| Lung cancer is a malignant tumour with fairly high morbidity and mortality rates.Approximately 15%-20%of all lung cancer patients are eventually diagnosed with small cell lung cancer(SCLC),which has the clinical characteristics of rapid tumour cell proliferation,early metastasis,high vascular density,easy recurrence and poor prognosis.Indeed,the 5-year survival rate of this cancer is less than 7%.Targeted drug and immunoassay point inhibitor therapies represent new advances in the field of lung cancer treatment.Whilst these methods have been applied in clinical practice,they are only effective for some specific non-small cell lung cancer(NSCLC)cases.In addition,the available treatment modes present a number of problems,such as drug resistance and ambiguous screening indicators.No data on an effective therapeutic approach for SCLC is yet available.Moreover,no significant progress in the treatment regimen of SCLC has been achieved over the last 30 years,and the overall survival of patients has not significantly improved.Angiogenesis is highly related to the growth,metastasis and prognosis of various solid tumours.Thus,designing and developing anti-angiogenic tumour therapeutic drugs is a key target in this area.Anti-angiogenesis therapy is considered amongst the most promising tumour therapeutic methods currently available.Vascular endothelial growth factor(VEGF)is a highly functional endothelial cell-specific mitogen that plays an important role in the complex process of angiogenesis.Several anti-angiogenic tumour therapeutic drugs target VEGF.However,most of these drugs are beneficial only for NSCLC,and their effectiveness for SCLC has not been clearly established in clinical trials.Unfortunately,the angiogenesis of SCLC is more extensive than that of NSCLC.Thus,explorations of new therapeutic approaches that can significantly improve clinical outcomes are urgently needed.The development of new therapeutic drugs for SCLC is effective and target angiogenesis remains the key direction of these explorations.sFlt-1,a soluble Flt-1 protein,is a transmembrane receptor of the tyrosine kinase family and the extramembrane component of VEGFR-1.It is a selective and powerful endogenous inhibitor of VEGF.The protein has high affinity similar to VEGF but cannot transmit information because it lacks cross-membrane and intracellular components,thus preventing the initiation of intracellular signals.Previous studies confirmed that sFlt-1 could function as an anti-tumour molecule by inhibiting various tumour angiogenic mechanisms.Exosomes are extracellular vesicles secreted by many cells into the bloodstream that contain a variety of information-related substances for signal transduction,material transport and remote targeted regulation.The mechanism of exosome action in NSCLC and other solid tumours and non-neoplastic diseases has been explored.In fact,exosomes are considered potential biomarkers,therapeutic targets and therapeutic drug carriers in tumour-related studies.Thus,these molecules may be exploited as a new approach for tumour therapy.Exosomes may be designed as vehicles to transfer functional biomolecules,such as proteins,lipids and nucleic acids(e.g.DNA,mRNA and miRNA),into target cells,thereby affecting their life processes,such as inflammation,apoptosis or angiogenesis.Given this context,establishment of research models of exosomes derived from SCLC and verification of their reliability are necessary to provide valid methods and model tools for further research.In the present work,we explore the effects of SCLC exosomes on the proliferation and migration of vascular endothelial cells and then propose a possible mechanism through which these exosomes influence the growth and metastasis of SCLC.Subsequently,we determine whether sFlt-1 exerts an anti-angiogenic effect on VEGFR,construct a model to verify the overexpression and silencing of SFLT-1 and assess whether exosomes may be used as an SFLT-1 carrier that could act on target cells with strong anti-angiogenic effects for SCLC treatment.These studies are intended to explore new anti-angiogenic treatment regimens for SCLC and improve current treatment outcomes for SCLC.Chapter One:Establishment and identification of an exosome model of small cell lung cancerObjectivePrevious studies on exosomes from lung cancer sources rarely considered the exosomes of SCLC.Thus,the mechanism of action of exosomes in the angiogenesis of this type of lung cancer and whether these exosomes could be an effective therapeutic approach remain incompletely understood.We first constructed a cell line model for exosomes of SCLC.Then,we isolated these exosomes and evaluated their morphology,function and molecular markers as a basis for subsequent studies.Materials and MethodsThe BEAS-2B,NCI-H69,DMS153 and SHP-77 cell lines were selected as research models,and an ExoQuick-TC