Study On The Relationship Between Microvesicles Secreted By Multiple Myeloma Cells And Angiogenesis And Disease Progression

Multiple myeloma?MM?is a malignant tumor originated in plasma cells.At present,the incidence of MM in China is 1-2/100,000.The incidence of MM in hematological malignant tumors ranks second,and has an increasing trend year by year.The overall 5-year survival rate of MM patients was about45%?less than 2 years for high-risk patients?and the median survival rate was3-4 years.Present known treatment regimens for MM can not cure this disease.Therefore,it is urgent to explore the pathogenesis of MM more deeply in order to find more effective and better treatment.The significance of tumor microenvironment?TME?in tumer progression has been commonly accepted in recent years.Tumors are gradually formed in dynamic and complex microenvironment,and can continue to grow,invade and metastasize.The interaction between cancer cells and microenvironment can regulates the invasion and metastasis of cancer cells and angiogenesis.Tumor vessel is a significant constituent part of TME.Tumors have the ability to induce angiogenesis.Angiogenesis is very important for the development of tumors,and their growth,migration and invasion depend on angiogenesis.Extracellular vesicles?EVs?,including exosomes,microvesicles and apoptotic bodies,are formed when various cells are activated or apoptotic.Recent studies have found that EVs have a series of physiological and pathological effects.EVs can promote the formation of thrombus,regulate inflammation,transfer RNAs and proteins to cells,cause cell signal transduction,and regulate the function of cells.Tumor EVs participate in the development and proliferation of tumors;regulate immune response,affect carcinogenic signal pathways and then promote neoplasm metastasis;contain cytokines and growth factors to support neovascularization in TME.The information transmitted by EVs to target cells depends on the components of EVs,which in turn depends on the origin cells of EVs and the microenvironment surrounding the formation of EVs.EVs from different cell sources can transmit complicated message to endotheliocytes and induce signals that promote or inhibit angiogenesis.Due to the dynamic changes of microenvironment,EVs constantly change the composition of their cargo to adjust the angiogenesis process.The occurrence and development of MM strongly depends on the microenvironment of bone marrow?BM?.Continuous interactions of MM cells and bone marrow microenvironment are crucial for the pathogenesis,vitality,proliferation and chemoresistance of tumors.In previous years,the interactions between MM and BM microenvironment has been absolutely attributed to cytokines and growth factors including IL-6?interleukin 6?,vascular epithelial growth factor?VEGF?,basic fibroblast growth factor?bFGF?and other factors,but these factors are not enough to fully illustrate all the whole phenomena.Until recently,EVs derived from MM were identified as media of communication between MM cells and BM microenvironments and accelerants of cancer progression.Bortezomib and Lenalidomide are the main drugs for the treatment of MM at present,but there are still some cases of MM patients with drug resistance,disease progression and so on.In order to eradicate MM,it is not enough to target MM cells alone.MM is very complicated and highly depends on BM microenvironment.Therefore,except the treatment of MM cells themselves,it is anticipated that it will bring about more efficient therapy by the transformation of cells in BM microenvironment from supporting tumors to killing tumors.At present,in the study of MM angiogenesis mechanism,the relationship between EVs and angiogenesis is few,and the effects of bortezomib and lenadomide on EVs angiogenesis are rare.In this paper,we studied the relationship between MVs and angiogenesis in MM,and the effects of bortezomib and lenalidomide on MVs angiogenesis and their mechanisms.We further explored the related mechanisms of angiogenesis in MM,and provided a new theoretical basis for the treatment of MM.By identifying and quantifying circulating MVs in patients with MM at different stages and in normal controls,the correlation between malignant plasma cell-derived MVs and other