Introduction
Prostate cancer (PCa) is one of the most common malignancies in developed Western countries, with the highest incidence recorded in the United States [
1]. Due to population aging, lifestyle changes and the increasing popularization of PCa screening, the incidence and detection rate of PCa in Asian countries has also been increasing gradually [
2]. Androgen deprivation therapy (ADT) is still the principal treatment option for locally advanced and metastatic prostate cancers. However, most patients received ADT inevitably develop resistance to treatment and relapse with a more aggressive form of castration-resistant prostate cancer (CRPC) within 2–3 years. At present, there is a lack of highly specific and sensitive molecular markers to precisely predict the progression of PCa to CRPC, and to evaluate the treatment of advanced prostate cancer.
Prostate-specific antigen (PSA) testing have been used for PCa screening for more than 35 years [
3]. Although PSA shows moderate sensitivity and specificity, it cannot distinguish between dormant tumors and invasive malignancies and cannot be used to assess the clinical benefit in patients who receive ADT [
4,
5]. Moreover, PSA detection cannot be applied to infer the mortality associated with PCa, but instead reflects the trend in PCa incidence worldwide [
6]. Therefore, researchers have been attempting to identify novel biomarkers for PCa precise diagnosis. One such potential biomarker, prostate cancer antigen 3 (PCA3), is a prostate-specific non-coding RNA that has been proven to be a promising PCa biomarker and more accurate than traditional PSA evaluations, particularly for patients with ambiguous prostate biopsy results [
7]. However, the potential for over detection and overtreatment of indolent tumors on the basis of PCA3 assessments needs to be clarified, because the correlation between PCA3 and long-term overall survival remains debatable [
8]. Thus, more accurate evaluation of PCa patients using effective and specific biomarkers are required to facilitate risk-assessment and treatment options, and thereby maximize the benefits for patients.
Exosomes are extracellular vesicles with a phospholipid bilayer structure that are secreted by a variety of cells and carry various biomacromolecules, including nucleic acids, proteins, lipids, and metabolites [
9]. These biomacromolecules serve as significant mediators for intercellular communication in physiological and pathological processes. Exosomes have been recently shown to be key drivers of tumor progression and are associated with a series of tumor behaviors, including tumor growth, metastasis, and the tumor microenvironment [
10]. Based on their advantages of tumor specificity, non-invasiveness, and rapid detection, exosomal biomarkers have shown extremely high diagnostic value for the evaluation of malignant tumors [
11,
12].
Previous studies on tumor-derived exosomes have mainly focused on non-coding RNAs [
13]. However, with the rapid advancements in high-sensitivity mass spectrometry, detection of proteins and metabolites using more precise and quantitative approaches has become possible. In particular, proteins derived from exosomes offer unique advantages over traditional serological markers. First, partial exosomal proteins prefer to be released outside specific cells. For instance, the phosphorylated nuclear transcription factor X box binding 1 (NFX1) can be captured only in the exosomes of breast cancer patients, which may suggest that the functions of phosphorylated NFX1 are mediated under specific conditions [
14]. Second, tumor-derived exosomes show higher specificity than those obtained from healthy donors. The exosome-derived GPC1 was enriched in pancreatic cancer patients and showed excellent ability in comparison with carbohydrate antigen (CA) 19-9 or serum-free GPC1 in pancreatic cancer screening [
15]. Third, the natural barrier effect of lipid bilayers ensures that exosomal proteins are hardly degraded by external proteases and other enzymes. The resultant outstanding stability confers exosomes the potential to serve as the PCa biomarkers [
14].
Metabolic abnormalities have been widely characterized as a distinguishing feature of tumors [
16]. Metabolic changes are closely related to disease progression and directly reflect the tumor microenvironment, cellular status, and clinical drug response [
17]. Thus, in comparison with transcriptomics and proteomics, analyses based on metabolomics could yield findings closer to the actual cellular situation. Therefore, metabolites are rapidly emerging as the valuable biomarkers for early PCa screening [
18].