kit(System Bioscience,Palo Alto,CA,USA)was used to isolate exosomes from the cell culture medium.Transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western blotting were used to describe the characteristics of cell-derived exosomes.1.The SCLC cell lines BeAS-2B,NCI-H69,DMS153 and SHP-77 were selected as research models.The cells were resuscitated,cultured and frozen in accordance with the instructions of their suppliers.RNA extraction experiments were carried out on cultured cell suspensions,and mRNA levels of sFlt-1 in these cell lines were detected by RT-qPCR.Finally,the expression level of sFlt-1 in several cell lines was evaluated.2.Exosomes were isolated from the medium using the ExoQuick-TC kit and characterised by TEM,NTA and Western blotting.Result1.The mRNA levels of sFlt-1 in several cultured cell lines were detected by RT-qPCR.The mRNA expression level of sFlt-1 in the human normal lung epithelial cell line(BEAS-2B)was significantly higher than that in the SCLC cell lines(DMS153,NCI-H69,SHP-77).The mRNA expression level of sFlt-1 in the NCI-H69 cell line was relatively low.(*p<0.05,figure1.1)2.The ExoQuick-TC kit could reliably isolate exosomes,and the exosome-specific markers of CD63,CD9 and CD81 were confirmed by Western blotting of NCI-H69-exo,DMS153-exo,SHP-77-exo and BEAS-2B-exo(figure1.2).3.TEM was used to observe the morphology of the obtained exosomes.NCI-H69-exo and BEAS-2B-exo showed typical membranous particles,membranous structures around vesicles,a cup-shaped appearance,and diameters between 30 and 150 nm(Figure 1.3A).4.NTA analysis confirmed the uniorm particle size distribution of the exosomes(30-150 nm),and the concentration was(1.2±1.5)× 1011 particles/ml(Figure 1.3B).These results confirm the successful isolation of the selected SCLC cell line and BEAS-2B exosomes.ConclusionssFlt-1 mRNA could be extracted from the BEAS-2B,NCI-H69,DMS153 and SHP-77 cell lines.The expression of sFlt-1 in normal lung epithelial cells was higher than that in the SCLC cell lines.Exosomes from the cultured cell lines were successfully isolated with the ExoQuick-TC kit and analysed by Western blotting.The morphological characteristics of NCI-H69-exo BEAS-2B-exo were further verified by TEM and NTA.The developed model could be used for the separation of exosomes from SCLC to explore new therapeutic approaches for this type of cancer.Chapter Two:Effects of exosomes on endothelial cell proliferation and migration in small cell lung cancerObjectiveOn the basis of the above exosome separation results,representative NCI-H69 and BEAS-2B exosomes were selected for subsequent studies.The exosomes were applied to human umbilical vein endothelial cells(HUVECs),and cell proliferation assay,plate transfer assay and in vivo matrix glue suppository tests were carried out to explore the effect of exosomes on the proliferation and migration of HUVECs.We also studied differences between these two exosomes in terms of formation of new blood vessels under in vivo conditions and the mechanism of action of SCLC exosomes and explore new therapeutic targets for SCLC.Materials and Methods1.HUVECs were digested and isolated using standard collagenase and preserved in endothelial cell culture medium(ScienCell,San Diego,CA,USA)supplemented with 5%foetal bovine serum(FBS),1%penicillin/streptomycin,and 1%endothelial cell growth additive.2.HUVECs was cultured with NCI-H69-exo or BEAS-2B-exo at different concentrations(25,50 or 100 μg/ml)for 48 h.Cells were treated with 1%bovine serum albumin(BSA)as a negative control.The proliferation of HUVECs was determined using Cell Counting Kit-8(CCK-8)reagent(Dojingdo Molecular Technology,Japan).3.An improved membrane system(Costar,Corning,NY,USA)with an aperture size of 8.0 μm was used to evaluate the migration capability of HUVECs.4.NCI-H69-exo and BEAS-2B-exo were separately mixed at low temperature with a low growth factor matrix gel(350 μl,BD Biosciences)and then subcutaneously injected into thymo-free nude mice(male,6 weeks old,BALB/C).The nude mice were sacrificed,and the matrix rubber plugs were harvested and fixed in 4%formaldehyde phosphate buffer for histopathological detection.Immunohistochemical analysis was performed on paraffin sections,and the optical density(OD)of CD31-positive cells was read to evaluate the extent of neovascularisation.Result1.NCI-H69-exo and BEAS-2B-exo were labelled with Dil dye and co-cultured with HUVECs.Exosome incorporation into the HUVECs was observed by fluorescence microscopy,and no difference in survival rate between the two groups was noted(Figure 2.1 A).2.Cell proliferation tests were performed using CCK-8 reagent.In the proliferation assay,HUVECs were co-cultured with NCI-H69-exo(25,50 or 100μg/ml),BEAS-2B-exo(25,50 or 100 μg/ml)or 1%BSA of the same quantities(negative control).After 48 h,HUVECs proliferation was analysed by the