Application of proteins in small extracellular vesicles as diagnostic markers for prostate cancer

Abstract

The present invention discloses the use of proteins in small extracellular vesicles as diagnostic markers for prostate cancer, characterized in that the proteins in small extracellular vesicles are plasma sample sEV protein LAMB1 and / or urine sample sEV protein Histone H4, the use of the plasma sample sEV protein LAMB1 as a diagnostic marker for metastatic prostate cancer, and the use of the plasma sample sEV protein LAMB1 in combination with prostate-specific antigen as metastatic prostate cancer; the use of the urine sample sEV protein Histone H4 as a prostate cancer grading diagnostic marker, the use of the urine sample sEV protein Histone H4 as a high-risk prostate cancer diagnostic marker, and the use of the urine sample sEV protein Histone H4 in combination with prostate-specific antigen as a high-risk prostate cancer diagnostic marker, with the advantages of high sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention belongs to the technical field of diagnostic markers, and in particular relates to the application of a small extracellular vesicle protein as a diagnostic marker for prostate cancer. Background Art

[0002] Prostate cancer is often difficult to detect in its early stages. Studies have shown that early detection can improve prostate cancer patient outcomes and ultimately reduce mortality. The current challenges in treating prostate cancer lie in the lack of accurate grading and early diagnosis. Survival is largely correlated with tumor grade. Risk grading can be used to assess the overall risk of metastasis from in situ prostate cancer, determine optimal treatment options, and predict the likelihood of recurrence after treatment. Therefore, accurate grading and early diagnosis of prostate cancer are crucial.

[0003] The primary tumor marker currently used for prostate cancer diagnosis and risk stratification is prostate-specific antigen (PSA). PSA levels in the diagnostic gray zone of 4.0-10.0 ng / mL exhibit low specificity and poor sensitivity (25-40%). PSA cannot differentiate between benign prostatic hyperplasia, indolent tumors, and early- and late-stage prostate cancer. A serum PSA level of 4 ng / mL is often used as a screening standard in clinical practice, but data demonstrate that diagnosis based solely on PSA is inaccurate. Furthermore, due to the histological multifocality and heterogeneity of prostate cancer, even with multi-point biopsy, there is still a certain false-negative rate, resulting in missed detection of prostate cancer, missing the optimal time for treatment, and failing to dynamically monitor tumor development and progression. Therefore, improving the early diagnosis of prostate cancer and assisting with scientific staging and grading of prostate cancer are crucial for improving prognosis and extending survival for prostate cancer patients.

[0004] Small extracellular vesicles (sEVs) are rich in proteins and nucleic acids, encapsulated by a protective double phospholipid membrane. The complex signaling network they mediate between tumor cells and the tumor microenvironment is a key factor in all stages of cancer progression. Studies have found that sEVs play an important role in tumor immune regulation, microenvironmental reorganization, angiogenesis, invasion, metastasis, and survival. Studies have shown that sEVs carry integrin α3 and integrin β1, which can promote epithelial cell migration and invasion. Currently, no sEV protein markers for the early diagnosis of prostate cancer have been reported. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a small extracellular vesicle protein with high sensitivity and strong specificity for use as a diagnostic marker for prostate cancer.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: the use of small extracellular vesicle proteins as diagnostic markers for prostate cancer, wherein the small extracellular vesicle proteins are plasma sample sEV protein LAMB1 and / or urine sample sEV protein Histone H4.

[0007] Furthermore, the plasma sample sEV protein LAMB1 is used as a diagnostic marker for metastatic prostate cancer.

[0008] Furthermore, the plasma sample sEV protein LAMB1 is used in combination with prostate-specific antigen as a diagnostic marker for prostate cancer.

[0009] Furthermore, the plasma sample sEV protein LAMB1 is combined with prostate-specific antigen as a marker for metastatic prostate cancer.

[0010] Furthermore, the PRM verified peptide sequence of the plasma sample sEV protein LAMB1 is YFQMSLEAEER.

[0011] Furthermore, the sEV protein Histone H4 in the urine sample is used as a prostate cancer grading diagnostic marker.

