| [Background]Cervical cancer(CC)is one of the most prevalent malignancies among women worldwide,with a incidence of about 15%and is the leading cause of cancer-related mortality for women in the developing countries.Although the combination of HPV vaccine,screening and surgery has been effective in improving the prognosis of cervical cancer patients,it is difficult to achieve a complete cure of metastasis and recurrence,which is also a major risk factor for cancer-related death.Lymph node metastasis(LNM)is the main route of the metastasis in cervical cancer and an independent indicator of treatment and poor prognosis.Therefore,it is of great scientific significance to strengthen the basic research on the mechanism of LNM,and carry out early prediction,diagnosis,targeted prevention and treatment and prognosis evaluation of cervical cancer metastasis,so as to improve the cure rate of cervical cancer and reduce the mortality.Tumor microenvironment(TME)is a microecosystem on which tumor cells live and develop,and its unique biological characteristics play an important role in tumor progression and metastasis.Tumor-associated macrophages(TAMs)are the main components of TME stromal cells in most solid tumors,and are "accomplices" of tumor progression,helping to produce "soil" that promote tumor metastasis.However,its regulatory mechanism is still unclear.Our previous results showed that TAMs promote tumor invasion and metastasis by inducing EMT changes in cervical cancer cells and upregulation of MMP9 expression(Cancer Prevention Research.2019,46(03):212-217).Patients with higher levels of TAMs invasion had higher rates of metastasis and shorter survival(J Cancer.2017.8(18):3868-3875).Local hypoxia is the basic feature of TME,and it is a common phenomenon in solid tumors.By establishing an in vitro hypoxic TME model of cervical cancer,we confirmed that hypoxic TME is a decisive factor in inducing the transformation of macrophage phenotype and function(Mol Carcinog.2019.58(3):388-397).High expression of ZEB1 in tumor cells was induced by local hypoxia in cervical cancer tissues,and the expression of CCL8 was up-regulated by transcription.The recruitment of TAMs in hypoxic TME was promoted by CCL8-CCR2 signal axis(Cell Death Dis.2019.10(7):508).The interaction between hypoxic TME and TAMs not only promotes the improvement of tumor cell proliferation and invasion,but also induces the activation of other stromal cells,and the formation of local blood vessels and lymphatics breaks the homeostasis to adapt to the survival and metastasis of tumor cells.LNM was a complicated process affected by many factors,and lymphatic vessels are the only and key way for tumor cells to metastasize to the lymph nodes.Recent studies suggest that lymphatic vessels are heterogeneous in hypoxic TME and contain different subsets with distinct phenotypes and functions,which hinders their application in the diagnosis and targeted therapy.Studies have identified lymphatic vessel subsets secreting different cytokine profiles in a variety of cancers.Therefore,It is of great clinical significance to accurately target the metastasis-related subsets of lymphatic vessels in cervical cancer,to understand their role and mechanism in LNM and to intervene them.However,studies on the function of lymphatic vessels associated with metastasis in cervical cancer are still lacking.As one of the abundant cells in hypoxic TME,the effect of TAMs on the function of lymphatic vessels has not been explored.The main objective oft his study is to explore a new lymphatic vessel pattern surrounded by TAMs in the hypoxic area of cervical cancer tissue,which can promote LNM by inducing recruitment of tumor cells and TAMs.At the same time,the specific molecular mechanism of this new metastasis-associated lymphatic pattern was discussed.This provides a theoretical basis for us to understand the LNM of cervical cancer,and also provides some help for us to find the potential therapeutic target genes of cervical cancer.Part Ⅰ.LVEM represents a novel lymphatic pattern that is correlated with lymphatic metastasis of cervical cancer[Objective]To investigate the effect of new lymphatic vessel pattern on lymph node metastasis in the hypoxic TME of cervical cancer.[Materials and Methods]The expression levels of CAIX,LYVE-1 and CD 163 in 75 cervical cancer tissues(35 with LNM and 40 without LNM)were detected by polychromatic fluorescence staining,and the average fluorescence intensity was calculated and the correlation was analyzed.