The Anti-tumor Effect Of Gene Transduced Oncolytic Adenovirus Sg655-sgm To Cholangiocarcinoma

BackgroundCholangiocarcinoma is one type of malignant disease originated from bile duct epithelialcells. Investigations have revealed that the occurrence rate of cholangiocarcinoma has beencontinuously increasing during last few decades. Due to the lack of typical symptoms,diagnosis of cholangiocarcinoma was often realized at its late stage which bringsinconvenience for surgical operation. Moreover, the effect of chemo-or radio-therapy at thisstage is also limited. So, it is of importance to find new treatment methods forcholangiocarcinoma.CD47is a ubiquitously expressed moleculewhich could generate negative regulatorysignals when binds to its ligand SIRPα.The CD47-SIRPα interaction builds an importantmechanism which maintains the cell renewal and stabilization. The binding of SIRPα toCD47inhibits phagocytosis of macrophageswhich can protect normal CD47-expressing cellssuch as erythrocytes frombeing killedby the immune system.However, tumor cells could harness this negative regulation from CD47-SIRPα as animportant mechanismfor immune-surveillance evasion.Many tumor cells aberrantly expressCD47which can ligate with SIRPα on the surface of macrophages and suppress the functionof macrophages and facilitate the escape. Previous studies have confirmed that the interactionbetween CD47and SRIPα could be anti-tumor target by blocking CD47or SIRPα. But theapplication of this strategy is limited by the distribution characteristics of CD47and SIRPα.GM-CSF is a major hematopoiesis factor which functions to recruit immune cells suchas DC, macrophages, and T cells. Previous study showed that GM-CSF also plays animportant role in antitumor immune responses. Tumor vaccines which transduced withGM-CSF gene have showed encouraging results in both preclinical and clinical trials. Besides,oncolytic adenovirus carrying GM-CSF gene also showed certain antitumor effect with goodsafety in bladder cancer patients.Method and ResultsIn this study, we constructed fusion gene SF gene by fusing the Fc region of human IgG1coding sequence with the coding sequenceof binding region of highly affinitive mutantSIRPαCV1. Then, we assembled SGM by linking SF to mGM-CSF through2A linker,inserted SGM to the genome of type35oncolytic adenovirus, and named it as oncolyticadenovirus SG655-SGM. Later, we investigated the anti-tumor activity of SG655-SGMagainst cholangiocarcinoma in vitro. Major methods and results:（1）Construction and identification of oncolytic adenovirus SG655-SGM and relevantcontrol virus SG655-SF and SG655-GMWe constructed oncolytic adenovirus SG655-SGM and relevant control virus SG655-SFand SG655-GM via technology of molecular cloning. PCR results confirmed the existence ofdesired genes SGM, SF, and GM, while absent of wild viruses.（2）Expression of therapeutic gene SGMResults of Western Blot assay confirmed the expression of fusion protein SF by bothoncolytic adenovirus SG655-SGM and SG655-SF. ELISA assayshowed that both oncolyticadenovirus SG655-SGM and SG655-GM could effectively express mGM-CSF.（3）Functions of oncolytic adenoviruses SG655-SGM, SG655-SF, and SG655-GM invitroResults of indirect immunofluorescence assay confirmed that fusion protein SF couldspecifically bind to CD47.Results of virus proliferation assay showed that all of the oncolytic adenovirusSG655-SGM, SG655-SF, and SG655-GM could replicate in cholangiocarcinoma cell lineEH-CA1-T and EH-CA1-Ywith a peak time48hours, and the peak volume of replication isabout1,000times. The half killing MOI of oncolytic adenoviruses SG655-SGM, SG655-SF,and SG655-GM to EH-CA1-T cells are50,20, and10respectively. For EH-CA1-T cells,thehalf killing MOI of oncolytic adenoviruses SG655-SGM, SG655-SF, and SG655-GMare20,10, and10.Oncolytic Adenovirus SG655-SGM and its control viruses could obviously inhibit thegrouth of cholangiocarcinoma in vivoTreatment nude mouse model of cholangiocarcinoma by using SG655-SGM and itscontrol viruses, results showed that all of these oncolytic adenoviruses could inhibit thegrouth of tomor.ConclusionOur results showed that SG655-SGM could effectively inffect cholangiocarcinoma cellline EH-CA1-T and EH-CA1-Y, and mediate the expression of exogenous genes SF andmGM-CSF as well as facilitating the phagocytosis of macrophages to cholangiocarcinomacells which can inhibit the growth of cholangiocarcinoma.