immunotherapy is a new class of cancer treatment that works to harness the innate powers of the immune system to fight cancer .
major conceptual and technical advances in immunology have led to a new understanding of cellular and molecular interplays between the immune system and a tumor over the past decades [ 1 - 3 ] .
because of the immune system 's unique properties , these therapies may hold greater potential than current treatment approaches to fight cancer more powerfully , to offer longer - term protection against the disease , to come with fewer side effects , and to benefit more patients with more cancer types . since
recent therapies for cancer are based on drugs or radiotherapy which can kill proliferating cells or suppress cell proliferation , these treatments have several side effects on normal dividing cells .
the potential roles of immunologic approaches for treating of cancer patients are specific for tumors [ 1 , 2 ] . targeting neoplastic cells can improve the therapeutic effects of gene delivery by preventing healthy tissues injury and reduce the risk of germ line transduction [ 3 , 4 ] .
immunotherapy strategies consists of cancer vaccines , antitumor monoclonal antibodies , adoptive transfer of ex vivo activated t and nk cells , and administration of recombinant proteins and antibodies or that either co - stimulate immune cells or suppress immune inhibitory pathways ( immune checkpoints ) .
the aim of cancer immunotherapy is to boost the weak host immune response to develop tumors .
gene therapy prepared a novel strategy of therapeutic intervention for lots of genetic and non - genetic disorders . with recent progress in gene therapy field
, a wide variety of viral and non - viral vectors have emerged that can transfer genetic materials to target cells .
non - targeted delivery of transgenes often causes undesirable effects , low tumor transduction , and decreased therapeutic effects [ 8 , 9 ] .
cancer - specific cytokine cascade is one of the manifestations of the underlying paraneoplastic systemic disease , and this hypothesis links the stage of cancer with both the functional status of the immune system and the patient 's prognosis .
cancer gene immunotherapy is a delivery of cytokine genes to tumor cells to modify the local tumor environment in order to induce anti - tumor immune responses . in comparison to the therapeutic protein therapy ,
delivery of cytokine genes avoids the necessity of production and purification of large quantities of recombinant proteins . moreover , gene immunotherapy is capable of delivering cytokines in a more efficient and safe manner .
interluekin36 ( il36 ) is a 74 kda heterodimeric cytokine that consists of 35 kda ( p35 ) and 40 kda ( p40 ) subunits [ 13 - 15 ] .
it was characterized as a natural killer - stimulating factor ( nksf ) and cytotoxic lymphocyte maturation factor ( clmf ) by trinchieri s group in 1989 and gately s group in 1990 .
it is produced by macrophages , monocytes , and dendritic cells [ 14 , 16 ] .
investigations have shown that il36 posse s superior antitumor effect compared with different cytokines . also the researches demonstrated that il36 can be efficacious in prevention of primary tumor growth .
il36 is a multifunctional cytokine which can develops the multi - functional effects such as rising the proliferation and cytotoxic activity of t cells and nk cells , regulating the production of ifn- , inducing cytokine production , progressing the cd4 + th1 cells development and also promoting the activity and generation of ctls , via activation of stat4 .
ifn- up regulates the expression of mhc class i and ii molecules , adhesion molecules such as intracellular adhesion molecules ( icam)-1 and transcription factors such as t - box expressed in t cells ( t - bet ) with inducing by il36 [ 15,18 - 19].also , il36 possesses potent anti - angiogenic activity produced by neuotrophils , macrophages and dendritic cells .
the endogenous production of ifn- is needed for the antitumor effect of il36 in most cases .
il36 is one of the potent cytokines for cancer immunotherapy [ 18 - 19 ] .
the aim of this study was to investigate the effects of gene therapy with il36 in the regression of tumor masses in fibro sarcoma mouse model .

 plasmid amplification and isolation 
  an il36 expression vector , pumvc3-mil36 , was purchased from ( aldevron company , uk.london ) . the plasmid dna ( 6247 bp )
pumvc1-il36 was amplified in escherichia coli dh5 strain which was obtained from drug applied research center ( tabriz , iran ) .
the dna concentration was quantified by measuring the uv absorbance at 260 nm using uv spectrophotometer ( shimadzu , japan , tokyo ) . 

