The Mechanisms Of Notch Signal Regulating The Functions Of Macrophages And The Corresponding Effects On The Tumor Progressing

Tumors,referred to as neoplasms formed by dysplasia of local cells losing the normal control of growth at the genic level through stimulation by carcinogen,present in most organs or tissues of humans.Traditional therapies contain surgerys,chemotherapies and radiotherapies,which,to a large extent,inhibit the growth of the tumor but exhibit obvious shortcomings as follows.On the one hand,these methods cannot guarantee complete elimination of tumor cells in the body,and the recurrence is therefore the tough problem not yet solved in clinical practices;on the other,the resistance to drugs after repeated treatments severely affects the efficacy of common therapies.Thus,illuminating the mechanisms of both the initiation and the progression of tumors and locating the best target in tumor therapies has ever been one of the hotspots of cancer researches.As a canonical population of host immune cells,macrophages present in all tissues of an organism,playing an critical role in multiple biological and pathological processes such as maintaining homeostasis,defending pathogens and restoring from tissue injuries.Macrophages are the very plastic cells in the hematopoietic system and possess various fuctions.They can be divided into the classically activated macrophages(M1) and alternatively activated macrophages(M2) based on the functional states.The former participates in Th1 response,not only fueling the inflammation to kill tumor cells,but also producing IL-6 and TNF-a which facilitate the proliferation and the growth of tumor cells through JAK-STAT3 and NF-κB signaling pathway.The latter takes part in Th2 response countering the inflammation,strengthening tissue repair and promoting immunoregulation, which secretes EGF and VEGF influencing the cell proliferation and the neovascularization and thus the growth,invasion and metastasis of tumors. The different functions of macrophages,however,are based not only on the diverse stimulatory states,but on the disparate origins.The latest researches have revealed that most of the tissue macrophages,which can self-renew by in situ proliferation,derive from yolk sac or hematopoietic stem cells(HSCs) during embryonic development,rather than from the monocytic progenitor cells from the adult bone marrow as the traditional concept supposed.The contribution of tissue resident macrophages and monocytic proginitors derived macrophages to various diseases and the corresponding regulatory mechanisms are the front-burner issues in the research area of macrophages.In particular,the origins and the regulatory mechanisms of Tumor-associated macrophages(TAMs) in tumors remains unclear.Tumor-associated macrophages(TAMs)exist in all tumors and is one of the chief subpopulations of the immune infiltrate in the tumor.Recently,a number of researches demonstrated that macrophages could participate in the development of tumors in multiple ways and they could impact differently on almost every aspect of tumors,such as the stem cells,the neovascularization,the invasion and the metastasis.Clinical researches have also shown that the increase of the density of TAMs in most human tumors is intimately related to the poor prognosis of patients.Tumor cells,in turn,can also exert effects on the tumor microenvironment,transforming the phenotype of macrophages into M2-like and eventually promoting the growth of the tumor.Due to the high plasticity of macrophages,whether TAMs is tumor-prompting or tumoricidal remains a tough question.Taking advantage of the plasticity of macrophages,namely enlarging the the beneficial effects while preventing the disadvantageous ones,however,represents a promising therapy attracting the researchers’ attention in the treatment of tumors including the hepatocellular carcinoma and the lung carcinoma.It is the unclarity of the precise regulatory mechanisms of both the differentiation and the functions of TAMs that immensely hinders the development of new therapeutical technologies and methods.Thus,probing deeper into these mechanisms is the requisite of effective immunotherapy for patients with tumors.Notch signaling is a highly conservative pathway regulating the differentiation and the development of cells.When the Notch ligand and receptor come in contact with each other, γ-secretase cleaved the Notch intracellular domain(NIC) which enters the nucleus,integrates with and activates the recombination signal-binding protein Jk(RBP-Jk),thus regulating the transcription of Hes and Hey which determine the cell differentiation,development,proliferation,apoptosis,and so forth.Researchers have found that Notch signaling not only determines the differentiation of T lymphocytes versus B lymphocytes,but that of macrophages(MΦs) versus dendrocytes(DCs) during the fate selection of monocytes.Our research group also found that Notch signal directly dertermined the polarization state of macrophages,thus affecting their functions in antitumor response.How Notch signaling modulates the differentiation and the functions of TAMs in vivo and the undermining molecular mechanisms,however,are not yet clear.The tissue resident macrophages in subcutaneous location may be relatively less,which means that tumor inoculated here can embody the contribution of recruited monocytes to TAMs.Tissue resident macrophages in the liver,namely the Kupffer cells,however,are quite abundant to represent the resident macrophages’ contribution to TAMs as they account for 80%-90% of the total tissue macrophages in the body.We therefore made use of the mice with specific notch-signal deficiency in macrophages, Lyz2creRBPJflox/flox mice(KO mice),and Wildtype control mice(WT mice)to construct both the model of the subcutaneous Lewis Lung Carcinoma(LLC) and the orthotopic hepatoma,to illustrate the regulatory mechanisms of both the differentiation and functions of TAMs by Notch signal and the corresponding effect on tumor progressing.The main experimental outcomes are as follows: 1.Subcutaneous LLC model(1)Tumor phynotype:We continuously measured the volumes of the tumors and found that the LLC of KO mice growed slower than that of WT mice.Both the size and the weight of the LLC of KO mice were lower than that of WT mice after excision.(2)Histopathological changes of tumors: HE staining and Immunofluorescent staining of CD31 demonstrated that the density of the vessel-like structure in the LLC of KO mice was lesser than that in WT mice,which was consistent with the tumor phynotype.