| Research background and objectives:Study cetuximab in KRAS mutations can be induced immunogenicity in colorectal cancer cell death and play a role of inhibiting tumor growth,but this effect benefit from fewer patients,cetuximab in KRAS mutations induced immunogenicity colorectal cancer death in the process,However,the effect is not good,which may be related to the factors that limit the immunogenic cell death of the tumor itself,resulting in the limited ability of cetuximab to induce immunogenic death of tumor cells,weakens the effectiveness of its inhibition of tumor growth.Therefore,to find the factors limiting the immunogenic death of KRAS mutation colorectal cancer is conducive to solving the dilemma of poor clinical efficacy of cetuximab in the treatment of KRAS mutation colorectal cancer.In the tumor microenvironment,the activation of immune cells directly determines the immune properties around the tumor,and determines whether the tumor cells go to immunogenic death or escape from the immune system.The activation of tumor macrophages into M2-type macrophages is the key to effectively change the immune properties in the tumor microenvironment and help tumor cells escape immune killing by the body.It has been reported in the literature that GRP78 is a key factor in the regulation of tumor cells.GRP78 can inhibit the glycolytic metabolic pathway to induce the activation of macrophages into M2-type macrophages.Moreover,GRP78 has been detected in exosomes secreted by various tumors and has favorable conditions for transport from tumor cells to macrophages.In our previous studies have shown that ART1 expression in colorectal cancer group increased intestinal mucosa,and type KRAS mutations in colorectal cancer cells KRASG13 D LOVO cells and KRASG12 D CT26 cells are expressed,inhibition of LOVO cells and the level of ART1 CT26 cells can inhibit tumor cell proliferation and invasive ability and promote the apoptosis of tumor cells,at the same time,the study found that ART1 can catalytic GRP78 R470 and single ADP ribose R492 base modification.This allows us to speculate that in KRAS mutations in colorectal cancer,ART1 can occur through catalytic GRP78 single ADP ribose base,thereby promoting GRP78 package into the large intestine cancer cells secrete in the body to release to the tumor microenvironment,inhibit the expression of TLR4 huge bite cell membrane surface,thus inhibiting TLR4 signaling in macrophages in ROS production,reduce HIF1 alpha gene transcription level,reduce HIF1 alpha and PKM2 expression,eventually leading to M2 macrophage polarization increases,suppress the immune cell death that reduce cetuximab for colorectal cancer treatment effect of KRAS mutations.This study could help identify a factor that helps cetuximab to work in colorectal cancer patients with KRAS mutations.Expand the number of patients with colorectal cancer who can be treated with cetuximab.Methods:1.Correlation analysis of ART1 expression and M2 macrophage activation in KRAS mutated colorectal cancer(1)KRAS mutation(KRS MUT)and KRAS wild type(KRAS wild type,KRAS WT)in colorectal cancer tissues and the distribution of M2 macrophages and the expression and correlation of ART1Immunohistochemistry was used to detect the protein expression of CD68,the key indicator of monocyte activation into macrophages in KRAS MUT colorectal cancer tissues and KRAS WT colorectal cancer tissues,as well as the M2 macrophage marker proteins CD163,CD206 and ART1,and to analyze the correlation between them.(2)The effect of KRAS MUT and KRAS WT colorectal cancer cell lines on activation induction of M2 macrophagesLOVO cells(KRAS G13D),SW480 cells(KRAS G12V),CT26 cells(KRASG12D),HT-29 cells(KRAS WT)and MC38 cells(KRAS WT)were co-cultured with THP-1 or RAW264.7 derived macrophages to observe the activation of M2 type macrophages.Flow cytometry was used to detect the positive rate of cells co-expressing M2 macrophage marker proteins CD68 and CD206 to confirm the induction of M2 macrophages.