[0012] Furthermore, the sEV protein Histone H4 in the urine sample is used as a diagnostic marker for high-risk prostate cancer.

[0013] Furthermore, the sEV protein Histone H4 in the urine sample is used in combination with prostate-specific antigen as a diagnostic marker for high-risk prostate cancer.

[0014] Furthermore, the PRM-verified peptide sequence of the sEV protein Histone H4 in the urine sample was DAVTYTEHAK.

[0015] The above-mentioned small extracellular vesicle protein is used in the preparation of a drug for treating prostate cancer, wherein the small extracellular vesicle protein is the plasma sample sEV protein LAMB1 and / or the urine sample sEV protein Histone H4.

[0016] Compared with existing technologies, the present invention offers advantages: The application of small extracellular vesicle proteins as diagnostic markers for prostate cancer reveals for the first time that LAMB1 plays a crucial role in cancer cell communication and in mediating PCa metastasis through sEVs. The role of LAMB1 in basement membrane and tumor-derived sEVs may indicate communication between PCa cells and the tumor microenvironment. Combined with our results, this demonstrates that the small extracellular vesicle protein LAMB1 has a higher area under the curve (AUC) for diagnosing metastatic prostate cancer than the existing marker PSA, demonstrating higher specific sensitivity. Furthermore, when combined with PSA, the AUC reaches 0.9378, further enhancing its diagnostic value.

[0017] Furthermore, we have discovered for the first time the small extracellular vesicle protein Histone H4, which can distinguish low-risk from high-risk prostate cancer. The small extracellular vesicle protein Histone H4 has a higher area under the curve (AUC) for diagnosing high-risk prostate cancer than the existing marker PSA, demonstrating higher specificity and sensitivity. When combined with PSA, the AUC reached 0.9733, further enhancing its diagnostic value. This will aid in the early, non-invasive diagnosis of prostate cancer and risk stratification. In summary, our findings provide a new avenue for the detection of early metastatic prostate cancer, risk stratification, and treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Characterization of small extracellular vesicles extracted from cell supernatant and clinical plasma samples; A is the size distribution of small extracellular vesicles in cell supernatant (CCM, left), plasma (Plasma, right) and urine (Urine, bottom) measured by NTA; B is a representative TEM image of isolated small extracellular vesicles with lipid bilayer and cup-shaped structure (red arrow); C is a Western blot of Flotilin-1, Syntenin-1, CD81 and Calnexin in small extracellular vesicles isolated from CCM, plasma and urine compared with whole cell lysate, whole plasma and whole urine lysate, the loading amount of whole cell lysate and sEV samples from cell supernatant is 2.5µg protein; the loading amount of whole plasma lysate and sEV samples from plasma is 4.2µg protein; the loading amount of whole urine lysate and sEV samples from urine is 1.7µg protein;

[0019] Figure 2 To identify and discover sEV proteins in prostate cancer cell lines and normal prostate epithelial cell lines using a label-free proteomic approach; A shows the proteomic identification and quantification results, B shows the number of proteins expressed in sEVs of five different cell lines, and C shows the visualization of PCA principal component analysis of protein differences between samples from five different cell lines;

[0020] Figure 3 Figure 3 is the result of sEV-based proteomic analysis, where A is a heat map of differential expression of sEV proteins in prostate cancer cell lines and normal prostate epithelial cell lines; B is the biological processes enriched in the top 10 GO terms of sEVs secreted by five different cell lines; C is a Venn diagram of proteins detected in sEVs from five different cell types, where the overlapping part shows the number of shared proteins, the side part shows the proteins expressed by single cell sEVs, the sEV proteins from each cell line are displayed in one color, and the boxes are the EV protein markers found in the sEV samples;

[0021] Figure 4 To quantify the expression of sEV plasma and urine proteins LAMB1, PXDN, URP2, DNAJA1, and Histone H4 using targeted PRM proteomics, A indicates LAMB1, B indicates PXDN, C indicates URP2, D indicates DNAJA1, and E indicates Histone H4. C indicates control, LR indicates low risk, H indicates high risk, M indicates metastasis, sEV-p indicates plasma-derived sEV, and sEV-u indicates urine-derived sEV.