transformation of e. coli 
  pumvc3-m il36 plasmid was transformed into host strain e. coli , dh5 , bl21 , respectively which was obtained , amplified and extracted according to tens protocol .
the dna concentration was quantified by measuring the uv absorbance ( biorad company product , usa , illinois ) . 

determination of transfection efficiency 
  the transfection efficiency of mil36 was measured by enzyme - linked immunosorbent assay ( elisa ) kit ( koma biotech company , china , zhenjiang ) according to the manufacturer s instructions .
balb / c mouse fibro - sarcoma cells ( wehi-164 ) were bought from pasteur institute , tehran .
the wehi-164 cells were cultured in rpmi1640 medium ( sigma , germany , frankfort ) supplemented with 10% fetal bovine serum ( sigma , germany , frankfort ) , in presence of penicillin ( 100 u / ml ) , streptomycin ( 100 g / ml ) , ( sigma , germany , frankfort ) , and incubated in humidified incubator with 5% co2 at 37 c . 
 in vitro transfection studies 
 
the cells were trypsinized and seeded into 6-well plate at a density of 4 105 cells / well ; the cells were carried out before 2 days of transfection procedure .
the cells were washed with phosphate - buffer solution ( pbs ) twice prior to the addition of 2 ml rpmi1640 without fbs and antibiotic .
six g of pumvc1-m il36 plasmid was diluted in 250  optimem in one micro - centrifuge tube .
ten  lipofectamine 2000 was diluted in 250  optimem in separate micro - centrifuge tube and incubated for 5 minutes .
the contents of micro - centrifuge tubes were mixed together gently and incubated at room temperature for 20 minutes .
after 6 hours , the complexes were aspirated and replaced with culture medium . after 48 hours of transfection
, supernatants were harvested and released il36 was confirmed with elisa , by mouse il36 elisa kit ( koma biotech , china , zhenjiang ) , following manufacturer 's instructions .
female balb / c mice ( 6 to 8 weeks old ) were purchased from the pasteur institute , tehran , iran .
one hundred and six of wehi-164 cells were inoculated into the right flank of the balb / c mice subcutaneously to establish a tumor model .
the viability of the cells used for inoculation was over 95% as determined by the trypan blue dye exclusion test .
tumor growth was monitored three times a week with calipers after tumor challenge until the experiment was completed .
tumor volume ( mm3 ) was calculated by the formula : 1/2 ( lengthwidth2 ) . 

