The Effect And Mechanism Of MGLL-enhanced Macrophage Activity In Colorectal Cancer Progression

Background:Solid tumors such as colon cancer seriously endanger human health.At present,the conventional cancer therapies such as surgery,radiotherapy and chemotherapy are not effective,so the emerging tumor immunotherapy brings new hope to patients.In the tumor stroma,macrophages account for the largest proportion and involve in multiple stages such as tumorigenesis,growth,metastasis and recurrence.Moreover,the tumor treatment targeting macrophages has achieved great progress in clinical trials,so it is extremely important to explore the anti-tumor immune mechanism of macrophages.Among them,reprogramming macrophages to regulate their own activities has attracted more attention in anti-tumor studies compared to the research strategies of depleting macrophages or blocking macrophage infiltration.Previous studies have shown that metabolic or signaling molecules associated with lipid metabolism can directly or indirectly act on cell receptors and regulate the inflammatory signals.For example,MGLL is an important factor of triglyceride catabolism,which can regulate the malignant progression of tumors by regulating the release and oxidation of fatty acids.Interestingly,our previous screening revealed an abnormally low expression of monoacylglycerol lipase?MGLL?in tumor associated macrophages?TAMs?in colon cancer.However,the effect of MGLL on macrophage activity and malignant progression of colon cancer remains unclear.ObjectiveTo investigate the effect and mechanism of MGLL on self-activation of TAMs and malignant progression of colon cancer.Methods:?1?Establishing the models of subcutaneous transplantation tumor,hematogenous metastasis and peritoneal carcinomatosis of colon cancer in mice.?2?TSMs and TAMs were collected by flow sorting,and their lipid storage was detected by immunofluorescence staining and flow cytometry.Macrophages were stimulated by tumor conditioned medium,and triglyceride-related metabolic gene changes were tested by microarray.MGLL mRNA level in TSMs and TAMs was detected by real-time PCR.Tg^MGLL mice were constructed and their TSMs and TAMs lipid storage were measured by flow cytometry.?3?Subcutaneous transplanted tumor models were established in WT and Tg^MGLL mice to observe the effect of MGLL on tumor growth and survival.?4?Subcutaneous tumor model was established in WT,Tg^MGLL,Rag1^ko and Tg^MGLL+Rag^ko mice to investigate the effect of lymphocytes on tumor growth in vivo.The expression of IFN-?,TNF-?and IL-6 mRNA was determined by real-time PCR in CD8+T cells and CD4+T cells.Furthermore,the influence of CD8 antibody on tumor growth and survival in mice was observed.?5?The proportion of M1-type and M2-type macrophages in the tumor of WT and Tg^MGLL mice was assyed with flow cytometry.The mRNA level of inflammatory cytokines was detected by real-time PCR in TSMs,TAMs and BMDM under different stimulation.?6?2-AG mRNA in TSMs and TAMs,CB2 mRNA in TSMs and TAMs at different time points in tumor progression,and inflammatory cytokines in macrophages of WT,Tg^MGLL,Tg^CB2 and Tg^MGLL+CB2 mice were detected by real-time PCR.The effects of CB2antagonist?AM630?and agonist?2-AG?on the TLR4 signaling pathway in abdominal macrophages were assyed by Western blot.The interaction between CB2 and TLR4 was detected by immunofluorescence staining and Co-IP.The mRNAs of inflammatory cytokines were determined by real-time PCR in macrophages in WT,TLR4^ko,Tg^CB2 and Tg^CB2+TLR4^ko mice.?7?The mRNA level of CD8+T cells in the tumor of WT,Tg^MGLL,Tg^CB2 and Tg^MGLL+CB2 mice was measured by real-time PCR.Subcutaneous transplanted tumor models were established in WT,Tg^MGLL,Tg^CB2B2 and Tg^MGLL+CB2 mice,and the effects of MGLL/CB2signal axis and CB2 antagonists AM-630 and JIE-907 on tumor growth and survival in mice were observed.?8?Clinical colon cancer tissue samples were collected,and the level of MGLL protein in TAMs was detected by Western blot.The levels of MGLL and CB2 mRNAs in TAMs were assayed by real-time PCR.The effects of MGLL and CB2 of TAMs on the prognosis of patients were analyzed.?9?The hematogenous metastasis model of colon cancer was established in WT,Tg^MGLL,Tg^CB2 and Tg^MGLL+CB2 mice,and then the metastasis rates in lung and liver,as well as the size of the metastasised lung tumors were observed.Results:?1?The models of subcutaneous transplantation tumor,hematogenous metastasis and peritoneal carcinomatosis of colon cancer in mice were successfully established.?2?Decrease or deficiency of MGLL in TAMs enhanced lipid accumulation.?3?MGLL in TAMs inhibited the malignant progression of colon cancer in mice.?4?MGLL in TAMs repressed the malignant progression of colon cancer in mice via increasing the activity of CD8+T cells.?5?MGLL promoted TAMs M1 polarization.?6?MGLL-CB2/TLR4 signaling axis regulated macrophage activity.?7?The MGLL-CB2 signaling axis in macrophages regulated the malignant progression of colon cancer in mice through influencing the activity of CD8+T cells.?8?The level of MGLL in TAMs could predict the survival of patients with colon cancer.?9?In the hematogenous metastasis model of colon cancer,the MGLL-CB2 signaling axis of macrophages also regulated its malignant progression.Conclusion:We for the first time demonstrated the role of macrophage MGLL-CB2/TLR4signaling axis in its own polarization,the maintenance of CD8+T cell activity and the malignant progression of colorectal cancer,which may pave a way to explore macrophage-based immunotherapy for cancer.