[0022] Figure 5 Figure 1: Receiver operating characteristic (ROC) curve analysis to verify the differential expression of LAMB1 protein in plasma samples extracted from the control and metastatic groups using ELISA. The red curve represents the diagnostic value of LAMB1 alone, the blue curve represents the diagnostic value of PSA alone, and the orange curve represents the combined diagnostic value of LAMB1 and PSA.

[0023] Figure 6 Receiver operating characteristic (ROC) curve analysis to verify the differential expression of LAMB1 protein in plasma samples extracted from in situ prostate cancer and metastatic prostate cancer groups using ELISA. The red curve represents the diagnostic value of LAMB1 alone, the blue curve represents the diagnostic value of PSA alone, and the orange curve represents the diagnostic value of LAMB1 and PSA combined.

[0024] Figure 7 Receiver operating characteristic (ROC) curve analysis to verify the differential expression of LAMB1 protein in plasma samples extracted from clinical control and prostate cancer groups using ELISA. The red curve represents the diagnostic value of LAMB1 alone, the blue curve represents the diagnostic value of PSA alone, and the orange curve represents the diagnostic value of LAMB1 and PSA combined.

[0025] Figure 8This image shows the differential expression of Histone H4 protein in sEVs extracted from urine samples from low-risk and high-risk prostate cancer groups using ELISA, and the receiver operating characteristic (ROC) curve analysis to distinguish the control group from the prostate cancer group. The yellow curve represents the diagnostic value of Histone H4 alone, the red curve represents the diagnostic value of PSA alone, and the purple curve represents the diagnostic value of Histone H4 and PSA combined.

[0026] Figure 9 This is a tissue microarray to verify the differential expression of protein markers between prostate cancer tissue and normal prostate tissue. A shows the differential expression of LAMB1 protein on the tissue microarray; B shows the differential expression of Histone H4 protein on the tissue microarray.

[0027] Figure 10 To analyze the role of LAMB1 and Histone H4 in the prognosis of prostate cancer patients through database. DETAILED DESCRIPTION

[0028] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. 1. Specific embodiments

[0030] 1. Clinical sample collection

[0031] Peripheral blood, urine, and tissue samples were collected from patients with histologically confirmed prostate cancer (PCa) or men aged 50 years or older without PCa at the First Affiliated Hospital of Ningbo University. Forty-seven clinical blood samples and 46 urine samples were collected for PRM-targeted proteomics, and 60 clinical blood samples and 60 urine samples were collected for ELISA protein marker validation. Tissue microarrays (Bioaitech, China) were used for immunohistochemistry to validate 79 tissue samples. Ethical approval (ethics number: 2021-R106) was obtained for both blood and urine samples, and each participant completed an informed consent form. Enrolled clinical subjects were stratified according to pathological diagnosis into in situ PCa, metastatic PCa, and a non-tumor group (control group). According to the NCCN guidelines (Mohler et al., 2019), the in situ PCa group was further divided into very high-, high-, intermediate-, and low-risk groups. Considering statistical significance and patient population balance, we combined very high- and high-risk patients into the high-risk group, and intermediate- and low-risk patients into the low-risk group. All biofluid samples were collected from fasting patients before any therapeutic intervention. Men with infectious diseases, those taking medications that affect serum prostate-specific antigen (PSA) levels, a history of other cancers, or those who had undergone surgical treatment within 3 months were excluded. The clinical characteristics of the participating patients are shown in Table 1, and the grouping criteria are shown in Table 2.

[0032] Table 1 Clinical group samples and corresponding clinical parameters in this study

[0033] .

[0034] Table 2 Clinical groups of patients

[0035] control group Age- and sex-matched subjects with no history of cancer, including patients with benign prostatic hyperplasia Low-risk prostate cancer group PSA ≤ 20 ng / mL, Gleason score ≤ 7, TNM: T1 to T2c High-risk prostate cancer group PSA > 20 ng / mL or Gleason score > 7 or TNM: T3a to T4 Metastatic prostate cancer group Local or distant metastasis of prostate cancer .

