The Mechanism Of MTOR Signal Dysfunction Blocks The Development Of Dendritic Cells In Colorectal Cancer

Colorectal cancer(CRC)is one of the most common malignant tumors in the world and has a high mortality rate.According to the Global Cancer Observatory(GCO)reported,there will be more than 1.9 million cases of CRC and 935,000 deaths in 2020.The incidence of CRC will reach the third place,but its mortality will rise to the second place.Surgical resection is the preferred treatment for CRC at present,and no effective treatment for metastasis and recurrence has been found yet.It is widely known that cancer is caused by the accumulation of genomic and epigenetic changes.Not only tumor cells,but also large numbers of stromal cells and inflammatory cells are contained in the tumor microenvironment.These inflammatory cells mainly include monocytes/macrophages,dendritic cells(DCs),mast cells,neutrophils,NK cells and T cells.These cells play a role through direct contacting with tumor cells or secreting some cytokines or chemokines.As an important bridge connecting the innate immune response and adaptive immune response,DCs plays an important role in the process of antigen recognition,antigen presentation and maintenance of autoimmune tolerance.DC subsets can be generally divided into conventional DCs(c DCs)and plasmacytoid DCs(p DCs).It has long been thought that CRC has little immunogenicity,but some findings suggest that there may be an anti-tumor immune response in patients.Like other malignancies,the tumor antigens of CRC can recruit DCs and promote their maturation.Mature DCs and their releasing cytokines result in the immune response that plays an anti-tumor role.DCs can also be directly migrated to CRC site and tumor infiltration DCs(TIDCs)can slow the tumor progression and metastasis.However,CRC inhibits the immune response at tumor sites in different ways which leads to immune dysfunction and immune escape.The developmental disorder and functional loss of DCs also contributes to this progress.Although we are aware of the potential role of DCs in cancer therapy,the mechanism by which DCs fail to effectively induce an immune response in tumors is not fully understood.Lots of reports have reported that m TOR signal plays an important role in the development and differentiation of DCs.Activation or reduction of m TOR signal activity can regulate the development and function of different DCs subsets.In addition,inhibitor of DNA binding or differentiation(Id2)is an important transcription factor,which also plays critical roles in the differentiation and development of DCs and has different regulatory effects on different subsets of DCs.Although it has been reported that m TOR signals can regulate Id2,the specific mechanism between them remains unclear.Therefore,understanding the interaction between DCs and tumors is of great significance for understanding the mechanism of immune escape of tumors,and provides a new idea for the treatment of tumors.ObjectiveThis study aims to explore the changes of DCs in vivo during the occurrence of CRC,and to clarify the mechanism of DC dysfunction.To explore the influence of m TOR signaling pathway on DC development,and clarify the specific mechanism of m TOR signaling pathway regulating DCs development.Methods1.The proportion of DCs and the expression of CD83,CD80 and CD86 in tumor and adjacent tissues of CRC patients were detected by flow cytometry.Human mo DCs was induced in vitro.The proportion of DCs and the expression of CD83,CD80 and CD86in healthy people and CRC patients were detected by flow cytometry.2.The expression of p S6K(T389)in peripheral DCs of healthy people and CRC patients was detected by flow cytometry.Human mo DCs and murine BMDCs were induced in vitro.The ratio of DCs and the expression of CD80 and CD86 were detected with or without rapamycin treated.3.The changes of Id2 protein in BMDCs,MEF^TSC2+/+,MEF^TSC2-/-,D2SC and DC2.4 cells were detected with or without rapamycin