phenotypic MVs and angiogenesis factors in patients with MM was explored,so as to study the clinical correlation between circulating MVs and angiogenesis and disease progression,and to explore the potential use of circulating MVs as a new diagnostic and monitoring condition.This study is divided into three parts:Part 1 Effect of microvesicles secreted by multiple myeloma cells on angiogenesisObjective:By extracting MVs from multiple myeloma cell lines and identifying their size,morphology,immunophenotype,and interaction with endothelial cells,the effects of MM MVs on endothelial cell proliferation,migration,invasion and tubule formation were examined,so as to explore the effect of MM MVs on angiogenesis of myeloma.Methods:1.RPMI8226,U266 and KM3 myeloma cells and umbilical vein endothelial cells?HUVEC?were cultured.2.MVs were extracted from the supernatants of three kinds of myeloma cell cultures.The size and morphology of MM MVs were observed by transmission electron microscopy.The size,integrity and phenotype of MM MVs were detected by flow cytometry and counted.Quantitative analysis of MM MVs was performed by BCA protein quantitative method.3.After co-culture of MVs and HUVECs,the uptake of MM MVs by HUVECs was detected by confocal microscopy and flow cytometry.4.CCK8 method was used to detect the effect of MM MVs on HUVECs proliferation.Scratch test,Transwell chamber invasion test and tubule formation test were used to detect the effect of MM MVs on HUVECs angiogenesis.Results:1 Morphological observation of MM MVs under transmission electron microscopyMVs were extracted from the supernatant of myeloma cells by differential centrifugation.The morphology of MVs from myeloma cells was observed under transmission electron microscopy.The MM MVs were complete vesicles with a diameter less than 1.0um.2 Detection of MM MVs in size,integrity,number and phenotype2.1 Flow cytometry showed that MVs were distributed in the lower left side of1.1um standard microspheres with diameter less than 1.0um.2.2 The positive rate of Calcein in the samples analyzed by flow cytometry was the percentage of MVs with intact membrane.The membrane integrity rates of MVs of three kinds of myeloma cells were as follows:RPMI8226 was89.90?±1.39%?,U266 was 86.77?±3.95%?and KM3 was 90.0?±4.33%?.2.3 The MVs shedding rate of three kinds of cellsThree kinds of myeloma cells in logarithmic growth phase were inoculated in culture flasks at a density of 5 x 105/ml,respectively.The cells were detected 24 hours after culture.2.3.1 The MVs protein was determined by BCA protein quantitative method.The results showed that MVs shedding from 5×10⁵cells of RPMI8226,U266and KM3 were 2.62?±0.74?ug,1.99?±0.59?ug and 2.70?±0.65?ug,respectively.KM3>RPMI8226>U266.2.3.2 Flow cytometry counted MVs:MVs produced by every 5×10⁵cells,RPMI8226 was?2.11±0.54?×10⁶,U266 was?1.60±0.51?×10⁶,KM3 was?2.17±0.62?×10⁶.KM3>RPMI8226>U266.2.4 Flow cytometry showed that MVs expressed the marker of origin cells.The positive rates of CD138 of MVs in three kinds of myeloma cells were24.17±3.70%in RPMI8226,23.43±4.71%in U266 and 20.53±2.59%in KM3.3 MVs were ingested by HUVECsAfter co-culture with HUVECs for 2,6,12 and 24 hours,the dynamic uptake of MM MVs by HUVECs was observed by confocal microscopy.With the prolongation of co-culture time,the uptake increased gradually.In addition,the expression rate of CD138 in HUVECs increased from 0.80%to 9.23%after 24 hours.4 MM MVs promotes HUVECs proliferationAfter 24 hours of co-culture with HUVECs at different concentrations of MVs?1ug/ml,5ug/ml and 10ug/ml?,MM MVs of the three cells could promote the proliferation of HUVECs,with 10ug/ml being the most significant.5 MM MVs promotes angiogenesis in HUVECs5.1 MM MVs promotes HUVECs migrationMVs?10ug/ml?shed by three MM cell lines were co-cultured with HUVEC cells.The healing rates were observed and photographed at 0 h and24 h after scratch,and then calculated by Image software.The results showed that MVs produced by three MM cell lines increased scratch healing rate and promoted HUVECs migration,with statistical significance.5.2 MM MVs Promote HUVECs InvasionMVs?50ug/ml?produced by three MM cell lines and HUVEC cells were co-cultured for 6 hours.The results showed that MVs from three kinds of myeloma cells