Study On The Effect Of 3-MA And PD-1 Inhibitors In The Synergistic Treatment Of Colon Cancer With Different Mismatch

Background:Programmed-death1/programmed-death ligands-1（PD-1/PD-L1）inhibitors immunotherapy has shown excellent clinical efficacy and has been endorsed by the FDA for many cancers.However,in the process of treating colorectal cancer（CRC）,only 3.5%to 5%of d MMR-m CRC patients have achieved superb results;compared with d MMR-m CRC,immunotherapy is extremely effective for a large population of p MMR-m CRC（95%）.Therefore,how to improve the clinical efficacy and prolong the survival of patients with p MMR-m CRC has become an urgent clinical problem to be addressed.Autophagy is a process of transporting damaged,degenerated or aging proteins and organelles into lysosomes for digestion and degradation.Autophagy also incorporates as a dual role in tumors,inhibiting cell canceration and promoting tumor growth.Compared with d MMR-m CRC,p MMR-m CRC has higher autophagy activity.We speculate that the high autophagy activity of p MMR-m CRC is the reason that has little effect on the efficacy of PD-1inhibitors;although studies have shown that the blockade of immune checkpoints PD-1/PD-L1 is closely related to different MMR types of CRC,it is determined that MMR CRC immunotherapy and cell autophagy leading to drug resistance have not been reported.Therefore,in this project,through the combined application of autophagy inhibitors and PD-1 inhibitors,we will investigate whether the inhibition of autophagy can improve the efficacy of PD-1 inhibitors on p MMR-m CRC,and achieve the similar effect of d MMR-m CRC.The mechanism of cellular autophagy and PD-1 inhibitor resistance is of great significance.This subject aims to explore the synergistic effect and the possible molecular mechanism of the combination of autophagy inhibitor 3-MA and PD-1 inhibitor to inhibit the proliferation of mouse colorectal cancer cell CT26 in vivo and in vitro.The feasible strategy of low efficacy of PD-1 inhibitors provides a new solution for improving the therapeutic efficacy and prognosis of patients with p MMR m CRC.Method:（1）In the natural state,there is no mismatch repair gene deletion genotype of mouse-derived CT26 cells.In this experiment,the Mlh1 gene in CT26was knocked out by CRISPR-Cas9 gene-edit technology,and the CT26-d MMR genotype cell line was successfully constructed for subsequent experimental research.（2）This experiment explored Akt agonists,3-MA（autophagy inhibitors）and TNF-α（simulating the effect of PD-1 inhibitors in vitro）by RT-PCR,Western blot（WB）and flow cytometry（FCM）in vitro treated the cell cycle,the expression levels of autophagy and apoptosis related genes and proteins of CT26-p MMR and CT26-d MMR cells,and understand the difference between the efficacy of d MMR and p MMR after the administration of autophagy and PD-1 inhibitor alone or in combination.（3）Animal experiments CT26-p MMR and CT26-d MMR cells were inoculated in Balb/C normal murine to construct a cell-line-derived xenograft（CDX）animal model,and 3-MA and PD-1 inhibitors alone or combination were used to detect the tumor growth inhibition of two types of CT26 cells subcutaneously bearing murine role and effect on immune cells.Randomly divided into four groups,group A:solvent control group（NC）,group B:3-MA single drug group,group C:PD-1inhibitors group,group D:3-MA+PD-1 inhibitors group.When the tumor volume was about 1500-2000 mm³,the tumor-bearing murine were murdered.Hematoxylin and eosin（H&E）staining of mouse internal organs to assess the safety of the two-drug treatment;Immunohistochemistry（IHC）detection of tumor tissue Ki-67,CD3,CD8,LC3,PD-L1 protein and TUNEL to assess tumor tissue proliferation,T cell infiltration and apoptosis.Results:（1）Western blot detected that Mlh1 protein was not expressed in CT26cells,indicating that the Mlh1 gene in CT26 was knocked out and successfully constructed into a murine d MMR cell line（CT26-d MMR）.