| Oncolytic virus therapy has become an important part of tumor immunotherapy.Oncolytic viruses can replicate,proliferate and lyse tumor cells,release progeny virus particles to infect surrounding tumor cells,induce systemic and local anti-tumor immune responses and activate the killing effect of immune cells by replicating in tumor cells and releasing tumor-associated antigens.In the current research on oncolytic viruses,adenoviruses are favored in clinical research due to their high transgenic efficiency,large capacity of foreign genes,and good safety.Oncolytic adenoviruses can also be genetically engineered to carry different exogenous genes to improve the killing,targeting and immune regulation functions.According to different tumor characteristics,oncolytic adenovirus is modified to carry different immune regulation genes or combined with other immunotherapy strategies,activate and promote anti-tumor immune response,maximize the anti-tumor effect,and finally achieve the goal of eliminating tumor.In the previous study,we designed and constructed a novel oncolytic adenovirus r Ad5-z TRAIL-RFP-Δ24E1A(CRAd5-TRAIL),which can express tumor necrosis factor apoptosis-inducing ligand protein(TRAIL)while specifically replicating in tumor cells.It can induce tumor cell apoptosis by binding to death receptors expressed on the surface of tumor cells.We evaluated the therapeutic effect of CRAd5-TRAIL in breast cancer,glioma and acute myeloid leukemia,and found that the tumor growth can be effectively inhibited in both solid tumors and non-solid tumors,showing a good application prospect.In order to further promote the application of CRAd5-TRAIL in tumor immunotherapy,the study of its anti-tumor immune mechanism in tumors has become our research focus.However,due to the limited ability of human adenovirus to replicate in murine tumor cells,there is no ideal animal model,and the anti-tumor immune mechanism of oncolytic adenovirus is currently unclear.In addition,recent studies have shown that,in addition to selectively inducing tumor cell apoptosis,TRAIL can also affect tumor growth by modulating immunosuppressive cells in the tumor microenvironment.Therefore,whether CRAd5-TRAIL has the function of regulating the tumor immune microenvironment while exerting the oncolytic virus itself to lyse tumor cells and induce tumor cell apoptosis,what role does CRAd5-TRAIL play in the anti-tumor immune response,and the specific mechanism?We need more in-depth research.On the basis of clarifying its anti-tumor immune mechanism,the anti-tumor effect of oncolytic adenovirus can be further enhanced by combining with other immunotherapy strategies.To select appropriate murine tumor cell lines to evaluate the antitumor immune effects and mechanisms of CRAd5-TRAIL,we first evaluated the infectivity,replication level,tumor cell killing activity,and ability to induce apoptosis of CRAd5-TRAIL in various mouse tumor cells in vitro.Since human adenovirus replicates at a low level in mouse tumor cells,we tried to construct a mouse breast cancer cell line4T1-100K stably expressing L4-100K to enhance the replication ability of CRAd5-TRAIL.But the results are not ideal.Then,according to the difference in the killing ability of CRAd5-TRAIL on different cells,we selected B16 cells with better killing effect and CT26 cells with relatively weak killing effect.These two types of cells were the main research targets for subsequent in vivo experiments.The anti-tumor effect and anti-tumor mechanism of CRAd5-TRAIL in mice were analyzed in CT26 and B16models respectively.At the same time,we found that CRAd5-TRAIL can induce a strong apoptosis in CT26 and B16 models,which indicated that the anti-tumor effect of the treatment is mainly caused by the apoptosis of TRAIL.Through the detection of adenovirus protein m RNA in tumor tissue,the replication level was limited to a certain extent,which was also one of the factors affecting the anti-tumor effect.For the infiltration of immune cells,CRAd5-TRAIL can increase the number of CD8~+T cells infiltrated in the CT26 model,and at the same time can increase the proportion of activated CD4~+and CD8~+T cells in B16,which was conducive to killing tumor cells to a certain extent and inhibiting tumor growth.In addition,the number of intratumoral immunosuppressive Tregs in both tumor models were significantly decreased after oncolytic adenovirus treatment,while TRAIL did not affect the reduction of adenovirus on Tregs cells.However,the specific immune molecular mechanism remains unclear.These results indicated that although CRAd5-TRAIL still had anti-tumor activity in an immunototipotent mouse model,its ability to activate immune cells in the tumor immune microenvironment was limited,which limited the effect of immunotherapy to a certain extent.As an immune activating molecule,CD40L can interact with a variety of immune cells,and CD40L/CD40 interaction has been shown to promote DCs maturation,provide B cells with proliferation or differentiation signals,stimulate Th1-type immune responses,and promote T cell activation,cytotoxicity and migration into the tumor microenvironment.At present,the adenovirus gene therapy vector(Ad5-CD40L)expressing CD40L has also played an active role in the clinical research of tumor immunotherapy.Therefore,the combination therapy of CRAd5-TRAIL and Ad5-CD40L is expected to further improve the effect of tumor immunotherapy by remodeling the tumor microenvironment.In combination therapy,on the one hand,oncolytic adenovirus releases more tumor antigens by efficiently killing tumor cells,on the other hand,CD40L enhances anti-tumor immunity by activating more immune cells,so it can mutually promote and inhibit tumor growth more effectively.Through in vitro analysis,we found that Ad5-CD40L can up-regulate the expression of TRAIL receptor on the surface of various tumor cells,thus promoting the apoptosis of CRAd5-TRAIL.Meanwhile,the lysate of Ad5-CD40L-infected mouse tumor cells can also activate DCs and mouse primary B cells,and this activation can enhanced after combined action in some cells.Afterwards,the antitumor effect and immune mechanism of CRAd5-TRAIL and Ad5-CD40L combination therapy were evaluated in vivo.We further analyzed the changes of intratumoral T cells,TAMs,MDSCs,Tregs,B cells,DC cells and NK cells in CT26,B16 and 4T1 models.The T cells and B cells response of mice were analyzed by ELISPOT experiment,and the concentration of tumor-specific antibodies,serum cytokine and intratumoral cytokine expression levels in mice were detected.The results showed that the intratumoral tumor cells had obvious apoptosis in CT26 and B16,indicating that the apoptosis still had a strong inhibitory effect on the growth of the tumor during the combined treatment.Combination therapy of CRAd5-TRAIL and Ad5-CD40L can enhance the infiltration and activation levels of T cells,B cells and DCs cells,reduce the infiltration number of Tregs,upregulate TRAILR expression,and generate tumor-specific T cell response and antibodies,producing cytokines favorable for antitumor responses.In summary,the combined therapy of CRAd5-TRAIL and Ad5-CD40L can better exert anti-tumor immunity by regulating the tumor immune microenvironment.In conclusion,this paper analyzed the anti-tumor effect and anti-tumor immune mechanism of oncolytic adenovirus carrying TRAIL in two mouse tumor models.Intratumoral and systemic antitumor immune responses are improved,and the therapeutic effect on tumors is improved.The mechanism of combination therapy includes:oncolytic adenovirus replicates and lyse tumor cells in tumors,and at the same time expresses TRAIL therapeutic gene to kill tumor cells,CD40L plays a role in regulating the immune response of the tumor microenvironment while promoting the apoptosis of TRAIL,enabling the combination therapy to achieve a better effect of oncolytic immunotherapy.Combination therapy in multiple ways has become a new trend in tumor treatment strategies.This combination virus therapy based on oncolytic adenovirus provides a new idea for future tumor immunotherapy. |
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