(3)The effect of Art1 knockdown in KRAS MUT colorectal cancer cells CT26 on activation induction of M2 macrophagesAfter CT26 cells(KRASG12D)ART1 was knocked down,CT26 cells were co-cultured with RAW264.7 cell-derived macrophages,and the co-expression positive rates of M2 macrophage marker proteins CD68 and CD206 were detected by flow cytometry.(4)Effect of ART1 knockdown on M2 macrophage distribution in subcutaneous transplanted tumor of colorectal cancer miceAfter CT26 cells(KRASG12D)ART1 was knocked down,CT26 cells without ART1 transfection and no loading were subcutanically inoculated into BALB/C mice to construct the transplanted tumor.After the tumor tissue was fixed,embedded and sected,the distribution of M2 macrophages in the transplanted tumor tissue was detected by immunohistochemistry.2.The molecular mechanism by which KRAS MUT ART1 regulates M2 macrophage activation in colorectal cancer(1)Identification and extraction of exosomesExosomes from CT26 cells in ART1 knockdown,no-load and untransfected groups were extracted,Exosomes was identified by electron microscope and WB test,and the particle concentration and particle size distribution were identified by NTA.(2)Effects of MIBG on inhibiting GRP78 content in exosomes of ART1.GRP78 levels co-localized with Exosome after MIBG inhibited ART1 were detected by laser scanning confocal microscopy.(3)Effect of ART1 on GRP78 content in exosomes of KRAS mutant colorectal cancer cell linesThe level of GRP78 in the supernatant of CT26 cells in the ART1 knockdown,no-load and untransfected groups was detected by enzyme-linked immunosorbent assay.The content of GRP78 in the Exosome of CT26 cells in the ART1 knockdown,no-load and untransfected groups was detected by Western Blot assay.GRP78 levels in Exosome of CT26 cells in ART1 knockdown,no-load and untransfected groups were detected by laser scanning confocal microscopy.(4)ART1 interferes with the exosome GRP78 to regulate the activation of M2 macrophages through glycolysis pathwayExosomes of KRAS mutated colorectal cancer CT26 cells were extracted from ART1 knockdown,empty and untransfected cells and co-cultured with macrophages for 24 hours,then collected for analysis.Western blot and cellular immunofluorescence assay confirmed the effects of ART1 on TLR4,PKM2,HIF-1ɑ alpha and ROS levels in macrophages,respectively.(5)ART1 interferes with the secretion of IL-10 and TGF-β levels in exosomal GRP78 M2 macrophagesAfter ART1 was knocked down from CT26 cells,exosomes secreted by cells of ART1 knockdown,no-load and untransfected groups were extracted and co-cultured with macrophages derived from RAW264.7cells for 24 hours.The levels of IL-10 and TGF-β secreted by macrophages were detected by ELISA in the supernatant of macrophages collected.3.ART1 regulates M2 macrophage activation and participates in the adjuvant treatment of cetuximab in KRAS mutant colorectal cancer cells(1)Effect of CT26 cells knocked down by ART1 on cell proliferation after drug treatmentART1 knockdown CT26 cells and macrophages derived from RAW264.7 cells were treated with Cetuximab and FOLFOX4 regimen and combined treatment respectively,and the proliferation of tumor cells was detected by EDU assay.(2)Effect of CT26 cells knocked down by ART1 on cell invasion ability after drug treatmentART1 knockdown CT26 cells and macrophages derived from mouse monocytes RAW264.7 were treated with cetuximab and FOLFOX4 regimen respectively and combined with ART1 knockdown,no-loaded and untransfected CT26 cells.Transe-Well assesses tumor cell invasion ability.(3)Effects of ART1 knockdown on the growth of subcutaneous grafted tumor of colorectal cancer treated with cetuximabCT26 cells were subcutaneously inoculated into BALB/C mice after ART1 knockdown,no-load and untransfected CT26 cells,and treated with cetuximab respectively.The growth rate,size and weight of tumor formation and transplantation were observed.