[0036] 2. Plasma sample pretreatment

[0037] Venous blood was collected from age-matched males using EDTA-treated blood collection tubes (BD bioscience, China). All blood samples were processed within 2 hours of collection. Whole blood samples were centrifuged at 3,000 revolutions per minute (rpm) for 15 minutes to collect raw plasma. The collected plasma was immediately transferred to a clean tube and centrifuged at 2,000 × g for 20 minutes at 4°C to remove cellular debris. The supernatant was transferred to a new clean tube and centrifuged at 10,000 × g for 30 minutes at 4°C to remove large particles and aggregates. The preprocessed plasma was then aliquoted and stored at −80°C.

[0038] 3. Cell culture

[0039] Human normal prostate epithelial cell line (RWPE-1) and human prostate cancer (PCa) cell lines (PC3, DU145, LNCaP, and 22Rv1) were purchased from Fuheng Biotechnology (Shanghai, China) and the ATCC Cell Bank, respectively. RWPE-1 cells were cultured in Defined K-SFM (Life Technologies, China), while PCa cell lines were cultured in RPMI1640 (Life Technologies, China). Cells were cultured to a confluence of 60–70%, then gently washed twice with DPBS and incubated in fresh exosome-depleted culture medium for 48 hours. All cell lines were free of mycoplasma contamination and authenticated by STR.

[0040] 4. Pretreatment of cell supernatant

[0041] The cell culture medium was centrifuged at 300 × g for 5 minutes at room temperature to remove live cells. The supernatant was collected and centrifuged at 2,000 × g for 20 minutes at 4°C to remove dead cells. The supernatant was then centrifuged at 10,000 × g for 30 minutes to remove aggregated cell debris and other biopolymers with a buoyant density higher than that of small extracellular vesicles (sEVs). The supernatant was collected and filtered through a 0.22 µm pore size filter.

[0042] 5. Isolation of small extracellular vesicles from cell culture medium

[0043] sEVs from cell supernatants were isolated using the Total Exosome Isolation kit (Life Technologies, China). Cell culture medium was incubated with the reagent at a 2:1 v / v ratio overnight at 4°C. Following incubation, the cell culture medium was centrifuged at 10,000 × g for 1 hour at 4°C, and all supernatant was carefully removed. The pellet was resuspended in filtered Dulbecco's phosphate-buffered saline (DPBS). sEV protein samples were aliquoted and stored at -80°C until use.

[0044] 6. Ultracentrifugation to collect sEV proteins (referring to proteins contained in small extracellular vesicles sEV)

[0045] Fresh or thawed cell culture supernatant or plasma samples were diluted with cold PBS and centrifuged at 110,000 × g for 2 h at 4°C using a Beckman Coulter Type 70 Ti fixed-angle rotor (adjusted k factor 131, maximum acceleration, maximum deceleration). After the first ultraspin, the sEV pellet was resuspended in cold PBS and then subjected to a second ultracentrifugation at 110,000 × g for 2 h at 4°C using a Beckman SW41Ti rotor. The final pellet was resuspended in freshly filtered cold PBS to a final volume of 100–200 μL and then processed for protein quantification or frozen at −80°C.

[0046] 7. Western blot experiment

[0047] Total protein was extracted using RIPA lysis buffer (Cell Biolabs, China) containing 1× Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, China). sEV proteins were lysed by sonication in an ice-water bath for four 15-second bursts, followed by 10-second incubation on ice. Protein was quantified using a BCA protein assay kit (ThermoFisher Scientific, China). Equal amounts of protein (1.7–20 μg) from the same sample types were separated on 4–12% Bis-Tris protein gels (Bio-Rad, China) and blotted onto 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Germany). The membranes were blocked with 5% BSA for 1 hour at room temperature, followed by overnight incubation with primary antibodies at 4°C. The following day, after washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. After another wash with TBST, the membranes were imaged by chemiluminescence. Sequential EV characterization was performed by detecting relevant proteins (CD81, Syntenin-1, flotilin-1) and a negative marker (Calnexin). The polyclonal antibody LAMB1 was purchased from Affinity Biosciences (China), and HistoneH4 was purchased from Proteintech (China).