CCK-8 method.Compared with BEAS-2B-exo,NCI-H69-exo promoted HUVEC proliferation more significantly at 100 μg/ml(*p<0.05,Figure 2.1B).3.Transwell migration assay revealed that,compared with BEAS-2B-exo(100μg/ml),NCI-H69-exo increased the migration of HUVECs to a greater extent(*p<0.05,Figure 2.1C).4.Angiogenesis of nude mice was evaluated by the matrix gel plug method.The nude mice were sacrificed after 13 days,and the matrix rubber plugs were harvested.Vascular density was analysed histopathologically using paraffin sections.Compared with BEAS-2B-exo,NCI-H69-exo significantly increased the density of new blood vessels during gel embolisation(*p<0.05,Figure 2.1D).These data suggest that exosomes from SCLC cells induce angiogenesis primarily by enhancing the migration of HUVECs.ConclusionsCompared with BEAS-2B-exo,NCI-H69-exo increases the migration of HUVECs.NCI-H69-exo can increase the density of new blood vessels in colloidal suppositories in vivo.These data show that exosomes from SCLC cells induce and promote angiogenesis by enhancing the migration of HUVECs,thus providing a basis for the growth,migration and metastasis of tumour cells.Chapter Three:Soluble fms-like tyrosinekinase-1-enriched exosomes suppress small cell lung cancer growth by inhibiting endothelial cell migrationObjectiveThe expression of sFlt-1 in serum exosomes of clinical SCLC patients was first explored on the basis of the two experiments described above.The effects of sFlt-1 protein and NCI-H69 exosomes on HUVEC migration were verified by a Transwell migration experiment,and a cell model of sFlt-1 overexpression was constructed to explore whether enriched sFlt-1 exosomes could inhibit HUVECs migration.Knockdown of the expression of sFlt-1 was then performed to observe the above effect.In this chapter,we investigate whether sFlt-1-enriched exosomes and sFlt-1 have therapeutic effects on SCLC cell line xenografts in mices.Materials and Methods1.Serum samples were used to compare serum levels of SCLC and sFlt-1 in healthy subjects.Levels of sFlt-1 in exosomes of BEAS-2B and NCI-H69 were measured to conduct a preliminary study on sFlt-1.2.The effect of SFLT-1 protein and sFlt-1 in NCI-H69 exosomes on HUVEC migration was verified through co-culture.3.An sFlt-1 overexpression model was constructed by introducing lentivirus overloaded with the sFlt-1 gene to HEK293 cells.Exosomes enriched with sFlt-1 were obtained by overexpressing stable cell lines,and the effects of these exosomes on HUVECs were determined.4.The effects of knockdown of NCI-H69 sFlt-1 expression on endothelial cell apoptosis,proliferation and migration were investigated.5.An in vivo xenograft tumour mouse model was constructed,and the anti-SCLC effect of overexpressed exosomes in vivo and the relevant mechanism of this model were investigated.Result1.sFlt-1 expression in serum and different cell line exosomesThe serum level of Normal-serum-exo sFlt-1 in normal subjects was significantly higher than that in SCLC patients.The expression of sFlt-1 protein in BEAS-2B-exo was also significantly higher than that in NCI-H69-exo(*p<0.05,figure 3.1).2.Effects of sFlt-1 protein and exosomes containing sFlt-1 on HUVECsHUVECs in the control group were treated with 0.5%exosome-free FBS,whilst those in the experimental group were treated with 0.5%exosome-free FBS+500 ng/ml sFlt-1.The Transwell experiment was then conducted.The number of migrating cells in the experimental group was significantly lower than that in the control group,which suggests that addition of sFlt-1 protein could inhibit the migration of HUVECs.HUVECs were treated with NCI-H69-exo(100 μg/ml)or NCI-H69-exo(100 μg/ml)+sFlt-1(500 ng/ml),and Transwell assay showed that the cell migration ability of the latter was significantly reduced compared with that of the former(*p<0.05,figure 3.2).3.Construction of an sFlt-1 overexpression model to study the influence of overexpressed exosomes on HUVECsConstruction and evaluation of the overexpression modelStable cell lines overexpressing sFlt-1 were obtained by infecting HEK293 cells with lentivirus expressing Tet-sFlt-1.Western blotting revealed that the expression level of sFlt-1 in the exosomes of the overexpressing cell line was significantly higher than that in exosomes of the control group.RT-qPCR also demonstrated that the relative expression level of mRNA in the experimental group was higher than that in the control group.These results confirm that a stable HEK-293 cell line overexpressing sFlt-1 had been successfully constructed(*p<0.05,figure3.3).The characteristics of exosomes isolated from overexpressing cell lines were further identified to verify the correct separation of exosomes(figure 3.4).Effects of exosomes in the overexpression model on the proliferation and apoptosis of HUVECsCCK-8 was used to detect the proliferation of HUVECs.The proliferation activity