The Role Of Macrophage In Promoting The Formation Of High Endothelial Venules In Melanoma And Relative Research On Macrophage Targeting By Nanoparticles

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The influence of hypoxia on the phenotype and function of BMDMsBackground:Classical macrophages can be divided into pro-inflammatory macrophages(M1 type)and anti-inflammatory macrophages(M2 type).The former plays an anti-tumor role in tumors,while the latter has an anti-tumor effect.Hypoxia is a common feature of many solid tumors including melanoma.However,the role and mechanism of macrophages with different phenotypes and functions in tumor progression and metastasis under hypoxic microenvironment of melanoma are still unclear.Therefore,it is of great importance to deeply explore the influence of melanoma hypoxia microenvironment on the phenotype and function of macrophages and its regulatory mechanism.Objective:To study the effect of hypoxia on the phenotype and function of bone marrow derived macrophages(BMDMs)Methods:Both of the HIF-1? and HIF-2? expression in BMDMs after hypoxic culture in different time were tested by western blot.Flow cytometry analysis was performed to study the expression of M1 type surface marker molecules(CD86,CD 16/32,MHC-?),M2 type surface marker molecules(CD206)and immunosuppressive molecule PD-L1 on BMDMs after hypoxia exposure.The change of PD-L1 expression was tested after HIF-1? inhibitor being added.ELISA method was used to detect the level of M1-type cytokine TNF-?,M2-type cytokine IL-10 and the changes of TNF-? level after adding HIF-la inhibitor when under hypoxia culture.CCK8 method was also applied to test the toxicity of HIF-1?inhibitor with different concentration under normal or hypoxia conditions.Results:After hypoxia exposure,the expression of HIF-1? in BMDMs increased while HIF-2? was rarely expressed.Moreover,the expression of classic M1 macrophage molecule CD86(p<0.05),CD16/32(p<0.01),MHC-II molecule(p<0.01)and inhibitory molecule PD-L1(p<0.01)were up regulated on BMDMs while the expression of classic M2 macrophage molecule CD206(p<0.01)was down regulated after 24 h of hypoxia culture.The secretion of IL-10,which is one of the classical M2 macrophage cytokine,decreased by BMDMs after 24 h hypoxia exposure(p<0.05).The M1 type cytokine TNF-? secreted by BMDMs was significantly increased after 6 h(p<0.0001),12 h(p<0.0001),24 h(p<0.0001)and 48 h(p<0.001)of hypoxia exposure,which can be inhibited by HIF-la inhibitor(p<0.001).Also the HIF-la inhibitor showed no obvious effect on BMDMs viability under either hypoxia condition or normoxia condition.Conclusion:Hypoxia can induce HIF-la expression in BMDMs.It also can promote the secretion of M1-type cytokine TNF-? and the differentiation into M1-type phenotype.In the meantime,hypoxia can inhibit the secretion of M2-type cytokine IL-10 and the differentiation to M2-type macrophages.Part ? The transformation of VSMC into HEV induced by hypoxia cultured macrophagesBackground:Tertiary lymphoid structure(TLS)is ectopic lymphoid tissue with similar structure and function of secondary lymphoid organs(SLO,such as spleen and lymph nodes).TLS has been proved to exist in various solid tumors(such as NSCLC,colon cancer,melanoma,etc.).The prognosis of patients with TLS in better than that of patients without TLS.The prognosis of patients is better in those with a large number of TLS.High endothelial venules(HEVs)is the most important component structure in TLS and is the main portal for lymphocytes to enter tumor tissue.However,the origin and occurrence of HEV in TLS are still unclear.Vasular smooth muscle cells(VSMC)have been proved to simulate lymphoid tissue organizer(LTo)in secondary lymphoid organs,which can be transformed into HEV under the induction of lymphotoxin(LT)or tumor necrosis factor-?(TNF-?)secreted by lymphoid tissue-induced cells(LTi).HEV can secrete a large number of chemokines such as CCL19,CXCL13 and CCL21,to recruit T and B lymphocytes to aggregate and promote TLS formation.Here we selected VSMC as LTo mimicing cells,and incubated with the supernatant of the hypoxic BMDMs.We detected the secretion of cytokines CCL19 and CXCL13 by VSMC,so as to prove whether BMDMs can induce VSMC to differentiate into HEV under hypoxia.Objective:To study the induction of VSMC differentiating into HEV by BMDMs exposed with hypoxia.Methods:ELISA method was used to detect the effect of BMDMs,which were under normal oxygen or hypoxia culture conditions,on the secretion of CCL 19 and CXCL13 by VSMC.Results:BMDMs rarely secrete CXCL13,but can elevate the secretion of CCL 19 after hypoxia exposure.BMDMs could promote VSMC to secrete CCL19(p<0.01)and CXCL13(p<0.0001)under hypoxia,which can be inhibited by HIF-la inhibitor(p<0.05).Conclusion:Hypoxia exposed BMDMs can promote VSMC to secrete CCL19 and CXCL13 to gain the function of HEV.HIF-1? may participate in this induction process.Part ? The in vivo study of macrophages promoting HEV formation in melanomaBackground:HEV is an important portal for lymphocytes to enter tumor tissue.Studies have shown that melanoma patients with more HEV and TIL in their tumor tissue have better prognosis.Therefore,inducing HEV production in melanoma is the key factor to increase the number of TIL and improve the prognosis of the disease.In the first two parts of our in vitro experiments,we have proved that BMDMs after hypoxia have similar LTi effect.That is,they can secrete TNF-? in large