[0048] 8. Nanoparticle Tracking Analysis (NTA)

[0049] Dilute the sEV sample to a suitable range, with a particle concentration of 10 8 -10 9 For each measurement sample, the parameters were set at Camera Level 13 and Threshold 5 (NTA 3.4), and five 60-second videos were captured. The original particle concentration of the isolate was then calculated based on the measured concentration and dilution.

[0050] 9. LC-MS-based proteomics

[0051] For the extracted sEV proteins, SDT (4% SDS, 100mM DTT, 150mM Tris-HCl pH 8.0) buffer was used for sample lysis and protein extraction. DTT, SDS, and other low molecular weight components were removed by ultrafiltration (Millipore, 10kD) using UA buffer (8M urea, 150mM Tris-HCl pH 8.0). The samples were then treated with 100 mL of iodoacetamide (IAA) solution (containing 100mM IAA in UA buffer) in the dark for 30 minutes to block the reduced cysteine ​​residues, and then washed three times with UA buffer and NH4HCO3 buffer (25mM), respectively. The samples were digested with trypsin overnight at 37°C. The digested peptides of each sample were lysed at C 18 The sample was desalted on spin tips (Thermo Scientific, USA), concentrated using a SpeedVac centrifugal evaporator, and then dissolved in 0.1% FA. The tryptic digest was analyzed by LC-MS / MS using a hybrid quadrupole-Orbitrap tandem mass spectrometer (Q-Exactive, Thermo Fisher Scientific, USA) coupled to an Easy nLC. The MS raw data for each sample were identified and quantified using MaxQuant 1.5.3.17 software (Germany). Peptides and proteins were identified using the UniProt database.

[0052] 10. Targeted proteomics PRM detection

[0053] sEV proteins were extracted by PCT-assisted lysis in a lysis buffer containing 6 M urea (Sigma, USA) and 2 M thiourea (Sigma, USA), followed by reduction in 200 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 32°C for 30 minutes with gentle vortexing at 600 rpm. Proteins were then alkylated with 800 mM IAA for 30 minutes in the dark at 32°C with gentle vortexing at 600 rpm. An additional 150 μL of ammonium bicarbonate (ABB) solution was added, and digestion with trypsin at a 1:4 enzyme-substrate ratio was performed at 32°C for 16 hours. Finally, to terminate the digestion, 10% TFA was added to the solution at a final concentration of 1%. The solution was desalted using SOLAμHRP (Thermo Scientific, USA), then vacuum-dried and stored at −80°C until MS analysis. The peptide precipitate was solubilized with 0.1% FA and 2% ACN, followed by vortexing in ice water for 1 minute and sonication for 10 seconds. Peptide concentrations were measured using nanodroplets.

[0054] 11. Immunohistochemical analysis

[0055] Prostate cancer tissue microarrays (M079Pr01) were purchased from Bioaitech (China). Immunohistochemical (IHC) staining and analysis were performed as described below. Briefly, tissue sections were dewaxed in xylene and rehydrated in graded ethanol (95%, 75%, 50%, and distilled water). Slides were washed and immersed in 0.01 M citrate buffer at 95°C for 30 minutes to retrieve antigens. They were then sealed with goat serum for 1 hour at room temperature and incubated with the primary antibody at 4°C overnight. The following day, after incubation with the secondary antibody for 1 hour at room temperature, the slides were developed using diaminobenzidine (Agilent, USA). Staining intensity was measured using a light microscope (Leica, Germany). Both intensity and histochemical score (H-score) were used to evaluate IHC results. Intensity was defined as follows: 0 = none, 1 = weak, 2 = moderate, and 3 = strong. The H-score ranges from 0 to 300. Rabbit polyclonal antibodies LAMB1 (Affinity Biosciences, China) and Histone H4 were diluted at 1:400 and 1:2500, respectively.

