The Effects Of Poly(I:C)in The Interactions Between Cervical Cancer Cells And Tumor-infiltrated Macrophages And On Angiogenesis In Cervical Cancer

Cervical cancer is the fourth most common cancer among malignant tumors in women.Cervical cancer is thought to be a poor consrquence of long term high risk human papillomavirus infection.About 10%of infected patients develop precancerous lesions that later develop into cervical cancer.The prevalence of human papillomavirus vaccine and cervical cancer screening has effectively reduced the incidence of cervical lesions.But unlike some types of cancer data,cervical cancer survival rates are not ideal,especially in advanced patients,where concurrent chemoradiotherapy is the standard treatment of choice.The 5-year overall survival rate is less than 70%,and the disease-free survival rate was 58%.A therapeutic vaccine,however,would likely be a promising approach in the treatment of cervical cancer by modulating the host immune system in vivo and thus provoking an anti-tumour response.Poly(I:C)is a synthetic analog of dsRNA and one of the most widely studied adjuvants for anti-tumor vaccines.It can activate innate immunity of cells and regulate adaptive immunity through both intracellular and extracellular ways.It cause a series of immune inflammatory factors to release,and play the role of tumor inhibition.Poly(I:C)activates TLR3,Melanoma differentiation-associated Antigen 5(MDA5)and retinoic acid-inducible gene I(RIG-1)in different cells,and has a different effect.It mainly includes inhibiting the proliferation of tumor cells,inducing apoptosis and promoting necrosis.The tumor microenvironment is composed of tumor cells,fibroblasts,immune cells and non-cellular components(including ECM,cytokines and hormones).The infiltration of immune cells is one of the markers of tumor microenvironment.Various types of immune cells are found localized in the tumor,the most common of which is the macrophage population.Macrophages belong to innate immune system,whose main function is to recognize,phagocytose antigen and present it to t cells,which can regulate the activity of acquired immune response and be the bridge between innate immunity and acquired immunity.Cervical cancer is a kind of immune sensitive tumor.There are a lot of immune cells in tumor microenvironment.The role and mechanism of poly(I:C)immunotherapy in cervical cancer are still controversial and need to be further explored.Angiogenesis is the key factor of tumor progression and metastasis.Inhibition of Angiogenesis can effectively reduce the metastasis and recurrence of tumor.It is the hotspot of tumor targeting therapy.Previous studies have found that the microvessel density in cervical cancer is significantly higher than that in normal tissues,and has no significant correlation with lymph node metastasis,peripheral infiltration and infiltration depth.And the higher the microvessel density,the higher the recurrence rate.Poly(I:C)was recently reported to reduce the volume and vascular density of uterine Leiomyoma in nude mice.There is no detailed report on the effect of Poly(I:C)on Angiogenesis in cervical cancer.Therefore,we designed the following studies to investigate the effect and mechanism of poly(I:C)on the interaction between cervical cancer cells and tumor-infiltrating macrophages,and the effect of Poly(I:C)on Angiogenesis in cervical cancer.Part ? Effects of poly(I:C)on cervical cancer cells and tumor-associated macrophage recruitmentObjectiveImmune cell infiltration is one of the markers of the tumor microenvironment,including various lymphocytes,macrophages,and dendritic cells.Macrophages are the most common cell group among tumor infiltrating immune cells and belong to the innate immune system.Poly(I:C)as an effective adjuvant can enhance the specific antitumor immune response induced by polypeptide vaccine,but the mechanism of its action is unknown.In this study,we analyzed the effect of Poly(I:C)on the recruitment of cervical cancer cells and tumor-infiltrating macrophages by Transwell,in order to provide a new theoretical basis for the application of poly(I:C)in the treatment of cervical cancer,the changes of macrophage infiltration after poly(I:C)injection were observed.Methods1.To investigate whether poly(I:C)treatment affects the recruitment of macrophages by cervical cancer cells,we used Transwell chambers to establish an in vitro cell migration model.THP-1-derived macrophages were placed in the upper compartment of the chamber,and the lower chamber was filled with cervical cancer cell CM with/without poly(I:C)action.The number of migrated THP-1-derived macrophages was observed after 24 hours.2.A nude mouse model of cervical carcinoma was established and divided into two groups randomly.PBS and Poly(I:C)were injected into the tumor.The changes of macrophages were observed after dosing 5 times.Results1.Cervical cancer cell CM with poly(I:C)function significantly promoted the migration of THP-l-derived macrophages2.The number of F4/80 in the transplanted tumors of Poly(I:C)group was significantly higher than that of PBS group.ConclusionIn Vitro and in Vivo Poly(I:C)stimulation promotes