Considering these perspectives, we collected plasma from tumor-free controls (TFCs), PCa patients, and CRPC patients and isolated exosomes from the collected samples. Subsequently, we used a combination of proteomic and metabolomic techniques to analyze the exosome expression profiles in these groups. The aim of this study was to identify biomarkers that can distinguish disease classifications at the protein and metabolic levels and to further explore their value for PCa precise diagnosis. Moreover, combined multi-omics analysis was used to further understand the molecular profiles of PCa at different stages. Finally, the functional role of exosomal protein LRG1 was studied in prostate cancer.
Materials and methods
Clinical samples
All participants were recruited from Shenzhen Hospital of Southern Medical University (Shenzhen, China), Hainan General Hospital (Hainan China), Shenzhen’s People Hospital (Shenzhen, China), and The Second Hospital of Tianjin Medical University (Tianjin, China). The study was approved by the human ethics committees of these hospitals. Written informed consent and clinical information were obtained from all patients. Serum samples (3–4 mL) were obtained from TFCs and PCa and CRPC patients and stored at − 80 °C until processing. TFCs were age-matched normal individuals or men whose prostates were free of cancer (e.g., individuals with benign prostatic hyperplasia [BPH]).
Exosome isolation and identification
Exosomes derived from patient serum were obtained using the classical ultracentrifugation approach [
19]. First, living and dead cells were removed by low-speed centrifugation (300 ×
g for 10 min and 2000 ×
g for 10 min successively) at − 4 °C. Next, the cell debris was centrifuged and removed at 10,000 ×
g for 30 min. Then, the obtained supernatant was filtered by a 0.22-µm filter to further purify exosomes. The rest of the supernatant was ultracentrifuged at 100,000 ×
g/70 min with a TI70 rotor. Second, the supernatant was discarded again, purified exosomes were resuspended in PBS, and exosome pellets were obtained following ultracentrifugation at 100,000 ×
g/70 min with a TI70 rotor. After the supernatant was discarded, an appropriate amount of PBS was added according to the initial plasma volume to resuspend exosomes, and the protein concentration was measured after packaging and stored at − 80 °C.
Trypsin digestion
On the basis of the protein concentration, 8 M urea was added to equal amounts of total protein, and then adjusted to the same volume. Next, DTT was added to the protein solution to a final concentration of 5 mM, and the solution was incubated at 56 °C for 30 min. After cooling to room temperature, IAM was added to a final concentration of 11 mM, and the solution was incubated at room temperature for 15 min in the dark. The protein sample was then diluted by adding 100 mM TEAB to a urea concentration of less than 2 M. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4 h digestion.
Liquid chromatography–tandem mass spectroscopy analysis for proteomics
The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a home-made reversed-phase analytical column (length, 15 cm; internal diameter, 75 µm). The gradient consisted of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, from 23 to 35% over 8 min, from 35 to 80% over 3 min, and then holding the concentration at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. The peptides were subjected to a nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS by using the normalized collision energy (NCE) setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was performed that alternated between one MS scan followed by 20 MS/MS scans with a 15.0-s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The fixed first mass was set to 100 m/z.
The resuspended exosomes in PBS were added to 1000 μL of extract solution (acetonitrile:methanol:water = 2:2:1). After repeated freezing and thawing three times with liquid nitrogen, all samples were vortexed for 30 s before sonication for 10 min. After standing for one hour at 40 °C, the samples were centrifuged at 12,000 rpm at 4 °C for 15 min, after which 950 μL of supernatant was dried in a vacuum concentrator, and an extract solution (methanol:acetonitrile:water = 2:2:1) containing an isotopically labeled internal standard mixture was added in proportion. After 30 s of vortexing and 10 min of sonication, the obtained samples were centrifuged at 12,000 rpm at 4 °C for 15 min. Subsequently, the supernatant was obtained and transferred to a fresh glass vial for liquid chromatography–mass spectroscopy (LC–MS) analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatant from all samples.