(4)Effect of Cetuximab Treatment on ART1 knockdown of proliferation-related Index of Subcutaneous Transplantation Tumor in Colorectal CancerThe tumor tissues were fixed,embedded and sected for HE staining to detect the mitotic image and immunohistochemical detection of the expression of proliferation index Ki67.Results:1.Correlation analysis of ART1 expression and M2 macrophage activation in KRAS mutated colorectal cancer(1)KRAS mutation(KRS MUT)and KRAS wild type(KRAS wild type,KRAS WT)in colorectal cancer tissues and the distribution of M2 macrophages and the expression and correlation of ART1Immunohistochemical results showed that the number of CD68+ positive macrophages,CD163+ and CD206+M2 macrophages in KRAS MUT colorectal cancer tissues was significantly higher than that in KRAS WT tissues(P<0.05).The expression of ART1 in KRAS MUT tissues was significantly higher than that in KRAS WT tissues(P<0.05).In 20 cases of KRAS MUT colorectal cancer,The number of CD68+ macrophages,CD163+ and CD206+ M2 macrophages was positively correlated with the positive degree of ART1.(2)Effects of KRAS MUT and KRAS WT colorectal cancer cell lines on activation induction of M2 macrophagesColorectal cancer cells with KRAS MUT and KRAS WT were co-cultured with macrophages,respectively.M2 macrophages co-expressing CD206 and CD68 were detected by flow cytometry.The results showed that KRAS MUT colorectal cancer LOVO,SW480 and CT26 induced more M2 macrophages than KRAS WT HT29 and MC38cells(P<0.05).(3)Effects of ART1 knockdown on activation induction of M2 macrophages in KRAS MUT colorectal cancer CT26 cellsArt1 knockdown,untransfected and no-load CT26 cells were co-cultured with macrophages of RAW264.7,M2 macrophages co-expressing CD206 and CD68 were detected by flow cytometry.The results showed that the induction of M2 macrophage activation after ART1 knockdown was significantly lower than that of untransfected group and no-load group(P<0.05),there was no significant difference in the induction of M2 macrophage activation between the untransfected group and the no-load group(P>0.05).(4)Effect of ART1 knockdown on M2 macrophage activation in subcutaneous transplanted tumor of colorectal cancer miceAfter the mice were sacrificed for tumor removal,the tumor tissues were fixed,embedded and sected.Immunohistochemical staining was performed on the tumor tissues of each group for M2 macrophage marker protein CD206,CD163 and the key indicator of monocyte activation into macrophages CD68.The results showed that,The number of CD68+macrophages and CD163+ and CD206+ M2 macrophages in the transplanted tumor tissues of colorectal cancer in the ART1-SH group was significantly lower than that in the ART1-WT and ART1-NC groups(P<0.05),while there was no significant difference in the number of CD68+ macrophages,CD163+ and CD206+M2 macrophages in the transplanted tumor tissues of colorectal cancer in the ART1-WT and ART1-NC groups(P>0.05).2.The molecular mechanism by which KRAS MUT Art1 regulates M2 macrophage activation in colorectal cancer(1)Identification and extraction of exosomesThe results of transmission electron microscopy and Western-blot showed that the substances extracted from the supernatant of CT26 cells with ART1 knockdown,no load and no transfection were exosome with high purity.NTA detected the concentrations of EXOs in the ART1-WT,ART1-NC and ART1-SH groups were 6+1014/L-1,3.2+1014/L-1and1.6+1014/L-1,respectively,and the sizes were all in the range of80-150 nm.(2)Effects of MIBG on inhibiting GRP78 content in exosomes of ART1When MIBG was used to inhibit ART1,compared with the untreated(ART1-WT)group,the results of laser scanning confocal microscopy showed that GRP78 and CD81(exosome marker protein)had significant co-localization in CT26 cells in the control group ART1-WT,and this co-localization phenomenon decreased after MIBG treatment.In addition,the level of GRP78 protein co-located with exosomes in MIBG group was lower than that in untreated group(P<0.05).