The Construction Of Bacillus Calmette-guerin And Pyroptosis Based Oncolytic Strategy And Its Preliminary Application In Pet Tumor Treatment

Tumors have become one of the most important causes that threaten the health of pets,and the mechanisms of the occurrence and development of spontaneous pet tumors and human tumors are relatively conservative.Therefore,it is urged to develop pet tumor treatment methods based on the research of human tumors remedies.Immune checkpoint blocking therapy represented by PD-1/PD-L1 blockade has made surprising progress in clinical tumor treatment.However,some restrictions have limited the application of the therapy,including low patient response rates.This may be related to the state of immunosuppression in the tumor microenvironment(TME).Therefore,tumor microenvironment may be a potential target to overcome the limitations of immune checkpoint blocking therapy.Both Bacille Calmette-Guerin(BCG)and pyroptosis have been proven to provoke a strong anti-tumor immune response at the tumor site,which can reshape the immune status of tumor microenvironment.However,the only widely used clinical application of BCG is in non-muscular invasive bladder cancer,while it has relatively few clinical applications in other tumor types,such as triple negative breast cancer(TNBC).In addition,it is challenging to efficiently produce and deliver GSDM^NTinto cancer cells due to its high intracellular toxicity to different cells and even to bacteria.In this study,BCG or pyroptosis were applied to construct an oncolytic strategy to target TME,the oncolytic mechanism of BCG or pyroptosis to animal tumor models was studied,meanwhile the effect of the combined use of BCG or pyroptosis and PD-L1 inhibitor on TNBC mouse models was estimated.At the same time,preliminary explorations have been made in the clinical application of pet tumors.The main results of this research are as follows:1 Targeting tumor microenvironment with BCG vaccine to enhance anti-tumor immunity.1)It has been confirmed in the TNBC mouse model that single-dose BCG treatment can up-regulate the expression of chemokine-related genes and anti-tumor effect genes,down-regulate the expression of immunosuppressive-related genes,meanwhile increasing the number of tumors infiltrating lymphocytes,furthermore reshaping TME Immunosuppressive state;triple-dose BCG treatment can significantly inhibit tumor growth,but there are also adverse effects of weight loss in mice.2)It has been confirmed by Bulk RNA-seq and q RT-PCR that single-dose BCG treatment can up-regulate the expression of PD-1 and PD-L1 in the tumor of TNBC mouse model.On this basis,an oncolysis program single-dose BCG combined with PD-L1inhibitor was developed,and confirmed to possess a significant oncolytic effect and no adverse effects of weight loss on TNBC mouse model.2 Construction of recombinant Adeno-associated Virus vector expressing pyroptosis protein to enhance anti-tumor immunity.1)A mammalian promoter m CBA that is not expressed in insect Sf9 cells was screened out,and it was confirmed that driving GSDM^NT with m CBA promoter can avoid the pyroptosis of insect Sf9 cells,thus developing a strategy of packaging recombinant adeno-associated virus vector expressing GSDM^NT using mammalian promoter m CBA and baculovirus/insect Sf9 cells.The oncolytic adeno-associated virus vector P1(Oncolytic AAV P1,o AAV-P1)was also obtained,which can mediate the pyroptosis of tumor cells.2)It has been confirmed that reversing the GSDM^NT sequence and adding two pairs of reverse lox P sequences on both sides can avoid the pyroptosis of HEK 293T cells,and the addition of Cre recombinase can restore the expression of GSDM^NT and mediate the pyroptosis,thus developing a strategy of packaging recombinant adeno-associated virus vector expressing GSDM^NT using Cre recombinase and three plasmids/HEK 293T cells.The oncolytic adeno-associated virus vector P2 was obtained(Oncolytic AAV P2,abbreviated as o AAV-P2).3)In some tumor cells and the rat model of Glioblastoma Multiforme(GBM),it has been confirmed that o AAV-P2 has a better and faster oncolytic effect than o AAV-P1.It has been explored in this study that this phenomenon is related to the weak infectivity of AAV packaged by insect Sf9 cells and the relatively weak activity of m CBA promoter.4)In the GBM rat model,it has been confirmed that o AAV-P2-mediated pyroptosis can temporarily open the Blood-Brain Barrier and destroy the TME of GBM,recruit lymphocytes to kill tumors,consequently prolong the survival time of the GBM rat;and in the TNBC mice model,o AAV-P2 therapy result in up-regulation of the chemokine-related genes and anti-tumor effect genes expression,meanwhile downregulates the expression of immunosuppression-related genes,increase lymphocyte infiltration in tumors,and change the TME immunosuppressive state.5)Through Bulk RNA-seq analysis of TNBC mouse model tumors,it can be observed that o AAV-P2 treatment can increase the expression of PD-L1 and PD-L2 in tumors.Based on the phenomenon we developed an oncolytic program of o AAV-P2 combined with PD-L1 inhibitor.It has been verified in a TNBC mouse model,which proved that the program has a good tumor treatment effect and can significantly inhibit tumor growth in a TNBC mouse model.This study selected TME as the target and developed an oncolytic program of BCG combined with PD-L1 blocker.This program can effectively treat TNBC and provide an effective remedy for breast cancer with high clinical incidence in pets.Meanwhile,this study constructed two packaging strategies for recombinant adeno-associated viruses expressing GSDM^NT,and obtained o AAV-P1 and o AAV-P2 with promising oncolytic effects.Moreover,these two packaging strategies have good scalability and safety,and can also be used for the packaging of recombinant adeno-associated virus vectors of other toxic proteins,providing more alternative tools for the treatment of pet tumors targeting TME.