Fiber-modified Hexon-chimaeric Oncolytic Adenoviral Vectors Targeting Gastric Cancer-associated Fibroblasts

Background:Prevalence and mortality of gastric cancer in china are much higher than mean value inthe world. At present all therapies, like surgery and chemotherapy are not effective enough,and lack of specific treatment has become the major problem, which made it essential todevelop new tumor targeted therapy.Tumor stroma, as environment for tumor to live, is made up of extracellular matrix,endothelial cells, immune cells and cancer associated fibroblasts (CAFs). CAFs are the majorcomponent in stroma and they can secrete cytokines, growth factors and adhesion moleculesfor tumor growth; they can secrete many kinds of protease to promote tumor cells proliferateand metastasize; they also can help tumor cells escape from immune system and enhance theirresistance for radiotherapy and chemotherapy, which become the root for tumor recidivation.Besides, studies have indicated that CAFs also play an important role in survival of tumorstem cells. In summary, CAFs have a close relationship with tumor genesis, development,recidivation and metastasis.Two specific features made CAFs different from normal fibroblasts: first, in mostcancers CAFs express fibroblast activated protein (FAP), which possess serine proteaseactivity. There is positive correlation between FAP level and tumor malignancy. And FAP isonly detected in activated fibroblasts, while normal tissues and cells doesn’t express FAP.Second, CAFs highly express chemotaxin CXCL12. CXCL12and its specific receptors(CXCR4,CXCR7) play a very important role in tumor cell proliferation, activation,neovascularization and metastasis. Studies on the two aspects mentioned above are very hot inthe tumor treatment. So CAFs targeted therapy can make tumor no support environment andit’s a very promising strategy. Previous studies focused on small molecule drugs ormonoclone antibodies. The techniques are complicated and the target is monotonous, whichmade limited therapeutic effect. So, in this study we introduced tumor stroma therapy, that isspecific killing CAFs through oncolytic adenovirus.Recombinant oncolytic adenovirus is a satisfactory viral vector with many good features,such as low pathogenicity, no integration with host genome (low carciongenic potency), able to transfect cell in or not in stationary phase and to accept exogenous big DNA fragments,good knowledge of gene expression and protein function, as well as easy for preparation andpurification. So far, it has been widely used in gene therapy, but some limitations still exist inclinical use, including:1.adenovirus vector mostly used at present (Ad2, Ad5in subgroup C)can transfect a big range of hosts, including almost all kinds of mammalian cells, but lackgood targeting ability;2. Because of different receptor and integrin levels in cell surface, bigvariations can be observed for adenovirus to transfect different tissues, and especially lowefficacy for CAFs;3. Ad2and Ad5，although easily produced, are liable to assemble in liverwhen systemic administered and cause hepatotoxicity. Meanwhile, it is hard for Ad2and Ad5to get to the tumor site;4. Existence of CAFs would decrease the curative effect ofadenovirus;5. Neutralizing antibody of Ad2, Ad5exist in most people. When systemicadministered, adenovirus is easily cleared by immune response. So on the basis of adenovirus,we managed to keep the merit and at the same time, reconstruct the viral vector usingadvanced techniques such as fiber protein modify, transcription regulation and hexonchimaeric. After reconstruction the oncolytic adenoviral vector can specific and effectivelytransfect and kill CAFs and possess low hepatotoxicity, low immunogenicity. This studyconstructed4FAP targeting oncolytic adenoviruses ex vivo and evaluated their ability ofspecific killing CAFs. Furthermore, treatment effect was observed in gastric cancerimplanted nude mice after different ways of drug administration. We hope through this study anew