(3)Effect of ART1 on GRP78 content in exosomes of KRAS mutant colorectal cancer cell linesThe supernatant of CT26 cells knocked down,unloaded and untransfected by ART1 was extracted for ELISA detection.The results showed that the content of GRP78 in the supernatant of CT26 cells in the ART1-SH group was lower than that in the ART1-WT group and the ART1-NC group(P<0.05),there was no significant difference in the content of GRP78 in the supernatant of CT26 cells between ART1-WT group and ART1-NC group(P>0.05);Exosomes from the supernatant of CT26 cells knocked down,unloaded and untransfected by ART1 were extracted for Western Blot assay.The results showed that The expression level of GRP78 protein in exosomes secreted by ART1-SH CT26 group(EXOS-CT26SH)was lower than that of exosomes secreted by ART1-WT CT26 group(EXOS-CT26WT)and exosomes secreted by ART1-NC CT26 group(EXOS-CT26NC)(P<0.05),there was no significant difference in GRP78 protein expression between EXOs-CT26 WT and EXOs-CT26 NC groups(P>0.05);Exosome marker protein CD81 was labeled with red fluorescence,and GRP78 was labeled with green fluorescence.LSCM observed that GRP78 and CD81 had significant co-localization in CT26 cells of the ART1-WT group and the ART1-NC group,However,this co-localization phenomenon decreased after ART1 knockdown.In addition,the level of GRP78 protein co-located with exosomes in the ART1-SH group was lower than that in the ART1-WT and ART1-NC groups(P<0.05),there was no significant difference in the level of GRP78 protein co-located with exosomes in CT26 cells of ART1-WT and ART1-NC groups(P>0.05).(4)ART1 interferes with the exosome GRP78 to regulate M2 macrophage distribution through the glycolytic pathwayAfter ART1 was knocked down in CT26 cells,exosomes secreted by ART1 knocked down,no-load and untransfected CT26 cells were extracted and co-cultured with macrophages derived from RAW264.7cells for 24 hours,macrophage proteins were extracted in each group for Western Blot experiment.The results showed that the expression level of ARG1 in M2 macrophage marker protein in CT26 cell and macrophage co-culture group(M2-ART1 SH)was lower than that in CT26 cell and macrophage co-culture group(M2-ART1 WT)and ART1 empty CT26 cells and macrophages co-culture group(M2-ART1 NC)(P<0.05),there was no significant difference in the expression level of M2-Art1 macrophage marker protein ARG1 between the M2-Art1 NC group and the M2-ART1 WT group(P>0.05).The expression levels of glycolytic signaling pathway indexes TLR4,HIF-1ɑ and PKM2 in M2-ART1 SH group were higher than those in M2-ART1 WT group and M2-ART1 NC group(P<0.05),the expression levels of glycolytic signaling pathway indicators TLR4,HIF-1ɑ and PKM2 were not significantly different between M2-ART1 WT group and M2-ART1 NC group(P>0.05).The level of ROS in M2-ART1 SH group was higher than that in M2-ART1 WT group and M2-ART1 NC group(P<0.05),there was no significant difference in ROS levels between M2-ART1 WT group and M2-ART1 NC group(P>0.05).(5)ART1 interferes with the effects of exosome GRP78 on the levels of IL-10 and TGF-β secreted by M2 macrophagesAfter ART1 was knocked down from CT26 cells,exosomes secreted by ART1 knocked down,unloaded and untransfected CT26 cells were extracted and co-cultured with macrophages derived from RAW264.7cells for 24 hours.The results showed that the levels of IL-10 and TGF-β in the supernatant of macrophage culture in M2-ART1 SH group were significantly lower than those in M2-ART1 WT group and M2-ART1 NC group.The results showed that the levels of IL-10 and TGF-β in the supernatant of macrophage culture in M2-ART1 SH group were significantly lower than those in M2-ART1 WT group and M2-ART1 NC group(P<0.05).There was no significant difference in the levels of IL-10 and TGF-β in the supernatant of M2-ART1 WT and M2-ART1 NC groups(P>0.05).3.ART1 regulates M2 macrophage activation and participates in the adjuvant treatment of cetuximab in KRAS mutant colorectal cancer cells(1)Effects of ART1 knockdown CT26 cells on cell proliferation after drug treatmentART1 knockdown,no-loaded,untransfected CT26 cells and RAW264.7 cells were treated with Cetuximab and FOLFOX4 regimen respectively and combined with ART1 knockdown,no-loaded,untransfected CT26 cells and RAW264.7 cells co-culture system.Compared with the control group without any drug,the DNA synthesis of tumor cells was examined by EDU method to reflect the proliferation of cells.The results showed that