[Results]1.Large amounts of TAMs infiltrate around lymphatic vessels in the hypoxic areas of cervical cancer,while TAMs are scattered around lymphatic vessels in the normoxic areas.We named this new TAMs-enclosed lymphatic vessel pattern in hypoxic TME as LVEM(lymphatic vessels encapsulated by macrophages).2.The expression of CAIX and LVEM in cervical cancer tissues with LNM is higher than that without LNM,and the difference is statistically significant(P<0.05).3.There was a positive correlation between CAIX and LVEM expression in cervical cancer tissues with LNM(r=0.5240,P=0.0023),but there is no significant correlation in cervical cancer tissues without LNM(r=0.1335,P=0.1804).[Conclusion]There is a new lymphatic vessel pattern surrounded by TAMs in the hypoxic area of cervical cancer tissue,which is positively correlated with lymph node metastasis.PartⅡ.Hypoxic TAMs promote LVEM formation and lymphatic metastasis[Objective]To investigate the role of hypoxic TAMs in LVEM formation and LNM.[Materials and Methods]1.Supernatants of macrophages were collected under different treatment conditions,namely Macrophage-Normoxia(MO-N),Macrophage-Hypoxia(M0H),TAMs-Normoxia(TAMs-N),and TAMs-Hypoxia(TAMs-H).2.Lymphatic endothelial cells were treated with the above-mentioned conditional macrophage supernatant,and the effects of hypoxic TAMs on the function of lymphatic endothelial cells were observed using the Transwell migration experiment.3.We constructed an animal model of popliteal lymph node metastasis in mice and observed the effects of hypoxic TAMs supernatant on LVEM formation and LNM.4.We construct a macrophage depletion model in vivo and observe the effects of macrophage depletion on LVEM formation and LNM.[Results]1.Hypoxic TAMs stimulated lymphatic endothelial cells can significantly promote the recruitment of macrophages and tumor cells,while the effects of other groups are not obvious.2.The supernatant of hypoxic TAMs can significantly promote the formation of LVEM in vivo,but the supernatant of macrophages treated with other conditions cannot induce the formation of LVEM.LVEM expression was statistically different between the groups(P<0.05).3.The popliteal lymph nodes of the mice in the hypoxic TAMs supernatant group were swollen,the CK7 expression was strongly positive,and obvious metastasis appeared,and the metastasis rate was statistically different between the groups(P<0.05).4.Macrophage in vivo depletion model mice can significantly inhibit the formation of intratumoral LVEM,and the expression of LVEM between groups is statistically significant(P<0.05).5.Mouse popliteal lymph node metastasis was significantly inhibited in macrophage in vivo depletion model,and the metastasis rate was lower than that of the control group(P<0.05).[Conclusion]1.The supernatant of hypoxic TAMs can promote the activation of lymphatic endothelial cells and recruit more macrophages and tumor cells.2.Hypoxic TAMs supernatant can promote the formation of intratumoral LVEM and LNM.Part Ⅲ.IL-10 derived from hypoxic TAMs is required to maintain LVEM pattern[Objective]Screen the differentially expressed cytokines secreted by hypoxic and normoxic TAMs and explore their functions.[Materials andMethods]1.RayBio’s Cytokines array GSM-CAA-4000(covering 200 cytokines),was used to evaluate the cytokines secreted by TAMs.2.qPCR and ELISA further validate the differential cytokines obtained from Cytokines array:IL-10,TREM-1,Decorin,Typtaseε,TGF-β1.3.The expression levels of CAIX,CD163 and IL-10 in 75 cervical cancer tissues(35 with LNM and 40 without LNM)were detected by polychromatic fluorescence staining,and the average fluorescence intensity was calculated and the correlation was analyzed.4.The supernatant of lymphatic endothelial cells treated with human recombinant cytokine IL-10 was collected,and the effect of IL-10 on the chemotactic function of lymphatic endothelial cells was observed using the Transwell migration experiment.5.We constructed an animal model of popliteal lymph node metastasis in mice and observed the effects of IL-10 on tumor LVEM formation and LNM.6.Matrigel plug experiments in mice to observe the effect of IL-10 on LVEM formation in vivo.[Results]1.According to the analysis of cytokine microarray,some cytokines were significantly increased in hypoxic TAMs,including IL-10,TREM-1,Decorin,Typtaseε,TGF-β1.2.The above differential factors were screened by qPCR and further verified by ELISA.The results showed that compared with macrophages and normoxic TAMs,the expression of IL-10 in hypoxic TAMs was most significantly upregulated.3.Trans well results show that lymphatic endothelial cells treated with human recombinant cytokine IL-10 enhance recruitment of macrophages and tumor cells.4.IL-10 is mainly expressed in the hypoxic area of cervical cancer and colocalized with hypoxic TAMs.Correlation analysis of CAIX,CD 163 and IL-10 found that IL-10 was positively correlated with the expression of CD163+TAMs in CAIX positive hypoxic regions of cervical cancer(r=0.5178,P<0.0001),but there was no correlation between them in normoxic tissues(r=0.1253,P=0.0550).5.LVEM expression was significantly increased in the IL-10 treated group compared with the control group(P<0.05).6.In the mouse model of popliteal lymph node metastasis treated with IL-10,the incidence of lymph node metastasis was significantly increased(P<0.05).