Cisplatin Nanoparticles Enhance The Sensitivity Of NSCLC To Radiotherapy And Boost Abscopal Effect

Background:Lung cancer is the most common malignancy and the most common cause of cancer death in the world.Non-small cell lung cancer(NSCLC)is the most common pathological type of lung cancer,which accounting for 85% of the total cases of lung cancer.Since most patients are already in the advanced stage with tumor metastasis when diagnosed,the 5-year survival rate of patients with NSCLC is very low.Therefore,it is of great clinical significance to explore new strategies for the treatment of NSCLC and find effective treatment modes to improve the prognosis of patients with advanced NSCLC.Radiotherapy(RT)as a local treatment for cancer has been widely used in clinical practice for more than a century.In 1953,RT was first found to have a systemic inhibitory effect on metastatic tumors outside of the radiation field besides the tumors receiving radiotherapy directly,which was referred to as the abscopal effect.Since the lacking of effective control on metastasis cancer is the main cause of death in cancer patients,abscopal recession of metastatic tumors is of clinical significance.However,it is a rare occurrence and has only been observed in some individual cases.Therefore,it is necessary to devise novel strategies to increase the frequency of abscopal effect of radiation and augment the therapeutic outcome of patients with advanced NSCLC.In recent years,researchers had found that the inhibition effect of RT on non-irradiated tumors was immune mediated and mainly dependent on the attacking of CD8~+ T cells on tumor cells.RT induces immunogenic cell death(ICD),which promotes immature dendritic cells(i DCs)activate into mature DCs(mDCs)and subsequent present tumor antigen to prime CD8~+ T cell.The activated CD8~+ T cells then infiltrate into the non-irradiated tumors and attack the tumor cells.Since the mechanisms of tumor evasion from the immune response became more and more clear and with the advent of immune checkpoint inhibitors,a large number of clinical trails have attempted to enhance the abscopal effect by combining immunotherapeutic strategies that target programmed cell death protein 1 and its ligand(PD-L1)with radiotherapy to mitigate the immunosuppressive environment and abolish the functional inhibition of CD8~+ T cells.However,less than 30% of the patients receiving RT along with anti-PD1 show the abscopal effect,one major limitation is the paucity of CD8~+ T cells in non-irradiated tumors.Therefore,explore effective measures that can simultaneously increase the sensitivity of cancer cells to radiotherapy to activate more CD8~+ T cells and effectively promote the infiltration of these activated CD8~+ T cells into the tumors without radiation has become an important prerequisite for increasing the abscopal effect of radiotherapy.Cisplatin(CDDP)is a kind of broad-spectrum cytotoxic chemotherapeutic agent which has been widely used in clinic.Studuies have confirmed that CDDP can increase the radiosensitivity of tumor cells by platinizing the DNA and thus prevent cells from repairing the radiation-induced DNA damage.In addition,CDDP also enhances secretion of chemokine(C-X-C motif)ligand 10(CXCL10)in the tumors,which plays a vital role in recruiting CD8~+ T cells.However,the systemic toxicity induced by CDDP,especially in combination with RT,significantly limits its clinical dosage to levels that are insufficient for enhancing the abscopal effect.Therefore,novel delivery systems are needed to minimize systemic toxicity of CDDP and effectively boost the abscopal effect.CDDP loaded poly(L-glutamic acid)-graft-methoxy poly(ethylene glycol)complex nanoparticles(CDDP-NPs)was developed by our research group independently and has been patented by State Intellectual Property Office Patent Office.Based on the EPR effect of nanoparticles,compared with CDDP,CDDP-NPs showed significantly stronger intratumoral enrichment and lower systemic toxicity as well as prolonged half-life.Objectives:In this study,we choose the Lewis lung carcinoma(LLC)murine NSCLC model,investigated the radiation sensitization effct of CDDP and CDDP-NPs under the premise of equivalent biotoxicity and compared the immunogenic of RT when combined with CDDP and CDDP-NPs,respectively.Further more,we also investigated the role of CDDP-NPs in boosting the abscopal effect and its mechanism in mice bearing bilateral tumors.Methods:1.Mouse bearing unilateral LLC tumor model was established,and the blood routine test,biochemical tests and H&E staining of important organs of tumor-bearing mice were conducted after different treatments to compared the systemic toxicity of CDDP and CDDP-NPs when used alone or combined with RT.2.Tumor inhibition esperiment in vivo was conducted on mice bearing unilateral LLC tumor to verify the radiation sensitization effect of CDDP and CDDP-NPs and the mechanism.3.Flow cytometry was used to detect the impact of CDDP and CDDP-NPs on the ICD induced by RT in vitro.4.Immune activation effect on tumor microenvironment of RT combined with CDDP or CDDP-NPs was reflectd by the population of mDCs and CD8~+ T cells within the tumor,which were detected by flow cytometry.5.Mouse bearing bilateral LLC tumors was established,and the levels of CXCL10 and CD8~+ T cells within both the tumors were detectd by enzyme-linked immunosorbent assay and flow cytometry,respectively,to verify the effect of CDDP and CDDP-NPs on recruiting CD8~+ T cells infiltated into tumors.6.The effects of CDDP and CDDP-NPs on boosting the abscopal effect were verified by tumor inhibition esperiment in vivo conducted on mice bearing bilateral LLC tumors.Results:1.CDDP-NPs at dosage of 10 mg/kg did not cause more severe biotoxicity than CDDP at dosage of 3 mg/kg,no matter when they was used alone or combined with RT.2.CDDP-NPs showed stronger DNA platinization than CDDP.At 24,48 and 72 hours after administration,the concentrations of Pt adducted to the DNA of tumor cells of mice treated with CDDP-NPs were significantly higher than those of CDDP.And tumor inhibition experiment in vivo showed that the tumor volume of mice treated with RT + CDDP-NPs was much smaller than that of RT + CDDP and other controls,and the difference was significant.3.CDDP-NPs significantly enhanced the capbility of RT to induced ICD,and RT+ CDDP-NPs resulted in the highest level of calreticulin(CRT)exposure compared with RT + CDDP and other controls.4.CDDP-NPs enhanced the activation of tumor immune microenvironment induced by RT significantly.The population of mDCs and CD8~+ T cells within the tumors of mice treated with RT + CDDP-NPs was significantly higher than those treated with RT + CDDP and other controls,and the differences were significant.5.CDDP-NPs significantly increased the levels of CXCL10 in both the irradiated and non-irradiated tumors,and RT + CDDP-NPs resulted in a significant increase of CD8~+ T cells in both the tumors than RT + CDDP and other controls,and the differences were significant.6.After the combination of RT and anti-PD1 with CDDP-NPs,the inhibitory effects on both the irradiated and non-irradiated tumors were all increased significantly.The volumes of the irradiated and non-irradiated tumors of mice treated with RT + anti-PD1 + CDDP-NPs were all significantly smaller than those treated with RT + anti-PD1 + CDDP and other controls,and the differences were significant.Conclusions:1.A higher dose of CDDP-NPs was tolareble compared to CDDP under the premise of equivalent biotoxicity,which made CDDP-NPs exhibited a stronger radiotherapy sensitization effect than CDDP by sustained high level DNA platinization.2.CDDP-NPs enhanced RT-induced ICD and immune activation within the tumor microenvironment more strongly than CDDP.3.CDDP-NPs promoted CD8~+ T cells infiltration into both irradiated and non-irradiated tumors through enhanced CXCL10 secretion more intensively than CDDP,thus CDDP-NPs boosted the abscopal effect significantly stronger than CDDP.