treated.4.m RNA levels of Id2 in BMDCs,MEF^TSC2+/+and MEF^TSC2-/-were detected with or without rapamycin treated.The changes of Id2 protein in MEF^TSC2+/+and MEF^TSC2-/-were detected with or without cycloheximide treated.The changes of Id2 protein in D2SC and DC2.4 with or without CQ,3m A and Baf A1 treated were detected.5.Co-IP assay was used to verify whether Raptor could directly interact with STAT3.The changes of STAT3 and its phosphorylation level in MEF^TSC2+/+,MEF^TSC2-/-,D2SC and DC2.4 with or without rapamycin treated were detected.6.The changes of Id2,p62 and LC3B protein in D2SC and DC2.4 with or without IL-6 or Stattic treated were detected.DC2.4 cells were transfected with ptf-LC3plasmid and the changes of green and red fluorescence with or without rapamycin and Stattic treated were observed by confocal microscopy.7.BMDCs were induced in vitro with or without Rapamycin and IL-6 treatment.The ratio of DCs and the expression of CD80 and CD86 were detected by flow cytometry.In vitro WT mice and DC-Raptor^?BMDCs were induced and the proportion of DCs and the expression of CD80 and CD86 were detected.8.Murine c DCs were induced in vitro.The ratios of c DCs and CD8⁺-like c DCs were detected by flow cytometry with or without rapamycin treated.WT and DC-Raptor^?c DCs were induced In vitro.The proportion of c DCs and CD8⁺-like c DCs were detected.9.The percentages of CD8⁺c DCs in the spleen,LNs and MLNs of WT and DC-Raptor^?mice.The ratios of CD103⁺c DCs in lung,liver,thymus,and intestinal lamina propria were detected by flow cytometry.10.MC38 cells were injected intradermally in the right fossa axillaries of mice.After14 days,left and right lymph nodes were collected and the proportions of CD8⁺c DCs were detected by flow cytometry.Results1.The proportion of DCs in CRC tumor tissues was decreased,and the expression of CD83,CD80,and CD86 were also decreased.The proportion of mo DCs from CRC was decreased.CD83,CD80 and CD86 of mo DCs had no significant changes compared with the healthy people.2.p S6K(T389)expression was decreased in peripheral DCs from CRC.Rapamycin inhibited the development of human mo DCs and mouse BMDCs,and repressed the expression of CD80 and CD86.3.Rapamycin inhibited the expression of Id2 protein in BMDC,MEF^TSC2+/+,MEF^TSC2-/-,D2SC and DC2.4.4.The expression of Id2 m RNA had no significant changes in BMDCs,MEF^TSC2+/+and MEF^TSC2-/-after rapamycin treated.m TORC1 slows Id2 protein degradation.Inhibition of autophagy can promote Id2 protein expression.5.m TORC1 directly and positively regulated STAT3 expression and phosphorylation through Raptor.6.STAT3 promotes Id2 protein expression.7.IL-6 could reverse the inhibitory effect of rapamycin on BMDCs Specific knockout of Raptor in DCs inhibited the development of BMDCs,and the expression of CD80 and CD86 was decreased.8.Rapamycin and Raptor specific knockout inhibited c DCs and CD8⁺-like c DCs development.9.In Raptor specific knockout mice,the proportions of CD11b^-CD8⁺c DCs the spleen,LNs and MLNs were reduced.The proportions of CD11b^-CD103⁺c DCs in the lung,liver,thymus,and lamina propria of the intestine were also reduced.10.The proportion of CD11b^-CD8⁺c DCs in lymph nodes was reduced after inoculated with MC38 compared with that in the other side.ConclusionThe proportion,maturity,and function of DCs infiltrating the tumor site were all reduced during CRC occurrence.The proportion of DCs in the peripheral blood was also decreased.In CRC patients,m TORC1 signaling activity was reduced in DCs,thus inhibiting the development of DCs.m TORC1 could directly interact with STAT3 to promote its expression and phosphorylation through Raptor,and then inhibit the level of autophagy.Repression of autophagy slowed down the degradation of Id2 protein which promoted the development of c DC1s.This discovery provided a new theoretical basis for the induction of immune tolerance during the occurrence of tumor,and provided a new idea for the optimization of DC vaccine in vitro.