macrophage recruitment in cervical cancer cellsPart ? The effects of Poly(I:C)in the interactions between cervical cancers and tumor-infiltrated macrophagesObjectivePoly(I:C)triggers the production and the expression of antigens in natural immune cells,including macrophages and dendritic cells,leading to antitumor adaptive immune system.In addition to its effects on immune cells,Poly(I:C)can also induce apoptosis through endogenous and exogenous apoptotic pathways.And these effects can not be separated from the tumor microenvironment,The effect of Poly(I:C)on the tumor microenvironment and the interaction between tumor infiltrating macrophages is almost 10 times that of dendritic cell,however,it has not been fully explored-This study will analyze the effect of poly(I:C)on cervical cancer cells and Tumor-associated macrophage secretion spectrum,and will further validate it in cervical cancer xenografts in nude mice.Methods1.Suspension array and RT-PCR were used to detect the effects of poly(I:C)on the secretion of proinflammatory and anti-inflammatory factors in cervical cancer cells.2.The regulation of Poly(I:C)on the expression of IL-6 in cervical carcinoma was detected by Elisa.3.3.The effect of Poly(I:C)on the expression of cytokines in cervical carcinoma by RT-PCR and Elisa.4.4.To further investigate whether IL-6 is involved in the secretion regulation of cervical cancer macrophages.First,the expression of IL-6 was inhibited by transfecting HeLa cells with control gene or IL-6 siRNA.36 H after transfection,25 g/mL poly(I:C)was added,and the conditioned medium(CM)was collected after 12 h culture.Then CM was added during THP-1-induced macrophage differentiation.The transcription and secretion of cytokines were measured after fully differentiated into THP-1-derived macrophages.5.To further investigate whether IL-6 is involved in the recruitment of macrophages by cervical cancer cells,we established an in vitro cell migration model using transwell cells.Thp-1-derived macrophages were placed in the upper part of the Lumen,and the lower chamber was filled with 50%of the HeLaCM treated/untreated by poly(I:C)after IL-6 knock-out.The number of migrating macrophages was observed after 24 hours in the control group.6.Detection of Poly(I:C)-related molecular pathways in cervical cancer cells by western and Elisa methods.7.The effects of poly(I:C)on the secretion of cervical cancer cytokines were examined in nude mice with cervical cancer.Results1.RT-PCR results showed that IL-6 mRNA transcription was significantly up-regulated after poly(I:C)treatment in both two cervical cell lines.Results showed that the expression profiles of cytokines detected in the two cervical cancer cell lines were consistent and IL-2,IL-4,IL-6,IL-8,IFN-?,MCP-1 and TNF-? were all expressed.The expression level of IL-6 was considered to be high(approximately 200 pg/mL),and the level of IL-1?,IL-10 and IL-12 in conditioned medium was below the minimum detection limit.After poly(I:C)treatment,IL-6 expression levels were significantly up-regulated in both cell lines,and HeLa cells had higher IL-6 expression than CaSki cells.2.poly(I:C)dose dependently promoted IL-6 expression in HeLa and CaSki cells,with the most significant effect at 25?g/mL.The up-regulation of IL-6 secretion was found after 2 hours of poly(I:C)treatment in both two cervical cancer cell lines.In HeLa cells,the level of IL-6 in conditioned medium became relatively stable after 10 hours of poly(I:C)treatment.The secretion of IL-6 in CaSki cells was continuously elevated until 12 hours of poly(I:C)treatment.3.To investigate the effect of poly(I:C)-treated cervical cancer cells on macrophage cytokine expression profiles,we collected conditioned medium(CM)with or without poly(I:C)treatment.The CM was added during the inducing differentiation of the THP-1 monocyte line into macrophages by PM A.The treatment procedure was described in the Methods section above.HeLa cell CM without poly(I:C)inhibited the expression of pro-inflammatory cytokines IL-1? and IL-6 in THP-1-derived macrophages,while the transcription and secretion levels of IL-10 and CCL22 were significantly higher.In contrast,12 hours of poly(I:C)treatment almost completely reversed the regulatory effect of HeLa CM on the secretion profile of THP-1-derived macrophages.4.We further explored whether IL-6 is involved in the regulation of macrophage secretion by cervical cancer.First,the HeLa cell line was transfected with control or IL-6 siRNA to inhibit the expression of IL-6.After transfection for 36 hours,poly(I:C)(25 ?g/mL)was added,and the CM was collected after culturing for 12 hours.The CM was then added during the process in which THP-1 induced differentiation into macrophages.The level of transcription and secretion of the cytokines were detected after complete differentiation into THP-l-derived macrophages.The results dispiay that cervical cancer cell CM transfected with control siRNA or IL-6 siRNA showed no significant difference in regulation of cytokine expression of THP-1-derived macrophages when there was no poly(I:C)effect.IL-6 silencing in cervical cancer cells significantly inhibited the transcription