The Effect And Mechanisms Of Notch Signal Regulating Tissue Resident Macrophages In Liver Cancer

Primary hepatic cancer is the fifth most common type of cancer and the second leading cause of the cancer-related death in the world,with hepatocellular carcinoma(HCC)accounting for nearly 90% of the cases.HCC is insensitive to chemotherapy and is often at an advanced stage upon diagnosis with limited treatment options and a quite poor prognosis.Therefore,it is imminent to further study the mechanism of the development and progression of HCC in order to explore new therapeutic targets or treatment strategies.Macrophages infiltrating tumors,known as tumor-associated macrophages(TAMs),participating in the whole process of HCC development and progression and closely related to the poor prognosis of HCC patients,are recognized as a promising therapeutic target.TAMs generally fall into the M2 or alternative activation catalog of macrophages because they promotes tumor growth,invasion and metastasis.In terms of the origins,the TAMs in HCC can be divided into the monocytes derived TAMs(mo TAMs)and the tissue resident macrophages(Kupffer cells,KCs)-like TAMs(kcl TAMs).Clarifying contribution of macrophages of different origins to HCC is critical for TAMs-targeted therapies.The RBPj-mediated Notch signaling plays a critical role in differentiation and functional plasticity of mo TAMs.Yet the role of Notch signaling in kcl TAMs remains unclear.In the study of my master thesis,we constructed orthotopic HCC model in Lyz2creRBPJflox/flox mice(RBPJ c KO mice,with myeloid-specific Notch deficiency)and the control mice(Ctrl mice)and found that myeloid-specific Notch deficiency led to increased tumor size,tumor weight,neovascularization and number of TAMs,the possible mechanisms of which was only preliminarily discussed.In this study,we further explore the mechanism of increased TAMs in orthotopic HCC models with emphasis on discussing the regulatory role of Notch in kcl TAMs and the corresponding molecular mechanisms,which are specifically divided into the following contents:1.We increased the sample size to further confirm the above phenotypic changes of orthotopic HCC model in the RBPJ c KO mice compared to the Ctrl ones,and analyzed the quantity changes of myeloid cells and their subpopulations between the two groups by flow cytometry with focus on the number and the functional status variations of TAMs,which were then verified by immunofluorescent staining and q RT-PCR.2.To undermine the cellular origin mechanism of increased TAMs in RBPJ c KO mice,flow cytometry or Ed U staining and cell cycle analysis were used to detect the changes of TAM subsets and the apoptosis status and the proliferation capacity of kcl TAMs.To further exclude the effects of mo TAMs and observe alone the regulation of Notch signaling on the proliferation of kcl TAMs,CCR2-/-Ctrl and CCR2-/-RBPJ c KO mice with orthotopic HCC model were utilized for analysis and verification.3.To explore the molecular mechanisms downstream of Notch,immunofluorescent staining,q RT-PCR and Western Blotting were used to detect the changes of signaling molecules possibly responsible for regulation of kcl TAM proliferation,with in vivo and in vitro rescue experiments performed for further confirmation.4.In order to verify whether the experimental conclusion of this study is applicable to other liver cancer models,we further constructed a metastatic liver cancer model of colorectal cancer CMT93 cells in Ctrl and RBPJ c KO mice,and carried out the detections similar to that in orthotopic HCC models.5.To further discover whether the conclusion of this research is of instructive significance for clinical work,we performed immunofluorescent staining with the collected specimens of HCC from patients and analyzed the correlation between Wnt activation in TAMs and Notch activation or disease grades of patients.The results are as follows:1.In orthotopic HCC model,the major effect of Notch deficiency on myeloid cells was the increase in the number of TAMs which were skewed to more M2-like.2.Myeloid specific Notch blockade in orthotopic HCC model impeded the differentiation of mo TAMs,but substantially promoted the proliferation of kcl TAMs to increase the total TAM quantity and accelerate HCC growth.3.Notch blockade promoted kcl TAM proliferation in orthotopic HCC by activating β-catenin dependent Wnt pathway.4.Wnt activation,downstream of Notch signaling,reinforced the immunosuppressive phenotype of kcl TAMs in orthotopic HCC through c-Myc.5.Myeloid specific Notch blockade promoted the expansion of kcl TAMs and the growth of CMT93 liver metastases.6.In patient-derived HCC biopsies,Wnt activation in TAMs correlated negatively with Notch activation and positively with HCC disease grades.Taken together,the conclusions are as follows:(1)We identified KCs as an alternative source of TAMs in liver.(2)Notch signaling exerts different effects on different TAM subpopulations in HCC.(3)Wnt signaling,downstream of blocked Notch signaling,promotes both proliferation and M2-like phenotype of kcl TAMs facilitating HCC progression.