of the overexpressed OV-sFlt-1 group was significantly lower than that of the control cells(*p<0.05,Figure 3.5).Flow cytometry indicated that the apoptosis rate of HUVECs treated with OV-HEK293-sFlt-l-exo is significantly higher than that of the control group(*p<0.05,figure 3.6).Effects of exosomes in the overexpression model on HUVEC migrationHUVECs in the OV-HEK293-exo control group were treated with NC-HEK293-exo.Scratch test was used demonstrated that the migration of HUVECs in the OV-HEK293-exo group is significantly lower than that in the NC-HEK293-exo control group(*p<0.05,figure 3.7).Transwell migration experiment was subsequently adopted.The combined action of sFlt-1 protein(500 ng/ml)and NCI-H69-exo reduced HUVEC migration compared with that in the NCI-H69-exo-only group,thus suggesting that sFlt-1 could inhibit HUVECs and angiogenesis.The OV-HEK293-sFlt-l-exo overexpressing sFlt-1 acted on HUVECs with NCI-H69-exo,and the number of migrating HUVECs in the combined exosome group(OV-HEK293-sFlt-1-exo+NCI-H69-exo)was further reduced compared with that in the control group.(*p<0.05,figure 3.8).4.Effects of knockdown of NCI-H69 sFlt-1 expression in exosomes on HUVECsAfter the three siRNAs(Si-sFlt-1#1,Si-sFlt-1#2,Si-sFlt-1#3)were transfected into NCI-H69 cells,RT-qPCR analysis showed that all three siRNAs could reduce the expression of sFlt-1 mRNA.Si-sFlt-1 group#3,which showed high knockout efficiency,was used for HUVEC studies,including CCK-8 detection,flow cytometry,scratch test,and other methods.The results indicated no significant differences between the knockout exosome group(Si-NCIH69-sFlt-l-exo)and the control group in terms of OD value,apoptosis rate(#p>0.05,figure 3.9-3.11).However,the results of scratch experiments showed that HUVECs migration ability was enhanced(*p<0.05,figure 3.12).5.Animal experimentsTumour weights in mice treated with OV-HEK293-exo overexpressing sFlt-1 decreased by 66%,on average,compared with those in the negative control group.sFlt-1 exosomes showed significant inhibition of NCI-H69 tumour xenograft growth and anti-tumour activity(figure 3.13 A-C).Pathological examination of transplanted tumours was carried out.CD31 staining showed that the microvascular density of tumour tissues after OV-HEK293-exo treatment was significantly lower than that in the control group.After Ki67 staining,the expression of Ki67 in tumour tissues after OV-HEK293-exo treatment was lower than that in the control group,thus suggesting that the proliferation rate of tumour cells decreased.TUNEL staining showed that the number of positive chromogenic cells in tumour tissues after OV-HEK293-exo treatment is higher than that in the control group,which indicates that the number of apoptotic cells in the former is higher than that in the latter(figure 3.13 D).Conclusions1.sFlt-1 expression decreased in patients with SCLC and its cell lines.2.sFlt-1 protein could inhibit the migration of HUVECs and reverse the migration of SCLC-exo in HUVECs.Exosomes containing sFlt-1 had a stronger inhibition of migration than the sFlt-1 proteome alone.3.Lentiviruses carrying the sFlt-1 gene fragment could be highly transfected into HEK293 cells.The overexpression model of sFlt-1 was successfully generated.Overexpressed sFlt-1 exosomes could be transferred into HUVECs to inhibit their migration and exert anti-angiogenic effects.4.No statistical difference in the proliferation and apoptosis of HUVECs before and after knockdown of sFlt-1 expression in the NCI-H69 cell line was observed,but the migration increased.5.Animal experiments indicated that the enrichment of exosomes after sFlt-1 could reduce the vascular density of the tumour,promote the apoptosis of tumour cells and inhibit the proliferation of tumour cells.This protein and exosome loading pattern may represent a potentially effective approach for the treatment of SCLC.SummaryExoQuick-TC was used to isolate exosomes from the serum and cell line cultures of SCLC patients.The expression level of sFlt-1 in the serum and exosomes of the bronchial endothelial cells of normal subjects was higher than that in malignant patients and cell lines.Compared with BEAS-2B-exo,NCI-H69-exo could better promote the migration of HUVECs.The migration of HUVECs was inhibited by sFlt-1 protein or sFlt-1 enriched exosomes.Compared with the same concentration of sFlt-1 protein,exosomes enriched with sFlt-1 had stronger inhibitory effects.Exosomes enriched with sFlt-1 could inhibit the growth of NCI-H69 tumour xenotransplants,thus showing significant anti-tumour activity by decreasing vascular density and cell proliferation and increasing cell apoptosis in the tumour.In conclusion,exosomes enriched with sFlt-1 could be used as an effective treatment model for SCLC.Future research should examine the mechanism and influencing factors of this protein. |
PDF Full Text Request