rna extraction and real time pcr 
  fallowing tumor mass extraction , total rna was extracted by accuzoltm reagent ( bioneer , daedeok - gu , daejeon , korea ) as described by the manufacturer .
complementary dna ( cdna ) was generated from 1 g of total rna by using oligo - dt primer and mmlv reverse transcriptase ( promega , madison , wi , usa , illinoise ) according to the manufacturer s recommendations .
qrt - pcr was performed with sybr premix ex taq ( takara bio , otsu , and shiga , japan ) in the rotor - genetm 6000 system ( corbett life science , mortlake , nsw , australia ) .
the pcr was done in a 20 l reaction system containing 12 l of sybr green reagent , 0.2 m for each primer , 1 l of cdna template and 6 l of nuclease - free distilled water .
the primer sequences were as followed : mouseil36p40 : 5- gagcactccccattcctact -3 ( as a forward primer ) and 5-gcattggacttcggtagatg-3 ( as a reverse primer ) , mouse ifn- : 5-tcagcaacagcaaggcgaaaaag-3 ( as a forward primer ) and 5-accccgaatcagcagcgactc-3 ( as a reverse primer ) and gapdh was used as an internal expression control : 5-cctcgtcccgtagacaaaa-3 ( as a forward primer ) and 5-aatctccactttgccactg-3 ( as a reverse primer ) .
the initial denaturation step was at 95c for 10 min and was followed by 45 cycles at 95c for 20 sec and 60c for 1 min .
relative gm - csf mrna expression was calculated with the 2- ( ct ) 17 , using gapdh as a reference gene .
ki67 is a nuclear protein and is considered as a tumor proliferation biomarker in tumor masses .
four micrometer frozen sections were cut , air - dried , fixed in acetone , and rehydrated in pbs con - taining 0.05% tween-20 .
non - specific binding sites were blocked by blocking buffer which is preformulated with tween-20 for 30 minutes .
slides were incubated by primary antibody ( purified anti - mouse ki67 ) , ( biolegend , uk , london ) , for 60 minutes .
subsequently , slides were washed in pbs containing 0.05% tween 20 and then slides were incubated with hrp labeled secondary antibody [ rabbit polycolonal secondary antibody to rat igg ( hrp - conjugated ) ] , ( abcam ) , for 30 minutes .
h2o2 was added to dab solution ( substrate solution ) and dab and h2o2 were added to the slides for 5 minutes .
the slides were consequently washed with pbs containing 0.05% tween-20 and studied by invert microscopy .
the purified anti - mouse il36 ( 1:1000 ) ( biolegend , uk , london ) , and anti--actin ( l : 1000 ) ( sigma ) were used as primary antibodies .
cell extracts were prepared in ripa - b buffer ( 0.5% non - idetp40 , 20 mm tris , ( ph 8.0 ) , 50 mm nacl , 50 mm naf , 100 m na3vo4 , 1 mm dithio - threitol , and 50 g / ml phenyl - methylsulfonyl fluorides . the protein concentration was quantified using a uv spectrophotometer .
protein samples were fractionated on a 12% polyacrylamide gel and transferred to the nitrocellulose membrane .
the membrane was blocked with 3% skim milk for 1 hour at room temperature and incubated with primary antibody at 4c overnight .
after extensively washing , the membrane was incubated with [ rabbit polyclonal secondary antibody to rat igg ( hrp - conjugated ) ] , ( abcam ) .
the results were visualized using the enhanced chemiluminescence ( ecl ) system ( amersham biosciences , piscataway , nj , usa ) and exposured to autoradiography film ( ko - dak xar film ) . 


 confirmation of il36 production by tumor transected cells 
  enzyme - linked immunosorbent assay ( elisa ) test was used for confirmation of il36 production by tumor transected cell .
the concentration of il36 in supernatant of tumor transected cell culture was assessed by spectrophotometer in 540 nm . the il36 concentration in the supernatant of the tumor transected cell culture was 1000 pg / m9l ( figure1 ) . 

antitumor effects of il36 in vivo 
  to test the antitumor response induced by il36 secreted in site , balb / c mice ( n=7 ) were injected subcutaneously with fibro sarcoma / il36 cells ( 110 ) , and monitored for tumor growth 3 times a week .
after 21 days of tumor inoculation the volume of the tumor masses was less than control group .
so gene therapy with il36 has effect in the regression of tumor masses ( figure 2 ) . 