[0056] 12. Statistical analysis

[0057] Statistical analysis was performed and visualized using GraphPad Prism 8.0 (GraphPad Software, USA). Data are presented as mean ± SEM. Parametric tests were performed using ANOVA with Turkey's post hoc test. Nonparametric tests were performed using the Mann-Whitney test. Data significance was assessed when the P value was < 0.05. Receiver operating characteristic (ROC) curves were used to assess the diagnostic performance of exosomal proteins and traditional tumor markers, and the area under the curve (AUC) was calculated. Kaplan-Meier survival curves were generated using the log-rank test.

[0058] 2. Results Analysis

[0059] 1. Identification and analysis of small extracellular vesicles (sEV) samples

[0060] sEV samples isolated from cell supernatant (CCM), plasma (Plasma) and urine (Urine) were evaluated by nanoparticle tracking analysis (NTA), and their particle size and distribution are shown in Figure 1A. Figure 1B shows a representative TEM image of the isolated sEV (red arrow). The particles from different biological fluids were all within the size range of sEV proteins (the majority of particles from CCM were 105.3±6.6 nm, 78.1±10.4 nm from plasma, and 139.9±4.1 nm from urine; the average particle size of CCM was 124.1±2.4 nm, the average size of plasma was 110.9±1.7 nm, and the average size of particles isolated from urine was 193.6±1.8 nm). The clear cup-shaped and lipid bilayer structures characteristic of sEVs were observed ( Figure 1 B) The cup-shaped structures indicate intact bilayer vesicles, but are dehydrated and therefore not perfectly spherical. Non-EV particles (i.e., lipid and protein aggregates) are also observed.

[0061] Immunoblotting (WB) also assessed protein expression in sEV samples from various biological fluids. Three sEV markers (CD81, Syntenin-1, and Flotillin-1) were used to define the presence of sEVs isolated from cell supernatants, plasma, and urine samples. As shown in Figure 1C, sEV samples isolated from plasma and urine of different clinical groups (including controls, low-risk, high-risk, and metastatic prostate cancer) were all positive for these sEV markers and negative for the non-sEV marker calnexin, indicating minimal contamination of the cell lysates and the recovery of high concentrations of pure sEV proteins.

[0062] 2. Preliminary screening of protein markers in small extracellular vesicles of prostate cancer

[0063] sEVs derived from four different prostate cancer cell lines (bone metastasis (PC3), brain metastasis (DU145), lymph node metastasis (LNCaP), and in situ tumor (22Rv1)) and a normal prostate epithelial cell line (RWPE-1) were evaluated by label-free proteomics. A total of 1570 proteins were identified, of which 878 proteins were quantified, as shown in Figure 2A; 384 sEV proteins were expressed in the RWPE-1 cell line, 473 sEV proteins were expressed in PC3, 480 sEV proteins were expressed in DU145, 597 sEV proteins were expressed in LNCaP, and 570 sEV proteins were expressed in 22Rv1. Figure 2 B. Furthermore, principal component analysis (PCA) by qualitative comparison highlighted the different protein composition of normal RWPE-1 sEVs compared with prostate cancer sEVs, as shown in Figure 2C. Analysis of sEV-based proteomics results is shown in Figure 3 As shown, the heat map shows that unsupervised hierarchical clustering shows that sEV proteins secreted by different prostate cancer cell lines and normal prostate epithelial cell lines can be clearly separated ( Figure 3 A). Figure 3 As shown in B, GO enrichment analysis showed that when all differentially expressed sEV samples were compared, “extracellular exosomes” and “extracellular vesicles” were enriched in sEV samples secreted by all five different cell lines.

[0064] To further explore the associations between sEVs from different cell lines, we used a Venn diagram to illustrate the overlap of sEV proteins across different groups, as shown in Figure 3C. We identified 219 shared sEV proteins, 133 sEV proteins expressed exclusively in prostate cancer, and 5 sEV proteins expressed exclusively in metastatic prostate cancer cell lines. Furthermore, comparative analysis with the EV database (Vesiclepedia) revealed a significant number of EV proteins identified in samples isolated from different cell lines, further demonstrating the efficiency of our EV extraction and providing a new foundation for subsequent studies of EV biological properties. Of the discovered sEV proteins, we further selected 20 for subsequent validation in clinical samples based on comparisons across different prostate cancer groups, as shown in Table 3.