LC–MS/MS analysis was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The analysis was conducted with the following elution gradient: 0 ~ 0.5 min, 95% B; 0.5 –7.0 min, 95% ~ 65% B; 7.0 –8.0 min, 65% ~ 40% B; 8.0 – 9.0 min, 40% B; 9.0 –9.1 min, 40% ~ 95% B; 9.1 –12.0 min, 95% B. The column temperature was maintained at 30 °C. The auto-sampler temperature was 4 °C, and the injection volume was 2 µL. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra in the information-dependent acquisition (IDA) mode under the control of acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluated the full-scan MS spectrum. The ESI source conditions were set as follows: sheath gas flow rate, 50 Arb; Aux gas flow rate, 10 Arb; capillary temperature, 320 °C; full MS resolution, 60,000; MS/MS resolution, 7500; collision energy, 10/30/60 in NCE mode; spray voltage, 3.5 kV (positive) or − 3.2 kV (negative).
Western blotting and semi-quantification
The detailed steps of western blotting have been described previously. Briefly, the cell pellet was collected in RIPA buffer with 0.1 M DTT. After 30 min of incubation in an ice-water bath, 5×loading buffer was added to the protein solution and heated for 5 min at 95 °C. To maintain the consistency of the loading control, 5 µg of total protein from resuspended exosomes was loaded for each experiment. For cell lines, BPH1, LNCaP, and LNCaPAI cell lysates were prepared using the same protocol. After centrifugation for 5 min at 12,000 × g, the obtained protein supernatant was subjected to SDS-PAGE, and the gel was then blotted onto a PVDF membrane. Membranes were blocked with 5% skim milk for 1 h at room temperature. The primary antibodies against CD9, TSG101, calnexin, and GAPDH were separately incubated with the membrane at 4 °C overnight. On the next day, HRP-conjugated secondary antibody (1:5000 anti-rabbit or 1:10,000 anti-mouse dilution) incubation was performed for 1 h, and the intensity was measured using an imaging system (Bio-Rad ChemiDocTM Imaging System).
Receiver operating characteristic curve
A receiver operating characteristic (ROC) curve was used to further assess the potential diagnostic value of proteins and metabolites derived from patients’ plasma. A combined protein and metabolite panel was constructed to improve the performance of the disease classifier.
Cell culture and lentivirus packaging
PC-3 and 293 T were grown in DMEM containing 10% FBS and 1% penicillin/streptomycin. DU145 and LNCaP were cultured in RPMI1640 medium with 10% FBS and 1% penicillin/streptomycin. Primary HUVEC cells were cultured in endothelial cell medium (ECM, Cat. #1001, ScienCell Research Laboratories, Inc). All the cells were maintained at 37 °C with 5% CO2. All the steps of lentivirus packaging were according to the previously described. LRG1 cDNA was obtained using Transcription High Fidelity cDNA Synthesis Kit and then subcloned into PCDH-CMV-Puro-EGFP vector.
IHC staining
Immunohistochemical (IHC) analysis was carried out to assess PCa tumor tissues and adjacent para-cancerous tissues. Briefly, the tissue microarray was put into a repair box filled with citric acid antigen repair buffer (pH 9.0) for antigen repair, and then it was cooled down to room temperature. LRG1 primary antibody were incubated at 4 °C overnight (Cat. #ab178698). The next day, secondary HRP-conjugated antibody were incubated for 1 h. Finally, counterstaining was performed with haematoxylin. The criteria of tissues scoring was performed as previously described.
Gene expression analysis by reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated with EasyPure RNA Kit (Transgene, Cat. #ER101). Immediately after, RNA reverse transcription was carried out using PrimeScript™ RT (TaKaRa, Cat. #RR047A). PCR amplification was performed using SYBR green fluorescent dye (Transgene, Cat. #AQ142-21). Primer sequences are provided as follow:
LRG1: F: 5'-GTTGGAGACCTTGCCACCT-3', R: 5'-GCTTGTTGCCGTTCAGGA-3';
GAPDH: F: 5'-ACAACTTTGGTATCGTGGAAGG-3', R: 5'-GCCATCACGCCACAGTTTC-3'.