expression of il36 & ifn- mrna and protein in tumor tissue 
  the extracted rna with 28s/18s ratios between 1.5 and 1.9 gp / ml as determined by agarose gel electrophoresis analysis , using ethidium bromide staining .
the median total rna recovery from tissue was 98% ( range , 90% to 105% ) . to investigate whether fibro sarcoma / il36 cells can express il36 in vivo
, tumor mass was harvested from mice on day 21 after inoculation with fibro sarcoma / il36 cells , respectively , total rna was extracted from tumor masses and cell lyses were prepared .
the results of real - time pcr indicated that the expression of il36 and ifn- was enhanced in group treated with il36 in comparison to the control group ( figure 3 ) .
the results of immunoblotting showed that the expression of il36 and ifn- was enhanced in group treated with il36 in comparison to the control group ( figure 4 ) .
the group treated with il36 were up - regulated ( compared to the control group ) by a mean factor of 1.90 ( s.e .
despite advances in our understanding of the processes involved in the development and progression of cancer , treatment options for many patients are limited and prognosis still remains poor .
gene therapy is the insertion of a functional gene into the cells of a patient to correct an inborn error of metabolism , to alter or repair an acquired genetic abnormality , and to provide a new function to a cell [ 2 , 5 ] .
cancer gene therapy by delivery of cytokine gene is an appealing strategy which can be efficiently transducer into the tumor cells and extracts potential immune responses .
cancer gene therapy consists of 2 main therapeutic strategies : one approach is to cause a direct effect on cancer cells by transferring suicide genes sirna for ontogenesis and proteins related to the cell cycle and apoptosis [ 21 - 24 ] . in this method ,
the second approach is indirect and activates antitumor immunity mediated by introducing a cytokine gene such as il36 [ 25 - 29 ] . in such cytokine gene therapies ,
the therapeutic gene does not need to be delivered into all the cancer cells , because the cytokine is secreted from the cells .. many anti - tumor effects are related to this method for il36 gene delivery [ 30 , 31 ] .
the high il36-producing cells show a nk- or t cell - independent antiangiogenic effect induced by ip-10 , although the final rejection in immunocompetent mice is totally dependent on t cells [ 32 , 33 ] . in prostate cancer ,
adenoviral - mediated il36 gene therapy had an antimetastatic effect in partial dependency on nk cells . in bladder carcinoma and colon cancer , adenoviral - mediated il36 gene therapy
is depend on t cell- local antitumor effects .in this study , we investigated that the effect of non - viral gene therapy by using plasmid dna expressing il36 , is a potent primer of anti - tumor immunity [ 35 - 37 ] .
in addition , a significant level of intratumoral ifn- was also observed as an inducer of 2 important antitumor chemokines , ip-10 and mig 37 .
no one denies that gene therapy for cancer holds extraordinary promise or that it will eventually yield results .
but critics have grown increasingly concerned that the initial excitement led to a premature rush to get unproved gene therapies for cancer out of the laboratory and into human patients .
researchers are still not sure which are the best methods to transport genes into cancer cells , nor have figured out how to stop a person 's own immune systems from rejecting what are , in effect , microscopic transplants of foreign materials . even more troubling
is the signs that indicate financial considerations may have replaced scientific rigor in determining how and when to use gene therapy for cancer .
the possibility of gene therapy opens a new area of therapeutics and hope for individuals afflicted with several types of cancers .
we demonstrated that the local il36 gene delivery into tumor tissue is an immune response to the tumor cells .
therefore , gene delivery by lipofectamine could be a useful non - viral vector system in cancer gene therapy .
the possibility of gene therapy opens a new area of therapeutics and hope for individuals afflicted with several types of cancers .
we demonstrated that the local il36 gene delivery into tumor tissue is an immune response to the tumor cells .
therefore , gene delivery by lipofectamine could be a useful non - viral vector system in cancer gene therapy .
 backgroundcancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors and is one of the cancer treatment strategies . 
 recently , interluekin36 ( il36 ) has been used as immunotherapeutic agents in cancer gene therapy . 
 present study investigated that the il36 gene therapy effects on the regression of tumor masses in mouse model . 
 aim of this study is determination of the gene therapy effects by il36 in the regression of tumor masses in mouse model.methodsto study the therapeutic efficacy of this cytokine , wehi-164 tumor cells were transected with mil36 plasmids . 
 elisa test was used to check cytokine production by transected cells . to establish fibro sarcoma mouse model , 
 tumoral transfected cells were injected subcutaneously to inoculate tumor in balb / c mice . 
 tumor volumes were measured by caliper . 
 mice were sacrificed and tumors were extracted . 
 the expression of il36 and ifn- was studied with real - time pcr and immunoblotting . 
 the expression of ki-67 ( a tumor proliferation marker ) in tumor masses was studied by immunohistochemistry staining . in this study 
 we had 2 groups which are treated with il-36 and untreated with il-36 as a blank.resultsthe group treated with il36 indicated decrease of tumor mass volume ( p<0.001 ) . 
 the results of western blotting and real - time pcr showed the il36 expression increased in the group treated with il36 ( with relative expression of 1.9).conclusionimmunohistochemistry staining indicated that the ki-67expression has been reduced in the group interfered with il36 . 
 il36 gene therapy has therapeutic effects on the regression of tumor masses in fibro sarcoma mouse model . 
Introduction
Materials and Methods
Results
Discussion
Conclusion