promoted the invasion of HUVECs in varying degrees at 6hours.Compared with PBS group,there were statistical differences in RPMI8226MV group and U266MV group?P<0.05 and P<0.01 respectively?.Although the invasiveness of KM3MV group also increased,it did not reach statistical significance?P>0.05?.5.3 MM MVs promotes tubule formation in HUVECsMVs?10ug/ml?produced by the three MM cell lines were co-cultured with HUVEC cells for 6 hours.The closed tubules were observed,photographed and counted under the microscope.The results showed that MVs from three kinds of myeloma cells promoted tubular formation of HUVECs in varying degrees at 6 h.Compared with PBS group,RPMI8226MV group and KM3MV group had significant difference?P<0.05and P<0.01 respectively?.Although tubular formation in U266MV group also increased,it did not reach statistical significance?P>0.05?.Summary:1.Complete MVs with diameter less than 1.0 um can be extracted by differential centrifugation,which carries the marker of origin cells.2.MVs and HUVECs co-cultured,MVs can be uptake by HUVECs.MM MVs can promote HUVECs proliferation and angiogenesis.Part 2 Effects of bortezomib and lenalidomide on proangiogenesis of microvesicles secreted by myeloma cells in vitroObjective:Angiogenesis plays an important role in the pathogenesis of multiple myeloma?MM?.Microvesicles?MVs?are extracellular vesicles and important participants in cell-to-cell communication.The first part of this study has confirmed that MVs secreted by normal cultured myeloma cells can promote angiogenesis.Bortezomib and lenalidomide are important drugs for the treatment of myeloma.Therefore,the aim of this study was to investigate the effects of bortezomib and lenalidomide on the proangiogenesis of MVs secreted by human myeloma cells and its mechanism.Methods:1.Cell culture:RPMI8226 human myeloma cells and human umbilical vein endothelial cells?HUVEC?.2.Grouping and treatmentRPMI8226 cells in logarithmic phase were seeded at a density of 5 x10⁵/ml and randomly divided into five groups.PBS?control group?,10nM bortezomib,100nM bortezomib,1uM lenadomide and 10uM lenadomide were added into the cell fluid respectively.After 24 hours of incubation,the MVs of the supernatant extracted by differential centrifugation were named N-MV,B10-MV,B100-MV,L1-MV and L10-MV,respectively.3.Flow cytometry was used to count the number of MVs in each group,and BCA protein quantitative method was used to quantify MVs.The MVs of each group were compared.4.CCK8 detection of proliferation ability of HUVECsHUVECs were seeded on 96-well plates at a density of 5 x 10⁴/ml and divided into six groups.PBS and the 5 different MVs?10ug/ml??N-MV,B10-MV,B100-MV,L1-MV and L10-MV?were added to the wells respectively,CCK8 was used to detect the cell viability after 24 hours of culture.5.Detection of angiogenesis in HUVECsHUVECs were seeded on 96-well plates at a density of 5 x 10⁴/ml and divided into six groups.PBS and the 5 different MVs were added as above.Scratch test,Transwell chamber invasion test and tubule formation test were used to detect the angiogenesis of HUVECs in each group.6.Detection of angiogenesis factor contents in MVs by QT-PCRThe expression levels of VEGF,IL-6 and bFGF in the above five groups of MM MVs were detected by QT-PCR.7.Detection of angiogenesis factor level in HUVEC cells by QT-PCR and ELISAHUVECs were inoculated at a density of 5 x 10⁴/ml and divided into 6groups.As mentioned above,PBS and 5 different MM MVs?10 g/ml?were added respectively.After 24 hours of incubation,the expression levels of VEGF,IL-6 and bFGF in HUVECs of different groups were detected by QT-PCR,and the expression levels of these factors in HUVECs culture supernatant were detected by ELISA.8.Detection of NF-?B pathway in HUVEC cellsSimilarly,HUVECs were randomly divided into six groups.After 24hours of co-culture with the above five 10 ug/mL MM MVs or PBS,the levels of NF-?B were detected by QT-PCR,Western blot and immunofluorescence staining,respectively.Results:1 Quantification of MM MVsThere were many MVs shedding in MM cells.MVs in bortezomib group?B10-MV and B100-MV?were significantly higher than those in control group,but lenalidomide had no significant effect on MVs secretion.2 Effect of MM MVs on proliferation of HUVECsCell proliferation