compared with the ART1 untransfected and no-loaded group,the DNA synthesis ability of CT26 cells in the ART1-SH group was significantly decreased in the no-dosed group,cetuximab,FOLFOX4 regimen,Separately,and the combined treatment group(P<0.05),there was no significant difference in DNA synthesis between CT26 cells transfected with empty vector and CT26 cells not transfected with ART1(P>0.05).The DNA synthesis ability of ART1-SH CT26 cells treated with cetuximab,the invasion ability of ART1-SH CT26 cells in FOLFOX4 group and combination group was significantly lower than that in non-drug group(P<0.05),and the invasiveness of ART1-SH CT26 cells in combination group was lower than that in single drug group(P<0.05).(2)Effect of ART1 knockdown CT26 cells treated with drugs on cell invasion abilityCT26 cells knocked down by ART1 were treated with cetuximab and FOLFOX4 regimen respectively and in combination with ART1,and compared with the control group without any drug,the cell invasion ability was examined by Transe-Well assay.The results showed that compared with the ART1 untransfected and no-load group,the invasion ability of ART1-SH CT26 cells was significantly decreased in the no-dosed group,cetuximab,FOLFOX4 regimen,respectively,and the combined treatment group(P<0.05),and there was no significant difference in cell invasion ability between ART1 untransfected CT26 cells and empty vector transfected CT26 cells(P>0.05);The invasion ability of ART1-SH CT26 cells treated with cetuximab,FOLFOX4,FOLFOX4 and combined treatment group were significantly lower than those in non-treatment group(P<0.05),and the invasive ability of ART1-SH CT26 cells in combined treatment group was lower than that in single treatment group(P<0.05).(3)Effects of ART1 knockdown on the growth of subcutaneous grafted tumor of colorectal cancer treated with cetuximabAfter treatment with cetuximab,the results showed that the transplanted tumor growth size and speed in the ART1-SH group were lower than those in the ART1-WT group and the ART1-NC group(P<0.05),However,there was no significant difference in tumor size and growth rate between ART1-WT group and ART1-NC group(P>0.05).The volume and weight of transplanted tumor in ART1-SH group were lower than those in ART1-WT group and ART1-NC group(P<0.05).There was no significant difference in tumor volume and weight between ART1-WT group and ART1-NC group(P>0.05).(4)Effect of Cetuximab Treatment on ART1 knockdown of proliferation-related Index of Subcutaneous Transplantation Tumor in Colorectal CancerAfter the mice were sacrificed for tumor removal,the tumor tissues were fixed,embedded and sected.After HE staining,the number of mitotic images in the tumor tissues of the ART1-SH group treated with cetuximab was lower than that in the ART1-WT and ART1-NC groups(P<0.05),while there was no significant difference in the image number of nuclear division in tumor tissues of ART1-WT and ART1-NC groups treated with cetuximab(P>0.05).After Ki67 immunohistochemical staining of tumor tissue sections in each group,the positive percentage of Ki67 in ART1-SH group was lower than that in ART1-WT and ART1-NC groups(P<0.05),and there was no significant difference in Ki67 positive percentage between ART1-WT and ART1-NC groups(P>0.05).Conclusion:(1)Both in vitro and in vivo experiments showed that the number of M2 macrophages and the expression of ART1 in KRAS MUT colorectal cancer tissues were higher than that in KRAS WT colorectal cancer tissues,and ART1 was involved in regulating the activation of M2 macrophages in KRAS mutant colorectal cancer tissues.The regulatory mechanism is related to ART1 in KRAS MUT colorectal cancer cells interfering with GRP78 encapsulation into tumor exosomes and release into tumor microenvironment,thereby regulating glycolytic signaling pathway to induce M2 macrophage activation.(2)Inhibition of KRAS MUT colorectal cancer cell ART1 induced M2 macrophage activation can assist cetuximab to inhibit the growth of KRAS mutated colorectal cancer cells.The detailed mechanism needs further study. |
PDF Full Text Request