[Conclusion]1.IL-10 is the highly expressed cytokine secreted by hypoxia TAMs.2.In cervical cancer tissues,IL-10 is mainly distributed in the area where hypoxic TAMs are located.The higher the percentage of total TAMs in IL-10+TAMs,the higher the probability of tumor metastasis.3.IL-10 can induce the functional activation of lymphocyte endothelial cells,promote the formation of LVEM and lymph node metastasis.Part Ⅳ.CCL1 mediates the migration of TAMs towards LECs in the formation of LVEM[Objective]Further explore the molecular mechanism of IL-10 secretion induced by hypoxic TAMs to activate lymphatic endothelial cells.[Materials and Methods]1.We screened a series of inflammation-related chemokines by qPCR and verified the secreted amount in the supernatant by ELISA.2.The chemotaxis of CCL1 on tumor cells and macrophages were observed by Transwell assay.3.We added human recombinant cytokine CCL1 into the culture of tumor cells,labeled tumor cytoskeleton with phalloidine,and observed the morphological changes of tumor cells at different time points by immunofluorescence(Omin,15min,30min,45min,60min).4.We constructed an animal model of popliteal lymph node metastasis in mice and observed the effects of CCL1 on tumor LVEM formation and LNM.[Results]1.IL-10-stimulated lymphocyte endothelial cells showed high expression of CCL1,CCL2,CCL8,CCL19,CCL21,CXCL12,among which CCL1 expression was the most significantly up-regulated.2.CCL1 can promote the recruitment of tumor cells and macrophages in a concentration-dependent manner.Interference with the expression of target cell receptor CCR8 decreased the recruitment capacity.3.CCL1 can induce tumor cell morphological changes and promote tumor cell migration.This chemotactic migration can be blocked by CCR8 on target cells.4.Mice treated with CCL1 were more likely to have popliteal lymph node metastasis than the control group(P<0.05).[Conclusion]1.IL-10 promotes the high secretion of CCL1 by lymphatic endothelial cells.2.CCL1 promotes the recruitment of tumor cells and macrophages.3.The CCL1-CCR8 signal axis promotes lymph node metastasis in cervical cancer.Part Ⅴ.Splhigh LECs are fundamental to LVEM formation and lymphatic metastasis[Objective]To investigate the role of IL-10 in up-regulation of CCL1 expression and in promoting the intermediate signaling pathway formed by LVEM.[Materials and Methods]1.The upstream transcription factors and possible binding sites of CCL1 were predicted online by JASPAR.2.The transcriptional regulation relationship between Sp1 and CCL1 was detected by double luciferase reporter gene.3.The secretion level of CCL1 in lymphatic endothelial cell supernatant was verified by ELISA.4.The expression levels of LYVE-1,CD 163,Spl and CCL1 in 75 cervical cancer tissues(35 with LNM and 40 without LNM)were detected by polychromatic fluorescence staining,and the average fluorescence intensity was calculated and the correlation was analyzed.5.Lentiviruses were used to construct silent or overexpressed lymphatic endothelial cell lines.6.Culture supernatants from the above constructed lymphatic endothelial cell lines that were silenced or overexpressed with Spl were collected and transwell chemotaxis was performed to observe their recruitment capacity for macrophages and tumor cells.7.Matrigel plug experiments in mice to observe the effect of Sp1 expression on lymphatic endothelial cells on LVEM formation in vivo.8.We construct an animal model of mouse popliteal lymph node metastasis and observe the effect of Sp1 expression on lymphatic endothelial cells on LVEM formation and lymph node metastasis in vivo.9.Western blot was used to detect the downstream signaling pathway of lymphatic endothelial cell s affected by IL-10.10.Transwell experiment was used to observe the effect of blocking IL-10R and STAT3 signaling pathways on the chemotaxis of tumor cells and macrophages.[Results]1.JASPAR website predicts that there are 242 upstream transcription factors with a score greater than 90 in the CCL1 upstream transcription factor.Among them,there were 57 transcription factors related to metastasis promotion,25 transcription factors related to metastasis inhibition and 160 transcription factors related to other pathways.Sp1 has the highest fraction of transcription factors associated with transfer promotion.2.The predicted target for CCL1 binding to Spl is TCCCCTCCCCC sequence.3.The results of the double luciferase reporter gene showed that the cotransfection of Sp1 and CCL1 significantly increased the fluorescence intensity of the vector,regardless of whether the tool cells were 293T or HDLECs,while there was no significant