells which were injected subcutaneously into Balb/c mice. Tumor sizes were measured by caliper. Mice were sacrificed and the tumors were extracted. The expression of GM-CSF was studied by real-time polymerase chain reaction (PCR) and immunoblotting. The expression of Ki-67 (a tumor proliferative marker) in tumor masses was studied by immunohistochemical staining.
Results: The group treated with GM-CSF indicated a decrease in tumor mass volume (P = 0.001). The results of western blotting and real-time PCR showed that GM-CSF expression increased in the group treated with GM-CSF (with a relative expression of 1.36). Immunohistochemical staining showed that Ki-67 expression has reduced in the group treated with GM-CSF.
Conclusion: Monotherapy with GM-CSF has therapeutic effects on the regression of tumor masses in the fibrosarcoma mouse model.

Keywords: Gene therapy, granulocyte-monocyte colony-stimulating factor, tumor


How to cite this article:
RaziSoofiyani S, Kazemi T, Lotfipour F, Mohammadnejad L, Hallaj-Nezhadi S, Shotorbani SS, Hosseini AM, Baradaran B. The effects of gene therapy with granulocyte-macrophage colony-stimulating factor in the regression of tumor masses in fibrosarcoma mouse model. J Can Res Ther 2017;13:362-6

How to cite this URL:
RaziSoofiyani S, Kazemi T, Lotfipour F, Mohammadnejad L, Hallaj-Nezhadi S, Shotorbani SS, Hosseini AM, Baradaran B. The effects of gene therapy with granulocyte-macrophage colony-stimulating factor in the regression of tumor masses in fibrosarcoma mouse model. J Can Res Ther [serial online] 2017 [cited 2019 Nov 13];13:362-6. Available from: http://www.cancerjournal.net/text.asp?2017/13/2/362/159083


 > Introduction Top


Sarcomas are relatively rare malignant tumors of mesenchymal origin, but often incurable at the late metastatic stage.[1] Gene therapy is a novel strategy for treatment of human disease via transferring genetic materials such as DNA, RNA, siRNA, and antisense oligonucleotides, with therapeutic activity in cells.[2] The idea of cancer gene therapy prepares the noble perspectives which is not associated with many of the drawbacks of routine therapies such as chemotherapy, radiotherapy, and surgery.[3]

It has been at least a century that the immune system has been used for cancer therapy.[4]

Cytokines are immune-modulating agents which secreted by specific cells of the immune system and various cells of the body. Cancer gene immunotherapy via delivery of cytokine genes to cancerous cells alters the local tumor environment to induce anti-tumor immune responses.[5],[6] Compared to the therapeutic protein therapy, delivery of cytokine genes avoids the necessity for the production and purification of large quantities of recombinant proteins. Moreover, gene immunotherapy is capable of delivering cytokines in a more efficient and safe manner; since the transfer of genes can cause more “natural” sustained protein levels in vivo and decrease troubles with immunotherapeutic agents being toxic at high doses while demonstrate short circulating half-lives.[7]

Granulocyte-macrophage-colony stimulating factor (GM-CSF), a 23 kDa single chain glycoprotein, was originally identified by its ability to generate both macrophage and granulocytes colonies from precursor cells as a result of differentiation and proliferation.[8],[9] The heterodimeric high affinity receptor of GM-CSF consists of two subunits, α-subunit, and β-subunit.[10],[11] GM-CSF is generated by many cell types, including T- and B-cells, macrophages, and endothelial cells.[12]

Although, GM-CSF regulates the proliferation and differentiation of hematopoietic progenitor cells, it plays a critical role in the maturation and activation of professional antigen-presenting cells (APCs) by up-regulating of MHC molecules via co-stimulating pathways. Furthermore, GM-CSF amplifies the T-cell proliferation and poses additional effects on the monocyte/macrophage system. Moreover, GM-CSF has the capacity to enhance antigen-induced immune responses; it can also modify the Th1/Th2 cytokine balance. Preclinical and clinical researches have shown that GM-CSF possesses the ability to initiate the anti-tumor cellular immunity through the mentioned mechanisms.[13],[14],[15],[16],[17],[18],[19]

Granulocyte-monocyte colony-stimulating factor has been investigated as an adjuvant factor for tumor immunotherapy. However, the consequences are controversial with anti-tumor effects in some studies and a tumor growth promotion effect in others.[20] The aim of this study was to investigate the effects of gene therapy with GM-CSF in the regression of tumor masses in the fibrosarcoma mouse model.