quantities and induce VSMC to differentiate into HEV.We further investigate the promoting effect of macrophages on HEV production in melanoma in vivo.Objective:To study the role of macrophages in promoting the formation of HEV in melanoma.To further explore the influence of tumor hypoxia microenvironment on macrophages and to study the effect of this process during HEV production in melanoma.Methods:Flow cytometry analysis was performed to detect and compare the CD31+PNAd+HEV quantity,TIL and lymphocyte subsets in tumor of mice with or without macrophage depletion.We also studied the changes of HEV quantity,CD45+TIL and lymphocyte subsets in tumor mice injected with BMDMs with hypoxia exposure or not in different periods by using flow cytometry analysis.Immunohistochemical staining method was applied to analyze the peripheral node addressin(PNAd,a characteristic surface marker of HEV)as well as degree of hypoxia in murine lymph nodes and tumors.Results:Compared with PBS control group,the CD31+PNAd+HEV proportion(p<0.0001)was significantly reduces in the tumor of macrophage depleted mice.The CD45+TIL in tumor was reduced(p<0.0001)as well as the proportion of CD3+B220T cells(p<0.001).PNAd positive cells were hardly observed in tumor after macrophage depletion.PNAd positive cells can be observed in none macrophage depletion PBS control group.Compared with tumor injected BMDMs under normal oxygen culture,the proportion of HEV(p<0.05)and total CD45+lymphocytes(p<0.05),especially the proportion of CD3+B220-T lymphocytes(p<0.05)increased in tumor injected with BMDMs under hypoxia culture.At the same time,more PNAd positive cells with lumen-like structure can be observed in hypoxia BMDMs injected group by immunohistochemical staining.In addition,the hypoxia probe combined with immunohistochemical staining showed that the degree of hypoxia in the tumor core was higher than that around the tumor.The degree of hypoxia in local draining lymph nodes of the tumor was higher than that in non-draining lymph nodes.And with the growth of tumor,the degree of hypoxia was further aggravated.Conclusion:In vivo study showed that macrophage depletion could lead to a sharp decrease in the number of HEV and TIL in melanoma tumor-bearing mice.Macrophage can significantly promote HEV production in melanoma through hypoxia,thus increasing the infiltration of lymphocytes in melanoma.Part ? The relative research on macrophage targeting by nanoparticlesBackground:Innate immunity mediated by macrophages,plays an important role in anti-tumor immune response.Due to the diversity of phenotype and function of macrophage,reprogramming M2-like macrophages with tumor-promoting function into M1-like macrophages with tumor-inhibiting function has important research value in tumor therapy.However,traditional drugs and drug delivery system are difficult to target macrophages specifically.Therefore,it is of great importance to design and synthesize a kind of nano-carrier that can target macrophage in realizing the functional transformation of macrophages.As professional phagocytes,macrophages specifically recognize apoptotic cells expressing phosphatidyl serine(PS)through their surface phagocytic related receptors(such as Mer and Tim-4,etc.).Due to their biological inertness,low cytotoxicity and unique photoacoustic imaging function,nanoparticles based on gold nanostructures(such as gold nanocages)are widely used in biomedical research.In addition,the gold nanocages also have hollow and thin-walled porous structures to load drugs and facilitate drug release.Therefore,they have wide application value in tumor imaging and targeting therapy.According to these,the drug-loaded gold nanocage modified by PS on the surface can specifically target macrophages and help to regulate their phenotype and function.Objective:To evaluate the targeting,specificity and high efficiency of nano-delivery system targeting macrophages.To study its effect on other immune cells in vivo,and to observe its imaging effect in vivo.Methods:Flow cytometry analysis was applied to detect the expression of phagocytosis-related receptors(mainly PS receptors)on the surface of macrophages.The phagocytosis of nanocarrier by macrophages after PS receptors blocking was tested by using flow cytometry analysis.We tested the change of surface activated molecules on T,B and DC cells after co-incubation with the nanocarrier by flow cytometry analysis.We detected the signals of nanocarrier in liver,spleen and kidney after tail vein injection by using photoacoustic imaging system.Results:The PS receptor Tim-4 and Mer are both expressed on the surface of macrophages.Their expression abundance is about 16.8%in former and around 80%in latter.Blocking either Tim-4 or Mer could impair the phagocytosis of nanocarrier by BMDM(p<0.0001).The targeted delivery carrier has no obvious influence on the expression of CD86 and MHCII molecules on DC and the expression of CD43,CD69 as well as CD25 on both T cells and B cells.Compare with none target nanoparticle,the photoacoustic signal of the target delivery system is much more obvious in liver,spleen and kidney after 12 h of tail vein injection.Conclusion:The PS modified nanoparticles designed and synthesized by us phagocytosed by macrophages mainly through PS receptors in the surface of macrophages.It realizing specific targeting delivery to macrophages.Meanwhile,the nanoparticles have good biological inertness and can be used for in vivo tracing.