method could be discovered for tumor stroma treatment and further decrease recurrence,mortality of gastric cancer and improve patients’ life quality and relieve their pressure.Methods and results:1. Construction, recombination and preparation of fiber-modified hexon-chimaericoncolytic adenoviral vectorpShE1、pENTR-GF-LA、pAd Rc/delLA vector systems were used to recombinateadenovirus genome. Shuttle vector (pSG502, constructed by our research team) of SG600system as template, its adenoviral E1A gene promoter (telomerase reverse transcriptionpromoter, hTERTp) was replaced by CXCL12gene promoter (after clone, sequencing andfunction verification) by molecular biology methods such as PCR, digestion, conjunction andso on. At the same time, E1B gene promoter (5×HR E and CMV) was reserved to construct anew shuttle vector pShSDF-E1. Using in-fusion site specific recombination technology, FAPspecific recognition peptide related base sequence was inserted into SpeI site of pENTR-GF-LA. At the same time, PTEN gene box was also inserted into the polyclone siteof the vector to get pENTR/FAP. Through molecular biology methods, such as PCR,digestion, conjunction and so on, original Ad5hexon gene was replaced by hexon-chimaericgene (H5HVR48, whole genome synthesis) to get hexon-chimaeric adenoviral plasmid vectorpAd5HVR48-Rc/delLA containing Ad48hypervariable region. Then pENTR/FAP andpAd5HVR48-Rc/delLA were recombinated by high-performance site specific recombinationtechnology (Gateway). CcdB toxicity and ampicillin resistance were used to select recon andright terminal sequence of reconstructed adenovirus genome was inserted back to plasmidvector to get fiber modified hexon-chimaeric recombination adnovirus plasmidpAd5HVR48/FAP/delE1E3. pShSDFpE1and pAd5HVR48/FAP/delE1E3cotransfectedE.coli BJ5183for homologous recombination and kanamycin resistance for recon selectionto get fiber modified hexon-chimaeric recombination adnovirus plasmidpAd5HVR48/SDFp/FAP which contained PTEN gene. E1A and E1B gene were separatelyregulated by CXCL12promoter and hypoxia response element (HRE). Inverted terminalrepeat (ITR) sequence in adenoviral genome terminals was exposed after fully digestion ofverified recon by restriction enzyme PacI. Then the recon was enwrapped by liposome totransfect adenovirus reconstructed cell strain HEK293A. Reconstructed viral particles werepurified by three times plaque-forming assay to get hexon-chimaeric oncolytic adenoviralvector pRCAdHVR48-SDF1p-Ad/EGFP，pRCAdHVR48-SDF1p-P9/EGFP,pRCAdHVR48-SDF1p-P9-4C/EGFP and pRCAdHVR48-SDF1p-GP/EGFP, which aretargeting CAFs and own the ability of replication and proliferation. These vectors werelargely expanded, density gradient centrifugated, virus titer purified by TCID50and finallystored in-80℃for future utilization.2. Effect of oncolytic adenoviral vector for CAFs infection, killing andreproductionSeparation and culture of GCAFs: gastric cancer and paraneoplastic tissue were gotfrom ten patients received operation therapy in first general surgery department, changhaihospital from April to August in2012. The tissues were digested by collagenase for GCAFsand human gastric mucosa fibroblast (HGMF) and then cultured by10%D/F12medium. FAP,cxcl12and integrin were detected by flow cytometric analysis (FACS).Detection for virus infectivity: CAFs in log phase growth were seeded in six wellplates with1×105each well. MOI=100replication-defective virus were added to each well and incubated with CAFs in4℃for1h. Virus not binding with cells were washed by coldPBS. Then cells were cultured in5%D/F12medium,37℃5%CO2for48h and fluorescencemicroscope was used to observe and photograph, FACS for EGFP detection. QIAamp Kitswere used to extract viral genome DNA and absolute quantitative PCR (SYBR Green) foradenoviral copies (gene in E4region) detection. GAPDH and normal fibroblast cell line BJwere used for negative control. Hexon protein expression on in GCAFs infected byreplication-defective virus was detected by Westernblot. PCR and westernblot showeddecreased