 > Materials and Methods Top


Plasmid amplification and isolation

A murine GM-CSF (m-GM-CSF) expression vector, pUMVC1-mGM-CSF, was purchased from Aldevron company (Aldevron, Fargo, ND, USA). The plasmid DNA is 4423 bp in size and contains CMV IE promoter.

PUMVC1-mGM-CSF was amplified in an Escherichia coli DH5α strain which was obtained from the Pasteur Institute (Tehran, Iran) and then extracted according to TENS miniprep protocol. The purified plasmid was detected by agarose gel electrophoresis. The DNA concentration was quantified by measuring the UV absorbance at 260 nm using the UV spectrophotometry (Shimadzu, Japan).

Cell culture

Balb/c mouse fibrosarcoma cells (WEHI-164) were purchased from the Pasteur Institute (Tehran, Iran). The WEHI-164 cells were cultured in RPMI-1640 medium (Sigma, Germany) supplemented with 10% fetal bovine serum (FBS) (Sigma, Germany), in the presence of penicillin (100 U/ml), streptomycin (100 μg/ml, Sigma, Germany), and incubated in a humidified incubator with 5% CO2 at 37°C.

In vitro transfection studies

WEHI-164 cells were seeded into a 6-wells plate at a density of 4 × 105 cells/well and incubated for 48 h at 37°C under 5% CO2. Then, the cells were washed with phosphate buffer solution (PBS) twice prior to adding of 2 ml RPMI-1640 without FBS and antibiotic. 6 μg of pDNA was diluted using 250 λ OptiMem in one Microcentrifuge tube. 10 λ Lipofectamine 2000 was diluted in 250 λ OptiMem in a separate Microcentrifuge tube and incubated for 5 min. Two microcentrifuge tubes were mixed gently and incubated at room temperature for 20 min. The cells were incubated at 37°C in 5% CO2. After 6 h, the complexes were aspirated and replaced with culture medium. After 48 h, for quantitative analysis of the m-GM-CSF expression supernatants of culture were harvested and the mouse GM-CSF enzyme-linked immunosorbent assay kit was used according to the manufacturer's instructions (Koma Biotech Company, Korea). For this purpose, the collected culture supernatants were analyzed for the measurement of m-GM-CSF. The amount of the protein was determined as picogram per ml.

Animal experiments

Balb/c mice (6–8-week-old, female) were purchased from the Pasteur Institute, Tehran, Iran (1 × 106) of WEHI-164 cells were injected subcutaneously into the right flank of the Balb/c mice to establish a tumor in the control group and (1 × 106) WEHI-164 cells transfected with pUMVC1-mGM-CSF were injected subcutaneously into the right flank of the Balb/c mice to establish a tumor in the group treated with GM-CSF (case group). The viability of the cells used for tumor inoculation was over 95% as determined by the trypan blue staining. Palpable tumors developed after 10 days. Tumor growth was monitored 3 times a week with calipers after tumor challenge since the experiment was completed. Tumor volume (mm 3) was calculated by following the formula: 0.5 × (length × width 2).

RNA extraction and real-time polymerase chain reaction

Following tumor mass extraction, total RNA was extracted by AccuZol reagent (Bioneer, Daedeok-GU, Daejeon, Korea) according to manufacture protocol and reverse transcribed to first strand cDNA by use of the Oligo-dt and Moloney murine leukemia virus reverse transcriptase system. QRT-polymerase chain reaction (PCR) was performed with using SYBR Premix Ex Taq (Takara Bio, Otsu, Shiga, Japan) using a Rotor-Gene 6000 system (Corbett Life Science, Mortlake, NSW, Australia).

Polymerase chain reaction was performed in a 20 μl reaction system containing 12 μl of SYBR green reagent, 0.2 μM of each primer, 1 μl of cDNA template, and 6 μl of nuclease-free distilled water. The primer sequences were as follows: 5'-ACCACCTATGCGGATTTCAT-3' as forward, 5'-TCATTACGCAGGCACAAAAG-3' as a Reverse for GM-CSF, 5'-CCTCGTCCCGTAGACAAAA-3' as a Forward, and 5'-AATCTCCACTTTGCCACTG-3' reverse, for GAPDH. The initial denaturation step at 95°C for 10 min was followed by 45 cycles at 95°C for 20 s and 60°C for 1 min. Relative GM-CSF mRNA expression was calculated with the 2−(ΔΔCT), using GAPDH as a reference gene. The primers were obtained from Bioneer.