[0065] Table 3. 20 potential sEV protein markers identified from prostate cancer cell lines

[0066] As shown in Table 3, protein markers in prostate cancer sEVs: RTN4, LARS1, LAMB1, LAMB2, PXDN, NRP1, ABI3BP, FLNC, and SPON2 are highly expressed in sEVs of metastatic prostate cancer cell lines, but are absent or lowly expressed in sEVs of in situ prostate cancer or normal prostate epithelial cell lines; URP2, DNAJA1, ADK, Histone H4, CLTC, AHCY, MYH9, Histone H3.1, RAN, FLNA, and CD151 are highly expressed in sEVs of prostate cancer cell lines, but are absent or lowly expressed in sEVs of normal prostate epithelial cells.

[0067] 3. Targeted proteomic analysis of sEVs from biological fluids from different sources

[0068] To validate the sEV proteins identified from the label-free proteomics, we also collected body fluids from clinical subjects, including blood and urine samples, and performed targeted quantification of the peptides of the target proteins by PRM targeted proteomics. Figure 4As shown in Figure 2, we first found that the protein profiles of a large number of sEVs in samples from different biological fluids were very different, or at least very different in cell supernatants, plasma, and urine. Figure 4 As shown in Figure AE, the expression levels and trends of the target proteins LAMB1, PXDN, URP2, DNAJA1, and Histone H4 were inconsistent in plasma sEV (sEV-p, left) and urine sEV (sEV-u, right), indicating that sEV results from different biological fluid sources cannot be generalized and vary significantly. The plasma sEV protein LAMB1 was expressed most highly in the metastatic group, while expression was relatively low in the control, low-risk, and high-risk groups. Significant differences were observed in the metastatic group compared to both the high-risk and control groups, while no similar trends or differences were observed in urine. The urine sEV protein Histone H4 was expressed most highly in the high-risk group, with significant differences compared to the control and low-risk groups, but no similar trends or differences were observed in the blood group. The PRM-validated peptide sequences for LAMB1 and Histone H4 were YFQMSLEAEER and DAVTYTEHAK, respectively.

[0069] 4. ELISA test to verify the differential expression of sEV protein LAMB1 in clinical plasma samples at different clinical stages

[0070] like Figure 5 As shown, in a comparison of the metastatic and control groups, plasma sEV protein LAMB1 was significantly overexpressed in metastatic patients (left panel). For distinguishing between metastatic prostate cancer and non-metastatic prostate cancer patients, the area under the curves for sEV LAMB1 protein alone, traditional PSA protein, and the combined sEV LAMB1 and PSA protein expression levels were 0.9556, 0.9422, and 0.9911, respectively (right panel).

[0071] like Figure 6 As shown, in a comparison of metastatic and in situ prostate cancer groups, plasma sEV protein LAMB1 was significantly overexpressed in patients with metastatic prostate cancer (left panel). For distinguishing between patients with metastatic prostate cancer and those with in situ prostate cancer, the area under the curves for sEV LAMB1 protein alone, traditional PSA protein, and the combined sEV LAMB1 and PSA protein expression levels were 0.8733, 0.8178, and 0.9378, respectively (right panel).

[0072] like Figure 7As shown, in a comparison of prostate cancer and clinical controls, plasma sEV protein LAMB1 was significantly overexpressed in metastatic patients (left panel). For distinguishing prostate cancer from non-cancer patients, the area under the curves for sEV LAMB1 protein alone, traditional PSA protein, and the combined sEV LAMB1 and PSA protein expression levels were 0.92, 0.8281, and 0.9363, respectively (right panel).