（2）The MTT method detected 3-MA and TNF-αafter treatment for 48 h for two types of CT26 cells,suggesting that 3-MA and TNF-αinhibited the proliferation of the two types of CT26cells in a concentration-dependent and time-dependent manner,3-MA and TNF-αwere IC₅₀=1 m M and IC₅₀=10 ng/m L after 48 hours of effect.The combined drug index EOB value of the two drugs also suggested that 3-MA and TNF-αexhibited a synergistic inhibitory effect in two types of CT26 cells,so this test was selected 3-MA(IC₅₀=1 m M)and TNF-α(IC₅₀=10 ng/m L)were used for subsequent in vitro experiments.（3）The cell cycle was detected by FCM and WB test showed that TNF-αcombined with 3-MA can block the expression of cyclin D1 and block the cell cycle,and then block the cell proliferation.（4）Real time PCR（RT-PCR）and WB detection of the expression of autophagy-related genes and proteins,suggesting that TNF-αcan promote the increase of cell autophagy and p MMR cells have higher autophagy activity than d MMR cells,and between p MMR and d MMR are significantly different（LC3,p<0.0001）;immunofluorescence and transmission electron microscope（TEM）observation of cell autophagy activity suggested that p MMR cells had higher autophagy levels than d MMR cells in NC group,TNF-αcould promote autophagy level of cells up-regulation,but TNF-αcombined with 3-MA down-regulated the expression levels of autophagy-related genes and proteins,which can reduce the differential expression of p MMR and d MMR cells produced by 3-MA and TNF-αin combination.（5）FCM Annexin V-APC/PI Apoptosis detection showed that after treatment with 3-MA combined with TNF-α,the CT26 two cells had a significantly increased apoptosis rate（p<0.0001）;RT-PCR and WB experiments indicate that TNF-αcombined with 3-MA can promote apoptosis more than TNF-αalone,induce the expression levels of Cleaved Caspase3 and Cleaved Caspase9 to be significantly up-regulated（p<0.001）,and make CT26-p MMR cells wither the degree of death is similar to CT26-d MMR cells.（6）Results of in vivo animal experiments,it was found that the inhibitory rates of PD-1 inhibitors alone on CT26-p MMR and CT26-d MMR murine were 48.75%and 69.64%,and the combination of 3-MA+PD-1 inhibitors tumor rates were 89.88%and 90.41%.H&E staining detected no pathological changes in the internal organs of murine,and the body weight of murine in the two-drug combination group did not change significantly,proving that the two drugs alone or combined administration have high safety.IHC detected tumor tissues in mice,and TUNEL showed that PD-1 inhibitors combined with 3-MA group significantly increased apoptosis than PD-1 inhibitor alone;PD-1 inhibitors and 3-MA in combination can significantly accumulated Ki67,CD3,CD8 and PD-L1 in p MMR and d MMR,but the expression of LC3 was down-regulated in p MMR and d MMR.（7）FCM detection of immune cells indicated that the PD-1 inhibitors combined with3-MA group（p MMR:0.623±0.055%,d MMR:0.373±0.265%,p<0.001）was better than the PD-1 inhibitor alone（p MMR:0.737±0.102%,d MMR:0.563±0.176%,p<0.001）,and could significantly reduced the number of Treg（p<0.01）.PD-1inhibitors and 3-MA in combination group（p MMR:35.367±13.294%d MMR:41.4±3.34%）can significantly accumulated CD3⁺T（p<0.01）than PD-1 inhibitors alone（p MMR:28.267±4.6%,d MMR:28.333±9.674%）.The combination PD-1 inhibitors with 3-MA group（p MMR:6.54±2.46%,d MMR:8.29±0.95%）could significantly up-regulated CD8⁺T than the PD-1 inhibitors alone（p MMR:4.67±1.76%,d MMR:8.75±0.46%）（p<0.05）.Conclusion:This subject successfully constructed unnatural d MMR CT26mouse-derived cells.The results demonstrate that the combination of autophagy inhibitors and PD-1 inhibitors can coordinate and synthesize to promote p MMR CT26tumor cell apoptosis and inhibit tumor proliferation,reversing the primary resistance of CT26-p MMR to PD-1 inhibitors immunotherapy.Those provide a novel idea for exploring the application of PD-1 inhibitors in the treatment of primary drug resistance,and provide a novel solution for reversing the intractable problem of primary p MMR solid tumor resistance to immune checkpoint therapy.