Immunohistochemical analysis

Immunohistochemical assays were performed to detect Ki-67 protein expression. 4 μm frozen section were cut, air-dried, fixed in acetone, and rehydrated in PBS containing 0.05% Tween-20. Nonspecific binding sites were blocked by blocking buffer which is preformulated with Tween-20 for 20 min. Slides were incubated with primary antibody anti-mouse Ki-67 (Biolegand) for 60 min. Subsequently, slides were washed in PBS containing 0.05% Tween-20 and then slides were incubated with HRP labeled secondary antibody (Rabbit polyclonal secondary antibody to Rat IgG (HRP-conjugated-abcam) for 30 min. DAB was added to the slides for 5 min and washed with PBS. Sections were viewed with an invert microscope. For studying of Ki-67 expression, image processing software was used to count both the total number of cell nuclei in each image, and the number of positively stained nuclei. Data were then expressed as percent of positively stained nuclei, derived from these counts. T-test was used for statistical analysis.

Western blot analysis

The primary antibodies, purified anti-mouse GM-SCF (1:1000) (biolegend), and anti-β-actin (l: 1000) (Sigma) were used. Cell extracts were lysated in RIPA-B buffer (0.5% NonidetP40, 20 mM Tris, [pH 8.0], 50 mM NaCl, 50 mM NaF, 100 μM Na3 VO4, 1 mM dithiothreitol, and 50 μg/ml phenylmethylsulfonyl fluoride).

The protein concentration was quantified using a Lowry's assay (Bio-Rad). Protein samples were fractionated on a 12% polyacrylamide gel and transferred to the nitrocellulose membrane. The membrane was blocked with 3% skim milk for an hour at room temperature and incubated with primary antibody overnight at 4°C. After extensive washing, the membrane was incubated with Rabbit Polyclonal secondary antibody to Rat IgG (HRP-conjugated), (Abcam). The results were visualized using the enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA) and exposure to autoradiography film (Kodak XAR film).

Statistical analysis

Results were evaluated by (SPSS) Software version 16 (SPSS, Chicago, IL, USA). Statistical analyses for tumor mass volume and real-time PCR and IHC results were performed using a unpaired t-test. P<0.05 was considered to be statistically significant.


 > Results Top


Confirmation of murine granulocyte-monocyte colony-stimulating factor expression by enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay test was carried out for confirmation of GM-CSF expression by tumor transfected cell. GM-CSF concentration in the supernatant of tumor transfected cell culture was assessed by spectrophotometry in 540 nm λ. The GM-CSF concentration in the supernatant of tumor transfected cell culture was found to be 800 pg/ml.

Antitumor effects of granulocyte-monocyte colony-stimulating factor in vivo

21 days after tumor challenge the volume of tumor masses of the mice injected with fibrosarcoma/GM-CSF cells reduced significantly incomparable in the mice injected with fibrosarcoma as a control group (mean volume of tumor masses in the control group is 84604.42857 and mean volume of tumor masses in the GM-CSF treated group is 38133.491 (P = 0.001). Hence, gene therapy with GM-CSF has an effect in the regression of tumor masses [Figure 1].
Figure 1: Tumor mass regression in group treated with granulocyte-monocyte colony-stimulating factor (GM-CSF). The results showed that the tumor mass volume was significantly reduced in group treated with GM-CSF (P = 0.001)

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Expression of murine granulocyte-monocyte colony-stimulating factor mRNA and protein in tumor tissue

To investigate whether fibrosarcoma/GM-CSF cells can express GM-CSF in vivo, tumor masses were harvested from mice treated with GM-CSF and control group on 21 days after tumor inoculation, respectively. Total RNA was extracted from tumor masses and cell lyset was prepared to confirm GM-CSF expression by immunoblotting. The results of real-time PCR indicated that the expression of GM-CSF was significantly enhanced in group treated with GM-CSF in comparison to control group (relative expression of GM-CSF: 1.36), (P = 0.001) [Figure 2] and [Table 1]. And the results of immunoblotting confirmed that the expression of GM-CSF was enhanced in the group treated with GM-CSF in comparison to control group [Figure 3].
Figure 2: Relative expression of granulocyte-monocyte colony-stimulating factor (GM-CSF). GM-CSF gene expression in the group treated with GM-CSF is UP-regulated (compared to the control group) by a mean factor of 1.360 (P = 0.001) (standard error range is 1.360–1.360)

