Extracellular Vesicle Protein Expression Profiles Reveal Features Of Prostate Cancer Progression From 3D Cell Model

Part I: Construction of 3D cell models for prostate cancerOBJECTIVE: To establish a prostate cancer cell model suitable for extracellular vesicles(EVs)study by selecting appropriate cell lines as well as 3D cell culture techniques and exploring appropriate culture conditions,and to compare it with the conventional 2D model.METHODS: BPH-1,LNCa P,C4-2,and C4-2B4 cell lines were selected for normal 2D culture,and 3D culture via 3D Co Seedis medium,respectively.EVs were isolated and purified using differential and density gradient centrifugation,briefly characterized by nanoparticle tracking analysis(NTA)and micro BCA,and the concentration,size,zeta potential,and protein concentration of EVs in both culture environments were compared.RESULTS: Cell viability and morphology were better under 3D culture conditions,and cell size was smaller than in 2D.Cells in 3D conditions produced a much higher number of EVs per cell than 2D,and the size of EVs was smaller than in 2D,but there were no significant differences in protein concentration and zeta potential.CONCLUSION: We developed a 3D cell culture model for EVs research in prostate cancer,which allows us to visually and conveniently observe and control cell status,proliferation,and simply collect and isolate EVs.it is better for EVs research than traditional 2D cell culture.Part II: Establish a method for isolation and purification of different size EVs subpopulations by TFF and SECOBJECTIVE: To combine Tangential Flow Filtration(TFF)and Size Exclusion Chromatography(SEC)for the efficient isolation and purification of different size EVs.And to compare EVs subpopulations obtained by conventional differential centrifugation method and our novel method.All parameters were optimized to make the method suitable for large-scale clinical applications.METHODS: Large extracellular vesicles(l EVs)were isolated using TFF,small extracellular vesicles(s EVs)were concentrated and extracted by TFF and SEC,and finally the samples were concentrated by ultrafiltration.The particle size distribution was characterized by ILM,NTA,and DLS for both subpopulations,morphological characterization was performed using TEM,and surface markers and lipid membranes were characterized by ELISA and fluorescent NTA.RESULTS: Both l EVs and s EVs had elliptical bilayer membrane structures under electron microscopy and expressed a certain amount of surface markers,and fluorescent NTA indicated that the number of EVs with lipid membrane structures accounted for more than 80% of the total number of particles,and there was a significant difference in particle size distribution between the two(P=2.07e-2).CONCLUSION: We innovated the separation of l EVs and s EVs using TFF.This method was able to effectively separate l EVs with higher recovery,less background impurity proteins,and collected EVs with more intact morphology and less damage compared to the traditional differential centrifugation combined with density gradient centrifugation.The new combined method balances efficiency,purity,convenience and economy for subsequent protein profiling as well as downstream functional studies,and is also more convenient for clinical applications.Part III: Extracellular vesicle protein profiling reveals molecular features of different vesicle subpopulations and prostate cancer progressionOBJECTIVE: EVs membrane protein microarray technology characterizes protein profiles of different EVs subpopulations derived from cancer cell lines with different malignancy,aiming to discover characteristic biomarkers of different size EVs subpopulations,as well as trends in protein expression and their characteristic markers during the shift of tumors from hormone sensitive to castration resistance.It helps us to model the risk of PCa,understand the mechanisms of its progression,and provide a powerful predictive tool as well as possible drug targets for clinical practice.METHODS: The protein abundance of four cell lines and two subpopulations of EVs were compared by EVs membrane protein microarray to screen out differentially expressed proteins,and using information from public databases,Lasso-Cox regression was used to screen out risk genes with significant effects on Progression Free Interval(PFI)and construct a risk model to evaluate their association with prognosis,clinical or pathological features,Androgen Deprivation Therapy(ADT)and androgen response,immune infiltration and treatment and PCa molecular characteristics.RESULTS: CD81,IL1R2,TLR3,LIF,and ICAM3 were identified as markers for s EVs,and LEUK,CRLF2,LYAM3,and B3GA1 were identified as markers for l EVs.The Lasso model based on CCL17,CYTL1,FCGR2 A,FGF2,IL13,and LTF divided PCa patients into high-risk and low-risk groups.The high-risk group was associated with poorer PFI,higher Gleason score,higher biochemical recurrence,and lower androgen response,and the risk score was an independent risk factor for biochemical recurrence in PCa patients.CONCLUSION: In this study,a series of novel EVs subpopulation markers,and 6 risk genes suggesting the progression of prostate cancer from hormone-sensitive to depot resistance were screened by EVs protein expression profiles in a 3D cell model of prostate cancer.A prostate cancer PFI-related risk model based on the six genes was also constructed,which was significantly correlated with androgen response pathways,immune response,and molecular typing,and could effectively predict biochemical recurrence in prostate cancer patients.