was detected by CCK8 assay.Compared with the control group?PBS?,normal MVs significantly induced HUVECs prolife-ration,but drug-treated MVs?including bortezomib and lenalidomide?had no significant effect on HUVECs proliferation.3 Drug treatment can significantly reduce the levels of VEGF,IL-6 and bFGF in MM MVsCompared with PBS group,the gene expression levels of VEGF,IL-6and bFGF in MVs of bortezomib and lenadomide groups were significantly lower,but there was no difference between bortezomib and lenadomide groups.4 The internalization of MM MVs changes the level of angiogenesis factors in HUVECsThe expression levels of VEGF,IL-6 and bFGF in HUVECs and conditioned medium were detected by QT-PCR and ELISA,respectively.The results showed that the above cytokines in MVs of normal cultured cells?NMVs?were significantly higher than those in PBS group.The expression levels of VEGF,IL-6 and bFGF in MVs of drug-treated cultured cells?DMVs?were significantly lower than those in NMVs groups.5 NMVs promoted the migration,invasion and tubule formation of HUVECs,but bortezomib and lenalidomide decreased the proangiogenic activity of MM MVs5.1 Wound healing test to detect cell migration:24 hours after NMVs treatment,HUVECs migration to scratch area was significantly improved.Compared with NMVs,the cell migration activity of DMVs group decreased overall,although there were no significant difference in B100-MV and L1-MV groups.5.2 Transwell invasion test showed that NMVs promoted cell invasion,and compared with NMVs group,the cell invasion ability of four DMVs groups decreased significantly.5.3 Tubular formation assay was used to detect angiogenesis activity.At 6hours,although there was an increasing trend in the NMVs group compared with the control group,it did not reach statistical significance,but tubule formation in all DMVs groups was significantly reduced compared with the NMVs group.At 24 hours,compared with the control group,NMVs significantly promoted tubular formation in HUVECs;compared with NMVs group,tubular formation ability in all DMVs groups decreased significantly.6 NMVs increased the activation of NF-?B in HUVECs,but bortezomib and lenalidomide inhibited the activation of NF-?B in HUVECs induced by MM MVs6.1 Quantitative real-time PCR was used to detect the expression of NF-?B p65 subunit.Compared with the control group,the expression of NF-?B p65increased in NMVs group,and decreased significantly in all DMVs groups compared with NMVs group.6.2 Western blot was used to detect the expression of p65 in total protein and p-p65 in nucleoprotein of HUVECsP-p65 protein expression level:NMVs group was significantly higher than the control group;compared with NMVs group,all DMVs group was significantly lower.However,there was no significant difference in p65 levels between groups.6.3 Detection of p65 intranuclear transport by immunofluorescence microscopyThe intranuclear translocation of p65 increased significantly in NMVs group and decreased significantly in DMVs group.Summary:1.Bortezomib can induce more MVs released by myeloma cells,but lenalidomide do not significantly change the number of MVs.2.Bortezomib and lenalidomide treatment reduced the levels of VEGF,IL-6 and bFGF in MM MVs,and decreased the content of angiogenesis factor in MM MVs.3.Bortezomib and lenalidomide treatment inhibited proangiogenesis of MM MVs.4.Bortezomib and lenalidomide treatment inhibited the activation of NF-?B in HUVECs induced by MM MVs and reduced the secretion of angiogenic factors in HUVECs.5.Bortezomib and lenalidomide may inhibit angiogenesis by reducing the content of angiogenic factors in MM MVs and inhibiting the activation of NF-kappa B in HUVECs,thus providing another mechanism for anti-angiogenesis therapy of MM.Part 3 Detection of circulating microvesicles in patients with multiple myeloma and clinical study of its correlation with diseaseObjective:To quantify the levels of circulating MVs and the levels of angiogenic cytokines in patients with multiple myeloma?MM?and normal controls,and to explore the correlation between the levels of MVs from malignant plasma cells and other phenotypic MVs and angiogenesis factors in patients with MM,and to explore the relationship between MVs and disease severity,clinical characteristics and