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Table 1: GM-CSF expression in tumors

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Figure 3: (a) The cytokine expression results of western blot: Granulocyte-monocyte colony-stimulating factor (GM-CSF) expression has been proved by western blotting analysis. One sample of each group has been showed in picture. A: Expression of β-actin, B: Expression of GM-CSF, A: Proteins were equalized by use of β-actin expression (b) Western blotting results showed that granulocyte-monocyte colony-stimulating factor (GM-CSF) expression was enhanced in group treated with GM-CSF incomparable to control group

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Expression of Ki-67 in tumor sections

The expression of Ki-67 in tumor masses was studied by immunohistochemical staining. The results showed that Ki-67 expression was significantly reduced in the group treated with GM-CSF, incomparable to control group (P < 0.05) [Figure 4].
Figure 4: (a) Expression of Ki-67 in control group (b) Expression of Ki-67 in granulocyte-monocyte colony-stimulating factor (GM-CSF) treated group Expression of Ki-67 was reduced in GM-CSF treated group in comparison to control group

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 > Discussion Top


Cancer gene therapy with cytokine gene delivery is an appealing strategy which provided that the gene can be efficiently transduced into the tumor cells and extracts potential immune responses.[21] GM-CSF as a multiple biological effector cytokine can increase the cytotoxic activity of NK cells or CTL. Previous studies have shown apparent antitumor and immunomodulatory effects of GM-CSF gene therapy in lung cancer, melanoma bladder cancer, renal cell carcinoma, and hematological malignancies.[22] To the best of our knowledge, the effects of GM-CSF gene therapy have not been studied on fibrosarcoma tumor models and according to a literature review, this is the first research that shows the effect of gene therapy with GM-CSF in the regression of tumor masses in a fibrosarcoma mouse model.

Other studies have shown that the local environment in different organs would impose the properties of tumors growing at those sites. Furthermore, different microenvironments might modify the induced effects.[23] The mechanisms involved in the boosting of tumor-specific immunity by locally produced GM-CSF are not clear until now. Local expression of GM-CSF at the tumor site may attract CD 4+ and CD 8+ T-cells from surrounding tissues. These T-cells would play prominent roles in the processing and presentation of tumor antigens released from GM-CSF/fibrosarcoma cells. Thus, they augment the induction of a specific antitumor immune responses. Armstrong et al. showed that GM-CSF could induce an anti-tumor effect by recruiting dendritic APCs and activate APCs resulting in induction of potent immune responses. Strong immune responses may cause tumor regression as our results displayed. Chang et al. showed that CD 8+ T-cells and NKT were important antitumor effectors for regressing orthotopic liver tumor.[24],[25] Song et al. indicated that gene therapy with GM-CSF is appreciable but has less effect on murine hepatocellular carcinoma.[13] Ki-67 is a protein associated with cell proliferation and is present in all other cell cycle phases except G0.[26]

Our results showed that the tumor volume in the group treated with GM-CSF reduced in comparable to the control group (P = 0.001). Furthermore, gene therapy with GM-CSF could decrease Ki-67 expression in the fibrosarcoma tumor masses and this finding showed that GM-CSF gene therapy could decrease cancerous cell proliferation. Tumor volume regression in the group treated with GM-CSF could indicate the strong immune response in the tumor micro-environment. These findings are in agreement with previously reported results.

On the whole, our results showed that gene therapy with GM-CSF regressed tumor in inoculated tumor models.


 > Acknowledgments Top


This work was supported by a grant from the Drug Applied Research Center, Tabriz University of Medical Sciences. We thank Dr. Hartoonian for her kind help in this project.

 
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Mahvi DM, Sondel PM, Yang NS, Albertini MR, Schiller JH, Hank J, et al. Phase I/IB study of immunization with autologous tumor cells transfected with the GM-CSF gene by particle-mediated transfer in patients with melanoma or sarcoma. Hum Gene Ther 1997;8:875-91.  Back to cited text no. 22
    
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