difference between the NC group and the blank group(P<0.05).4.ELISA results showed that the secretion of CCL1 in the supernatant was significantly down-regulated after Sp1 was knocked down in HDLECs.However,,the secretion of CCL1 in the supernatant was significantly up-regulated after overexpression of Spl in HDLECs.5.In cervical cancer tissues with LNM,LYVE-1+lymphatic vessels highly express Sp1 and peripheral CCL1 are highly expressed.In cervical cancer tissues without LNM,LYVE-1+lymphatic vessels do not express Spl,and peripheral CCL1 expression is also lower.6.Sp1+LYVE-1+lymphatic vessels were positively correlated with CCL1 expression in cervical cancer tissues with LNM(r=0.5078,P=0.0029),while no correlation was found in cervical cancer tissues without LNM(r=0.2168,P=0.0803).7.In cervical cancer tissues with LNM,LVEM expression increased and was positively correlated with Spl expression(r=0.5846,P=0.0009).However,in cervical cancer tissues without LNM,no LVEM was found,and there was no correlation with Spl expression(r=0.1018,P=0.2465).8.Transwell results show that knockdown of Spl expression on lymphatic endothelial cells can reduce its chemotaxis to macrophages and tumor cells(P<0.05);overexpression of Sp1 expression on lymphatic endothelial cells can enhance its chemotaxis to macrophages and tumor cells(P<0.05).9.In vivo mouse matrigel plug experiments,the formation of LVEM in the gel plugs of the Spl overexpression group was clearly observed,while the formation of LVEM in the Sp1 silent group and the control group was suppressed(P<0.05).10.Mouse popliteal lymph node metastasis model:Sp1 overexpressing lymphatic endothelial cell line supernatant treated mice were more likely to develop popliteal lymph node metastasis than Spl silenced and control mice(P<0.05).11.Western blot results showed that the expression of Sp 1 and phosphorylated STAT3 increased in time and concentration dependence of IL-10.With STAT3 pathway inhibitor DPP blocking the expression of STAT3 on lymphatic endothelial cells,the expression of Sp1 was correspondingly reduced,and it was no longer affected by IL-10 stimulation.12.Transwell results showed that IL-10-stimulated lymphoendothelial cells promoted chemotaxis of tumor cells and macrophages,respectively.However,after blocking the expression of IL-10R,STAT3 signaling pathway and Spl on lymphocyte endothelial cells,the chemotaxis decreased,and the difference was statistically significant(P<0.05).[Conclusion]1.Sp1 is an upstream transcription factor of CCL1.2.Sp1 is a key marker for metastasis-associated lymphatic vessels of cervical cancer.3.High expression of Spl in lymphoendothelial cells promotes LVEM formation and LNM in cervical cancer.4.IL-10 up-regulates Spl expression by activating the STAT3 signaling pathway in lymphatic endothelial cells.Part VI Clinical relevance of IL-10-induced upregulation of Spl and CCL1[Objective]The immunofluorescence results of cervical cancer tissues were analyzed,and the relationship between IL-10,Spl and CCL1 expression and the clinicopathological parameters of cervical cancer was discussed.[Materials and Methods]1.The mean fluorescence intensity of IL-10,Sp1 and CCL1 in cervical cancer tissues was analyzed.2.The correlation of IL-10,Spl and CCL1 expression in cervical cancer tissues was analyzed.3.The relationship between il-10,Sp1 and CCL1 and the prognosis of cervical cancer was analyzed by TCGA database.4.The expression of CAIX and LVEM detected in cervical cancer tissues was evaluated,and the correlation between CAIX and LVEM and the clinicopathological parameters of cervical cancer was analyzed.[Results]1.IL-10,Sp1 and CCL1 expression were significantly increased in cervical cancer tissues with LNM(all P<0.05).2.In cervical cancer tissues,IL-10 was positively correlated with Spl expression(r=0.4257,P<0.0001),IL-10 was positively correlated with CCL1 expression(r=0.3527,P<0.0001),and Sp1 was positively correlated with CCL1 expression Correlation(r=0.4126,P<0.0001).3.TCGA results indicated that IL-10(P=0.0324),Spl(P=0.0259),and CCL1(P=0.0436)were positively correlated with poor prognosis of cervical cancer.4.In cervical cancer tissues,the high expression of CAIX and LVEM was positively correlated with cervical FIGO staging(P=0.038,P=0.011)and lymph node metastasis(P=0.011,P=0.008),respectively.The expression of CAIX was significantly correlated with vascular infiltration(P=0.002).There was no significant correlation between the expression level of CAIX and LVEM and age,pathological grade or paratactic infiltration(P>0.05).[Conclusion]1.High expression of IL-10,Spl and CCL1 in cervical cancer tissues indicates LNM and poor prognosis.2.Increased expression of LVEM in hypoxic microenvironment is associated with poor prognosis in patients with cervical cancer. |
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