| Background;Gastric cancer is one of the most common digestive system malignancies in China.Incidence rate and mortality rate are second of malignant tumors,accounting for about half of the total incidence and mortality of gastric cancer.At present,the treatment of gastric cancer is surgery mainly.The main treatment measures of gastric cancer patients who can not be treated by surgery include radiotherapy and chemotherapy,immunity therapy,etc.Advanced gastric cancer patients can not tolerate radiotherapy and chemotherapy.Photodynamic therapy（PDT）is an effective treatment for these patients.PDT is a physical therapy.It has strong ablation effect and minimal invasive.It can be used alone or in combination with other treatments.It is an effective Treatment strategy to prolong the survival period of patients with advanced gastric cancer,which has been recognized and popularized by more and more people.In our previous study,we found that the endoscopic observation and pathological examination after PDT showed acute inflammatory in tumor tissue,which was consistent with the characteristics of pyroptosis.Therefore,the purpose of this study is to explore the mechanism of photodynamic induced pyroptosis of gastric cancer cells and its effect on the efficacy of immunotherapy,so as to provide a new strategy for the treatment of gastric cancer.Materials and methods:（1）Gastric cancer patients who received endoscopic PDT in our department from November 2019 to June 2021 were collected.Gastric cancer tissue and peripheral blood samples were collected.The clinical data of all patients were recorded and followed up.To observe the relationship between clinical profiles,laboratory test index changes and prognosis,the survival benefit and related risk factors of gastric cancer patients after PDT were preliminarily predicted and evaluated through univariate and multivariate analysis;（2）Pathological methods were used to detect the changes of necrosis and inflammation of gastric cancer before and after PDT,and the changes of inflammatory indexes in peripheral blood.Immunohistochemical staining（IHC）and Western blotting were used to detect the expression levels of CRT and HMGB1,the key components of DAMPs,in gastric cancer tissue before and after PDT,and to detect the changes of T cells and their subtypes in gastric cancer before and after PDT;ELISA detect IFN-γin tissue homogenate before and after PDT.The changes of immune cells in peripheral blood of gastric cancer patients before and after PDT treatment were detected by flow cytometry;Western blotting and luciferase luciferase system were used to detect the changes of CRT,HMGB1 and ATP in gastric cancer cells and culture medium before and after PDT;（3）The morphological changes of cell death after PDT intervention were observed by transmission electron microscope and microscope,and the level of GSDMD,the key protein of pyroptosis,was detected by Western blotting,.The inflammatory factor IL-1βreleased after pyroptosis was detected by ELISA、The changes of IL-18 and LDH levels were detected by the above three methods.In addition,the death of gastric cancer cells AGS and MKN-45 was observed by calcein AM/PI live cell or dead cell double staining.Western blotting was used to detect the GSDMD in gastric cancer and Paracancerous tissue,and normal gastric cell GES and different gastric cancer cell lines MKN45,AGS,MKN28,HGC27,BGC-823,SG-7901 and MKN-74.（4）In vitro,IHC was used to detect the expression levels of pyroptosis and related proteins NLRP3,CASP1 and GSDMD in gastric cancer before and after PDT.DCFH-DA Fluorescence probe detection and Western blotting technology were used to detect the expression level and activation of key protein of the classic activation pathway ROS-NLRP3-CASP1-GSDMD in gastric cancer tissue and paracancerous tissue pyroptosis before and after PDT intervention.The expression of GSDMD in gastric cancer cells was down regulated by specific GSDMD sh RNA lentivirus,and the changes of cell death and pyroptosis were detected.Gastric cancer cells were pretreated with ROS,NLRP3 and CASP1 inhibitors NAC,mcc950 and vx765 respectively to detect the changes of gastric cancer cell death and pyroptosis;（5）sh-GSDMD MKN-45 and the control group were further divided into PDT group and non PDT intervention group.The changes of gross view,volume and weight of subcutaneous tumors in nude mice and the expression of pyroptosis key protein GSDMD in tumor tissues were compared.At the same time,the effects of sh-GSDMD on tumor local tissue necrosis and acute inflammatory microenvironment were detected.Results:（1）the PDT was well tolerated in patients with gastric cancer;The total survival time（OS）prolonged.patients with advanced gastric cancer treated with immunotherapy,PDT and PDT combined with immunotherapy were 5.0,8.2 and 14.3 months respectively,suggesting that better survival benefits can be obtained after PDT treatment,and PDT combine immunotherapy have synergistic effect;It was found that PDT treatment could significantly increase the proportion of leukocytes,monocytes,lymphocytes and neutrophils in peripheral blood and induce the inflammatory response;Similarly,after PDT intervention,gastric cancer tissue showed extensive necrosis,acute inflammatory and increased lymphocyte infiltration;（2）In order to further explore the effect of PDT on immunity,it was found that CD4+T and CD8+T cells in peripheral blood of gastric cancer patients were significantly up-regulated after PDT;Similarly,the infiltration of CD4+T and CD8+T cells in gastric cancer tissue increased significantly after PDT;In addition,The IFN-γlevel increased in tissue homogenate after PDT significantly.In conclusion,PDT leads to acute inflammatory,increased lymphocyte infiltration and activity in tumor tissues,suggesting the activation of adaptive immunity.In order to find the reasons for adaptive immune activation,it was found that the expression levels of CRT and HMGB1,the key protins of DAMPs increased after PDT.In conclusion,the activation of adaptive immune activation and immunogenic death in patients with gastric cancer after PDT is related to the increase of DAMPs level resulting in persistent antitumor effect.