[0073] In summary, the sEV protein LAMB1 has high diagnostic accuracy for metastatic prostate cancer, with higher specific sensitivity than conventional PSA and higher diagnostic value than serum PSA. Importantly, combined detection of sEV LAMB1 and PSA further improved the diagnostic value of metastatic prostate cancer compared with non-prostate cancer controls and primary prostate cancer (AUC = 0.9911 and 0.9378, respectively).

[0074] 5. Validation of differential expression of sEV protein Histone H4 in clinical urine samples at different clinical stages by ELISA

[0075] like Figure 8 As shown, in a comparison of the stratification of low-risk and high-risk prostate cancer patients, the urinary sEV protein Histone H4 was significantly overexpressed in high-risk patients (left panel). For distinguishing high-risk from low-risk prostate cancer patients, the area under the curves for protein expression of sEV Histone H4 alone, traditional PSA, and the combined sEV Histone H4 and PSA were 0.9422, 0.7244, and 0.9733, respectively (right panel). These results demonstrate that Histone H4 protein demonstrates superior diagnostic value in diagnosing high-risk versus low-risk prostate cancer, with higher specificity and sensitivity than traditional PSA. Combined detection of sEV Histone H4 and PSA further enhances the diagnostic value of high-risk versus low-risk prostate cancer, thereby assisting clinical prostate cancer stratification and treatment decisions.

[0076] 6. Clinical association analysis between LAMB1 and Histone H4

[0077] like Figure 9 As shown, through tissue microarray and immunohistochemistry experiments, we first found that LAMB1 ( Figure 9 A) and Histone H4 ( Figure 9 B) Significantly higher expression in prostate cancer tissues than in normal tissues, indicating consistency in protein expression of LAMB1 and Histone H4 in sEVs and tissues.

[0078] Further, if Figure 10 As shown in the results, through clinical database analysis, it was found that high expression of LAMB1 and Histone H4 in tumors was closely associated with low OS rate and poor prognosis of prostate cancer patients, indicating the potential therapeutic value of sEV LAMB1 and Histone H4.

[0079] In summary, this study established a PCa-associated circulating sEV detection system based on sEV proteomics. Targeted proteomics and ELISA were used to quantify sEV proteins, and the expression levels of sEV proteins in blood and urine were compared across disease groups (non-tumor group, low-risk prostate cancer group, high-risk prostate cancer group, in situ prostate cancer group, and metastatic prostate cancer group). We found that LAMB1 expression in plasma sEVs was significantly altered in the metastatic prostate cancer group compared with the in situ prostate cancer group and the control group, and showed a higher diagnostic value than PSA for distinguishing metastatic prostate cancer. The combination of LAMB1 and PSA had a higher area under the curve (AUC) than either marker alone. Furthermore, Histone H4 expression in urine sEVs was significantly altered in the high-risk prostate cancer group compared with the low-risk prostate cancer group, and showed a higher diagnostic value than PSA for distinguishing low-risk from high-risk prostate cancer, thereby aiding in prostate cancer staging. The combination of Histone H4 and PSA showed a higher AUC than either marker alone.

[0080] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. Use of the small extracellular vesicle protein LAMB1 in the preparation of a diagnostic kit for metastatic prostate cancer, characterized in that: The PRM-verified peptide sequence of the protein LAMB1 is YFQMSLEAEER, and the small extracellular vesicles are from plasma.

2. The use according to claim 1, characterized in that: Application of small extracellular vesicle protein LAMB1 in plasma samples combined with prostate-specific antigen as diagnostic markers for metastatic prostate cancer in the preparation of diagnostic kits.

3. Use of Histone H4, a small extracellular vesicle protein, in the preparation of a prostate cancer grading diagnostic kit, characterized by: The PRM-verified peptide sequence of the protein Histone H4 is DAVTYTEHAK, and the small extracellular vesicles are from urine.

4. The use according to claim 3, characterized in that: Application of small extracellular vesicle protein Histone H4 in urine samples in the preparation of a diagnostic kit for high-risk prostate cancer.

5. The use according to claim 3, characterized in that: Application of small extracellular vesicle protein Histone H4 in urine samples combined with prostate-specific antigen in the preparation of a diagnostic kit for high-risk prostate cancer.

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