(3)Phynotype of macrophages:Both the results of the flow cytometry and Immunofluorescence staining suggested that the TAMs in KO mice decreased compared with WT mice.(4)Functional phynotype of macrophages:①q-PCR results showed that,compared to WT mice, the expression of M2-related molecules,IL-10, Ym-1, Arg-1 was downregulated,while that of M1-related marker,IL-1β,was upregulated in the LLC tumor of the KO mice.ELISA experiments revealed that the serum level of IL-10 in the KO mice was significantly lower than that in the WT mice,further confirming the results of q-PCR. ②The flow cytometric analysis,however,showed that the amount of both T and B lymphocytes presented no significant difference between the two groups.(5)The mechanisms of TAMs decrease: ①The differentiation of TAMs:FACS analysis revealed that the percentage of Ly6c+ cells in the F4/80intCD11 bhi monocytic progenitor cells of the tumor,spleen and bone marrow(BM) in KO mice was all higher than that of the corresponding organ in WT mice,which suggested that their differentiation towards macrophages might be hindered. ②The proliferation of TAMs:Costaining of F4/80 and Ki67 by Immunofluorescent staining suggested that the proliferation capacity of TAMs in the LLC of KO mice was lower than that of WT mice.Both of the macrophage proliferation-associated factors, CSF1 and CSF2, were downregulated in the LLC tissue of KO mice. ③The chemotaxis of TAMs:The expression of the CCL7 chemokine and the endothelial cell receptor,VEGFR1,was reduced in the LLC of KO mice. 2.Othotopic hepatoma model(1)Tumor phynotype:We dissected the hepatoma and found that the size and the weight of the tumor of KO mice were both greater than that of WT mice.(2)Histopathological changes of tumors: HE staining and Immunofluorescent staining of CD31 demonstrated that the density of the vessel-like structure in the hepatoma of KO mice was greater than that in WT mice,also in accordance with the tumor phynotype.(3)Phynotype of macrophages:FACS Analysis and Immunofluorescence staining results displayed that the TAMs in KO mice increased compared with WT mice.(4)Functional phynotype of macrophages:①The expression of M2-related markers,IL-10, Ym-1,was increased while that of M1-related marker,IL-1β,was reduced in the hepatoma tissue of the KO mice.ELISA experiments showed that the level of IL-10 in the serum of KO mice was higher,in accordance with the above variation of IL-10 level. ②It was shown by FACS analysis that the amounts of CD8+ T cells in the tumoral region and CD4+ T cells in the peritumoral area of the KO mice were both less,which is consistent with the enlargenment of tumor,while that of the CD4+ T cells in the blood were greater in the KO mice,suggesting that the peripheral lymphocytes recuited for adaptive immune response might be accumulated when the tumor augmented.(5)The mechanisms of TAMs increase: ①The differentiation of TAMs:As revealed by FACS analysis,the percentage of Ly6c+ cells in the F4/80intCD11 bhi monocytic progenitor cells of the hepatoma in KO mice was still higher than that in WT mice,which still suggested an impeded differentiation towards macrophages. But the same didn’t go for those cells in the spleen and bone marrow(BM). ② The proliferation of TAMs:Demonstrated by Immunofluorescent staining of F4/80 and Ki67 was that the proliferation capability of TAMs in the hepatoma of KO mice was greater than that of WT mice.The expression of CSF1 was still reduced in the hepatoma of KO mice,while that of CSF2 wasn’t significantly different, suggesting that the rise of the proliferative capability of TAMs was mediated by other factors,IL-4 included. ③The chemotaxis of TAMs:The expression of the CCL9 and VEGFR1 in the hepatoma tissue of KO mice was upregulated. 3. The origin of TAMs in orthotopic hepatoma modelAs the tumor phynotype of the subcutaneous LLC model and the orthotopic hepatoma model are totally different,we speculated that the origin of TAMs in the latter was different from that in the former leading to the disparate regulation of TAMs by notch signaling.We therefore set up an orthotopic hepatoma model in mice with bone marrow reconstitution and found that only about 20% of the TAMs are GFP positive,which suggested that most of the TAMs of the orthotopic hepatoma model derived probably from Kupffer cells rather than from monocytes. The following conclusions can be drawn from the above outcomes: 1.Specific RBPJ deficiency in macrophages inhibits the progression of LLC in the subcutaneously inoculated tumor model,but promoted the development of hepatoma in the orthotopically inoculated tumor model.So we may conclude that Specific RBPJ deficiency in macrophages exerts different impact on the tumor growth inoculated at different location. 2.The TAMs in subcutaneous LLC model might derive mainly from bone marrow monocytes,whose differentiation into TAMs was impeded when notch signal was blocked leading to the decrease of TAMs.Accordingly,the expression of M1-related molecules were upregulated while that of M2-related markers downregulated in the LLC of KO mice.The neovascularization was also reduced,which together resulted in the attenuation of the LLC.The reduction of macrophage proliferation factors attenuated the proliferative capacity of the TAMs and thus the number of them.However,the TAMs of orthotopic hepatoma model might derive mainly from proliferative tissue resident Kupffer cells rather than myelomonocytes. Although the differentiation of monocytic progenitors into macrophages was still hindered under the circumstance of notch blockade,the proliferation of Kupffer cells didn’t rely on notch signal and increased instead(independent of M-CSF and GM-CSF) causing the accumulation of TAMs. Consequently,the M1-related molecules expression,the M2-related markers expression and the neovascularization were downregulated, upregutated and increased respectively,bringing about the enlargement of the hepatoma.All in all, this research detailed the changes of the histopathological features,the immune microenvironment and the functions of macrophages in a subcutanesous LLC model and a orthotopic hepatoma model with specific notch signal deficiency in macrophages.We also discovered that,apart from monocytes,tissue resident macrophages(Kupffer cells) could also be the origin of TAMs.The differentiation of monocytic progenitors into TAMs was dependent on Notch signal while the proliferation of tissue resident Kupffer cells was not.