binding level of adenovirus after block of FAP antibody.Virus reproduction experiment: Cells in log phase growth were added to six wellplates (1×105each well) and cultured in37℃5%CO2atmosphere for24h. Then the mediumwas replaced by serum-free medium and MOI=5virus added.2h later changed with5%bloodserum medium. Cells and supernatant in0h,6h,12h,48h96h were collected after virustransfection. Viral titre was examined following standard tissue culture infective dose(TCID50) method. Compared with viral titre in0h, we got different reproduction number.Viral reproduction observed by fluorescence microscope:4adenoviruses containinggreen fluorescence report gene EGFP were used to transfect normal BJ cells, gastric cancercells and CAFs cells and green fluorescence was observed by microscope after transfection1days,3days,7days later. Results showed in normal BJ cells green fluorescence persistentlyexisted, single and dispersed. In gastric cancer cell and CAFs green fluorescence changedwith time, single and dispersed at beginning, gradually to centered and fused. Meanwhile, all4recombinant adenoviruses can replicate, proliferate in gastric cancer cells and CAFs andfinally lyse cells.Cell killing ability detection: Cells in log phase growth were added to6-well plates(1×105each well) and MOI=1oncolytic adenovirus added.3,7and10days later cells werephotographed by fluorescence microscope.WST1detectedthe ability of oncolytic adenoviruskilling GCAFs.3.Effect of replication-competent adenovirus for transplantation tumor treatmentin nude micePrimary gastric cancer cells in log phase growth were digested by trypsin,resuspended by PBS, counted and finally attenuated to cell suspension (1×108/ml),subcutaneously subcultured to nude mice. After tumor formation, cancer samples werecollected and cut into pieces about1mm3under aseptic condition. These pieces were subcutaneously subcultured in nude mice once more to establish transplantation tumormodeling. When the tumor grew up to0.5cm3, the mice were divided into5groups randomly:RCAd5HVR48/SDFp/FAP-Ad/EGFP group, CAd5HVR48/SDFp/FAP-P9/EGFP group,RCAd5HVR48/SDFp/FAP-P9-4C/EGFP group, RCAd5HVR48/SDFp/FAP-GP/EGFP groupand control (injected with PBS） group. In all groups, the therapy concentration of oncolyticadenovirus was1×108pfu/100μl, and the experiment following singe blind principle.Oncolytic adenoviral vectors and PBS were systemic administrated. Unpaired t test was usedfo statistical analysis.Results showed that compared to control group, tumor growth in four treatment groupswere significantly inhibited (P<0.01) and the effect of GP, P9-4C, P9, Ad was decrement(P<0.05). Pathological section examination showed that compared with control group, intreatment groups much more cancer necrosis appeared. Immunohistochemistry detectedhexon protein expression in tumor of virus treatment groups, meaning replication-competentadenovirus could proliferate in tumor cells and inhibit tumor growth significantly.Conclusions:The study constructed fiber modified hexon-chimaeric oncolytic adenoviral vectorsuccessfully, including: pRCAdHVR48-SDF1p-Ad/EGFP,pRCAdHVR48-SDF1p-FAP-P9/EGFP, pRCAdHVR48-SDF1p-FAP-P9-4C/EGFP,pRCAdHVR48-SDF1p-FAP-GP/EGFP, which can specific and effectively kill CAFs. Alsothese vectors had low liver toxicity, low immunogenicity. Experiments ex vivo showed4recombinant adenoviruses could selectively not only proliferate in FAP positive GCAFs andgastric cancer cells, but also kill these cells, while in normal cells they didn’t. Transfectionand killing ability was decrement in GP, P9-4C, P9, Ad. Capacity of4adenoviruses forbinding GCAFs remarkably decreased after FAP blocked by its antibody. Recombinantadenovirus could inhibit tumor growth in nude mice SGC-7901after they are transplantedwith gastric cancer. All experiments mentioned above demonstrated recombinant adenovirustargeting FAP could specific kill GCAFs and gastric cancer cells. The new treatment is safeand also afforded a new and effective strategy for gastric cancer targeted therapy.