drug treatment,so as to explore the possibility of MVs as a potential use of diagnostic,disease assessment indicators and therapeutic targets.Methods:From March 2015 to September 2017,66 patients with multiple myeloma were selected from the Hematology Department of the Second Hospital of Hebei Medical University and the Affiliated Hospital of Hebei University,including 39 de novo patients,27 patients in remission stage and 21 non-cancer patients as control group.Patients in remission stage were divided into two groups according to whether or not bortezomib was used:15patients in bortezomib-free group and 12 patients in bortezomib-based group.The MVs in peripheral blood of MM patients were extracted by differential centrifugation,the morphology of MVs was observed under transmission electron microscopy,MVs were counted and the phenotype of MVs was detected by flow cytometry,and the serum levels of VEGF,IL-6 and bFGF were measured by ELISA.Results:1.The MVs in the peripheral blood of MM patients were also complete vesicles with a diameter less than 1.0um.2.The proportion of CD38⁺CD138⁺MVs in peripheral blood of MM patients was significantly higher than that of normal control group,and the de novo group was higher than that of remission group.Flow cytometry showed that the number of CD38⁺CD138⁺MVs in remission patients decreased significantly,and further decreased with the deepening of remission,while the number of CD38⁺CD138⁺MVs in remission patients tended to increase after the disease progressed.3.CD41 and CD45 positive MVs of MM patients were higher than the normal control group,and there was no significant difference between the initial treatment group and the remission group.In contrast,the average proportion of CD105⁺MVs in the initial treatment group was significantly higher than that in the remission group and the control group,but there was no significant difference between the remission group and the normal control group.CD38⁺CD138⁺MVs were positively correlated with CD41⁺MVs,CD45⁺MVs and CD105⁺MVs.4.Compared with the normal control group,the levels of VEGF,IL-6and bFGF in de novo MM patients were significantly increased.With the remission of myeloma treatment,the levels of these three angiogenic factors in remission patients were significantly decreased.CD38⁺CD138⁺MVs were positively correlated with levels of VEGF,IL-6 and bFGF.5.CD38⁺CD138⁺MVs in peripheral blood of de novo MM patients were positively correlated with the stage,bone damage,plasma cell ratio in bone marrow and the level of?₂-MG.6.MM patients in remission stage were divided into non-BTZ group and BTZ group.The level of bFGF decreased significantly in BTZ group,but there was no significant difference in the number of CD38⁺CD138⁺MVs,CD41⁺MVs,CD45⁺MVs,CD105⁺MVs and the levels of VEGF and IL-6between the two groups.Summary:1.The number of malignant plasma cell-derived MVs?CD38⁺CD138⁺MVs?in MM patients may be related to the tumor load,which increases significantly at the initial diagnosis and decreases with the decrease of the tumor load after treatment.CD38⁺CD138⁺MVs can be used as an index for diagnosis and evaluation of multiple myeloma.2.The level of CD38⁺CD138⁺MVs is positively correlated with the levels of CD41⁺MVs,CD45⁺MVs,CD105⁺MVs,VEGF,IL-6 and bFGF.CD38⁺CD138⁺MVs may be associated with angiogenesis in MM patients and may be a powerful target for treatment and intervention of multiple myeloma.3.CD38⁺CD138⁺MVs level was positively correlated with MM stage,bone damage,bone marrow plasma cell ratio and?₂-MG,suggesting that CD38⁺CD138⁺MVs may be an indicator of tumor load and prognosis in MM patients.Conclusions:1.Complete MVs can be extracted by differential centrifugation.MVs carry the marker of origin cells.MM MVs can be uptake by HUVECs,which can promote the proliferation and angiogenesis of HUVECs.2.Bortezomib can induce more MVs released by myeloma cells.Bortezomib and lenalidomide may inhibit angiogenesis by reducing the content of angiogenic factors in MM MVs and inhibiting the activation of NF-?B in HUVECs.3.The number of circulating CD38⁺CD138⁺MVs in patients with MM may be related to tumor load and angiogenesis.It can be used as an index for diagnosis and evaluation of MM,a prognostic indicator and a target for therapeutic intervention.