（3）To investigate whether PDT induces gastric cancer cell death.It was found that GSDMD was low expressed in gastric cancer tissues and gastric cancer cell lines MKN-45,MKN-74,MKN-28,AGS,SGC-7901,BGC-823 and MGC-803,while the expression of GDSMD was significantly increased after PDT intervention;Similarly,after PDT intervention on gastric cancer cells MKN-45 and AGS,the expression level of GSDMD protein also increased,and the increase of activated GSDMD-N protein was more obvious.In order to clarify the occurrence of focal death,typical morphological changes of focal death occurred in AGS and MKN-45 gastric cancer cells after PDT intervention;Transmission electron microscopy also showed that gastric cancer cells had typical focal death changes after PDT intervention;In addition,gastric cancer cells AGS and MKN-45 generally died 12 hours after PDT intervention;12hours after PDT intervention,IL-1 in AGS and MKN-45 medium of gastric cancer cellsβ、IL-18 and LDH increased significantly.It shows that PDT can induce coke death of gastric cancer cells;（4）In order to explore the activation pathway of PDT induced gastric cancer pyroptosis,ROS-NLRP3-CASPASE1-GSDMD was detected after PDT 12 hours in gastric cancer cells MKN-45 and AGS;It was found that the core proteins NLRP3,CASPASE1 and GSDMD increased,especially the activated cleaved-CASPASE1 and GSDMD-N;In addition,the level of ROS was also significantly increased in gastric cancer cells AGS and MKN-45;Using ROS inhibitor NAC,gastric cancer cells MKN-45 and AGS,after PDT 12 hours,the level of cell death decreased significantly,some cells survived,ROS decreased significantly,the expression levels of ROS,NLRP3,cleaved-CASPASE1 and GSDMD-N generally decreased,and the phenomenon of pyroptosis was also significantly lower than that of the control group;Using NLRP3inhibitor MCC950,the cell death level of gastric cancer cells MKN-45 and AGS decreased after PDT 12 hours,the cell death was still visible,the level of ROS did not change significantly,the expression levels of NLRP3,cleaved-CASPASE-1 and GSDMD-N generally decreased,and the phenomenon of pyroptosis was lower than that of the control group;Using CASPASE1 inhibitor VX765,the cell death level of gastric cancer cells MKN-45 and AGS decreased after PDT 12 hours,the levels of ROS and NLRP3 did not change significantly,the expression levels of cleaved-CASPASE1 and GSDMD-N decreased,and the phenomenon of pyroptosis decreased compared with the control group;In addition,the expression of GSDMD was down regulated,and the death level of AGS and MKN-45 gastric cancer cells decreased after PDT 12 hours,and the phenomenon of pyroptosis was lower than that in the control group.In conclusion,PDT activates pyroptosis through ROS-NLRP3-CASPASE-1-GSDMD pathway.（5）To verify that PDT induces the expression of DAMPs in gastric cancer cells through GSDMD mediated pyroptosis.Firstly,it was found that intervention on gastric cancer cells MKN-45 and AGS after PDT 12 hours,the expression levels of CRT and HMGB1,the main members of DAMPs,and ATP in the culture medium increased significantly,which was consistent with the increase of CRT,HMGB1 and ATP in gastric cancer tissue after PDT.In order to investigate the effect of GSDMD mediated pyroptosis on PDT induced DAMPs release,DAMPs were detected after PDT12 hours targeting GSDMD lentivirus infected MKN-45 and AGS cells.Compared with Sh Ctrl group,the increase of CRT and HMGB1,and the increase of ATP in culture medium were inhibited.It shows that inhibition of GSDMD mediated pyroptosis can affect the production and release of DAMPs by PDT.（6）Compared with Sh Ctrl group,there was no significant difference in the volume and weight of subcutaneous tumorigenesis in Sh GSDMD group,but after PDT intervention,the volume and weight of subcutaneous tumorigenesis in Sh GSDMD group increased.Conclusion:Through the above-mentioned clinical and related in vitro and in vivo experiments,we have proved that:（1）Patients with gastric cancer are well tolerated by endoscopic PDT.PDT can significantly improve the prognosis of patients with advanced gastric cancer and has a synergistic effect with immunotherapy.（2）After PDT therapy,gastric cancer tissue changed from the chronic inflammatory type to the acute"inflammatory type"with high response rate to immunotherapy.After PDT treatment,the number and function of CD4+T and CD8+T in gastric cancer tissue increased and activated,meanwhile,the expression of DAMPs increased.（3）GSDMD was low expressed in gastric cancer cells and gastric cancer tissues;PDT can induce pyroptosis of gastric cancer cells through ROS-NLRP3-CASPASE1-GSDMD pathway,and induce the production and release of DAMPs.After PDT therapy,gastric cancer cells pyroptosis release inflammatory factors and DAMPs,Meanwhile,not only killing tumor cells directly,but also form an"inflammatory"microenvironment conducive to anti-tumor immunotherapy response,recruitting mature adaptive immune cells infiltrate into local tumor tissue,and induce ICD,so as to improve the efficacy of anti-tumor immunotherapy and improve the clinical prognosis of patients with gastric cancer. |
PDF Full Text Request