and secretion of pro-inflammatory cytokines IL-1? and IL-6 by THP-1-derived macrophages,but had no significant effect on the expression of IL-10 and CCL22.5.We further explored the role of IL-6 in the promotion of macrophage recruitment by cervical cancer cells in poly(I:C).The recruitment of HeLa cell CM transfected with IL-6 siRNA to THP-1-derived macrophages was significantly inhibited compared with control siRNA.6.In this study,we found that the level of phosphorylated NF-?B in HeLa cells was up-regulated 5 minutes after poly(I:C)treatment,reaching a peak at 30 minutes,suggesting that the signalling pathway is activated.To investigate whether the activated NF-?B signalling pathway is involved in poly(I:C)-regulated IL-6 expression,we selected the common NF-?B signalling pathway inhibitor PDTC(4?g/mL)to pre-treat HeLa cells for 1 hour,then changed the medium and added poly(I:C).ELISA results showed that PDTC significantly reversed the effect of poly(I:C)on IL-6 secretion in HeLa cells.7.In vivo model,IL-6 secretion of nude mice in poly(I:C)group was significantly higher than that of PBS Control Group.ConclutionPoly(I:C)stimulated the expression of IL-1 and IL-6 in THP-1-derived macrophages and inhibited the expression of IL-10 and CCL22 in THP-1-derived macrophages,Then affected the immune status of the whole tumor microenvironment.Part ? The effect of Poly(I:C)on angiogenesis in cervical carcinomaObjectiveTumor growth,invasion and metastasis depend on the formation of new blood vessels,which not only provide sufficient nutrients for tumor cells,but also are necessary for tumor cells to invade and metastasize.Early invasion and metastasis of cervical cancer is the primary factor of poor prognosis,which has been troubling the clinic.Targeted anti-angiogenic drugs are widely used in clinical practice because of their small side effects,low drug resistance and wide action,Poly(I:C)has been studied as an adjuvant of anti-tumor drugs for decades,but its application in tumor angiogenesis is still lacking.In this study,we established a cervical cancer xenograft model,after poly(I:C)treatment,The effect of Poly(I:C)on Angiogenesis of cervical carcinoma was analyzed by immunohistochemistry and contrast-enhanced ultrasound.MethodsA total of 12 nude mice models of cervical cancer were established and randomly divided into two groups.10 days after injection of tumor cells,when the tumor size was about 0.6 cm,PBS and Poly(I:C)(include 50?g per head)were injected into the tumor respectively.Once every five days,five times in total.After administration of the drug,the general condition of the two groups of nude mice was observed every day and the tumor size of the nude mice was measured every three days.Contrast-enhanced ultrasound was performed four days after the last administration,and then Obtain peak intensity(PI),and offline analysis was performed using the Quantitative analysis software.The nude mice were then killed and the tumor was removed intact.The expression of CD31 and CD34 was detected by Immunohistochemistry,and the microvessel density(MVD)was calculated.Results1.General situation of nude mice:As the time of drug action prolonged,the xenografts grew up gradually in the PBS group,and the Diet,activity and spirit of the nude mice were weakened.There was no significant change in Poly(I:C)Group compared with the Control Group.2.Size changes of transplanted tumor in nude mice:After administration of the drug,we found that there was no significant change in the growth rate of the xenografts in the PBS group,but the growth rate of the xenografts in the poly(I:C)group decreased significantly.The growth curve was drawn according to the xenograft size measured after administration in PBS Group and Poly(I:C)group.At the end of the experiment,the tumor volume of the experimental group was significantly smaller than that of the PBS Control Group,and there was a significant difference between the two groups(p<0.05).3.CD31and CD34 immunohistochemical results of transplanted tumor in nude mice:Using CD31 and CD34 as a marker for Endothelium,these substances are stained claybank in the cytoplasm of endothelium cells.The average value of CD31 and CD34 MVD in PBS group and poly(I:C)group was counted.The MVD value of the two groups was significantly lower than that of the experimental group(p<0.05)4..Contrast-enhanced ultrasound software Quantitative analysis the vasculature of the tumor:Contrast-enhanced ultrasound showed that the xenografts in the PBS Group showed ring-like enhancement around the implanted tumors,but not in the poly(I:C)Group.The contrast-enhanced ultrasound intensity curves were drawn according to the changes of contrast-enhanced ultrasound intensity with time.Contrast-enhanced ultrasound showed that the peak intensity of contrast-enhanced ultrasound in PBS group and poly(I:C)group were-23.31±0.74 dB and-52.89±0.41 dB respectively.The peak intensity in PBS group was significantly higher than that in Poly(I:C)Group(p<0.05).ConclutionPoly(I:C)can effectively inhibit the growth and angiogenesis of cervical cancer in nude mice..Poly(I:C)is expected to become a new target for cervical cancer therapy.