Experimental Study Of Plasmodium Immunotherapy Combined With Radiotherapy For Solid Tumors

Background and objective:Cancer is currently one of the leading causes of human death,with nearly 10 million deaths of cancer in 2020,accounting for about one in six deaths worldwide.Solid tumors account for more than 90 percent of new cancer cases.Currently,the treatment of tumors mainly includes surgical resection,chemotherapy,radiotherapy,targeted drug therapy and immunotherapy,etc.,and the combined treatment strategy of multiple therapies is usually used in the clinical practice to prolong the survival of tumor patients and reduce the recurrence of tumor as far as possible.Plasmodium immunotherapy that uses benign Plasmodium infection,activates the immune system of the tumor-bearing host through multiple pathways and multiple targets,relieves the tumor immunosuppressive microenvironment and inhibits tumor angiogenesis,thus significantly inhibiting tumor growth and metastasis,and significantly extending the lifespan of the tumor-bearing hosts.However,a disadvantage of Plasmodium immunotherapy is that the direct killing effect on tumor cells is relatively weak,and a combination with a therapy that directly kills tumor cells may be needed to achieve better results.Radiotherapy is known to directly kill tumor cells and also induce immunogenic death of tumor cells,enhancing the anti-tumor immune response.Therefore,we hypothesized that Plasmodium immunotherapy combined with radiotherapy could produce a synergistic antitumor effect.Methods and results:In this study,we selected mouse tumor models with a typical cold tumor,brain glioma(GL261)and a typical hot tumor,non-small cell lung cancer(NSCLC,LLC),to test our hypothesis.We infected tumor-bearing mice with benign murine Plasmodium yoelii(Py17XNL,Py)for Plasmodium immunotherapy,and locally irradiated the tumor with X-rays for radiotherapy.We first demonstrated that Plasmodium infection significantly inhibited glioma growth and prolonged median survival of tumor-bearing mice in GL261 intracranial orthotopic and subcutaneous inoculation models.In GL261 subcutaneous inoculation model,Plasmodium infection significantly promoted the infiltration of effector CD8+T cells expressing perforin,and inhibited the infiltration of regulatory T cells(Treg)and myeloid-derived suppressor cells(MDSC)in the tumor tissues.Our previous studies have demonstrated that Plasmodium infection significantly inhibits the growth of Lewis lung cancer(LLC)in mice and improves the tumor immune microenvironment.In GL261 glioma and LLC lung cancer models,Plasmodium infection combined with radiotherapy significantly inhibited tumor growth and prolonged median survival in tumor-bearing mice compared with Plasmodium infection or radiotherapy monotherapy,demonstrating a synergistic anti-tumor effect of Plasmodium immunotherapy combined with radiotherapy.Importantly,in both intracranial orthotopic and subcutaneous GL261 models,70%of tumor-bearing mice in the combined treatment group achieved the goal of long-term tumor-free survival and produced sustained tumor-specific immune memory.By immunohistochemical detection of Ki-67 expression and Western blotting detection of caspase 3 expression,we found that the combination therapy had a synergistic effect on i nhibiting the proliferation of tumor cells and promoting the apoptosis of tumor cells.By flow cytometry analysis of infiltrating immune cells in tumor tissues,we found that the combination therapy significantly promoted the infiltration of effector CD8+T cells expressing perforin and inhibited the infiltration of immunosuppressive Treg and MDSC,improved the immunosuppressive microenvironment of tumor,and thus enhanced the anti-tumor immune response in tumor tissues.By flow cytometry analysis of immune cells in spleen and peripheral blood,we found that the combination therapy had a synergistic or complementary effect on the up-regulation of proportion or absolute number of CD8+T cells and down-regulation the proportion or absolute number of Treg or MDSC,thereby systemically enhancing the anti-tumor immune response of the body.In addition,combination therapy acted on STAT1/STAT3-TGF-β-PTEN-CDC25A-PI3K/Akt-MMP2/MMP9 pathways that were related involved to tumor growth and metastasis,which might also play a role in enhancing positive immunity and inhibiting negative immunity,as well as improving the microenvironment of tumor tissues.Conclusion:We have demonstrated that Plasmodium immunotherapy is effective in treating brain glioma in intracranial orthotopic and subcutaneous models,and preliminarily elucidated the immune mechanism.Meanwhile,we have demonstrated that Plasmodium immunotherapy combined with radiotherapy produces the synergistic antitumor effects in mouse glioma and lung cancer models,and preliminarily elucidated the possible immunological and molecular mechanisms of the synergistic effects.Based on the progress of the ongoing clinical trials of Plasmodium immunotherapy in the treatment of advanced solid tumors,this study provides a new experimental basis for combination therapy based on this therapy and a possible new solution for the treatment of solid tumors.Since brain tumors(including gliomas)are protected by the blood-brain barrier,few anti-cancer drugs can cross it,therefore,the clinical transformation of this study is worthy of expectation.