Cell proliferation and colony formation assay
Cells were seed into 96-well plates (5000 cells/well). Cell viability was measured by CCK8 reagent (FC101-01, TransGen Biotech Co., Ltd) at various time points (0 h, 24 h, 48 h, and 72 h). 10 μl CCK8 was added into culture medium. After 2 h incubation, the absorbance of each well was measured at 450 nm. For colony formation assay, cells were digested into single cells by 0.25 trypsin, which were further counted by a hemacytometer. Cells (1000 cells/well) in log growth phase were plated into 6 well plate. After 14 days, cells were washed, fixed in 4% paraformaldehyde for 30 min, and then stained with crystal violet solution.
Wound healing and transwell assay
Cells were plated into 6-well plate. When the cells grew to 100% confluence, a sterilized tip was used to gently draw lines on the plate. After 0 h, 24 h and 48 h, the wound healing area was calculated using Image-Pro PlusV1.8.0. For transwell assay, cells were cultured in RPMI 1640 medium without FBS. After 24 h, cells (2 × 105 LNCaP; 2 × 104 PC3) were seed into a 24-well plate. The upper chamber membranes (labselect, 8.0 μm pores #14342) were coated with matrigel (BD, #356230) and the lower chamber was supplemented with FBS. After a period of culture, cells on the bottom side of the chamber membrane were fixed, stained with crystal violet and photographed. Relative invasion ability was calculated as cell number per five random visual fields.
We performed pretreatment for removal of the exosomes derived from commercial fetal bovine serum. In brief, the exosomes of fetal bovine serum were eliminated by ultracentrifugation, then collecting the supernatant for cell culture. Subsequently, PCa cells were cultured in DMEM 10% FBS medium without exosomes. After 48 h, the exosomes derived from PCa cell lines were enriched by ultracentrifugation and applied for further angiogenesis experiment. HUVEC cells were cultured in RPMI1640 medium without FBS. Meanwhile, exosomes (20 μg/ml) derived from different cell lines were added to culture medium. After 24 h cell culture, HUVEC cells were harvested and counted, then 1 × 104 HUVEC cells were plated into Matrigel (BioCoat, #356231) coated 96-well plate. After 4 h and 8 h incubation, the number of branch points were measured to quantify tube formation.
Statistical analysis
Multiple group comparisons were performed using one-way ANOVA followed by Tukey–Kramer tests. P < 0.05, shown in the figures as “*”, was considered to indicate statistical significance. Consistently, P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****) indicated highly significant findings. Statistical analyses were performed using the GraphPad Prism 7.04.
Conclusions
The progression of PCa patients to CRPC varies widely among individuals. At present, there is still a lack of highly specific and sensitive molecular markers in clinical practice to predict the progression of PCa to CRPC and evaluate the therapeutic effect. Exosomes have provided a new direction for the precise diagnosis and treatment of malignancies in recent years for their natural advantage in liquid biopsy and therapeutic carrier. Urine is a very advantageous source of liquid biopsy markers because of its simple and easy access. Prostate cancer urine markers have been developed into commercial qualitative detective products.
e.g. PSA3 is the first FDA-approved urine RNA-marker, and ExoDx kit is based on three urinary exosome-derived gene (PCA3, ERG and SPDEF) combination [
25]
. Howerver, urine markers are also easily affected by urine volume, concentration and the presence of other substances. In terms of quantitative monitoring markers, blood is relatively more stable, and plasma-derived exosome markers have more advantage for early prediction and efficacy monitoring of CRPC.
In the present study, we collected plasma from TFCs and PCa and CRPC patients, and isolated and purified exosomes for proteomic analysis. Our data indicated that in terms of exosome abundance, there were no significant differences between various disease groups as a whole, but individual variations observed. Since we used 0.22-µm filtration, most of the larger vesicles were directly removed. Thus, good homogeneity was observed in all of the samples. In the comparison of PCa patient and TFCs, we identified 27 DEPs, including 18 upregulated and 9 downregulated proteins. The expression levels of LRG1, C7, SHBG, HRG, SERPINF1, and LUM in the PCa group were twofold or higher than those in the TFC group. Conversely, the expression levels of ExtL2, UGP2, RSU1, TUBB1, HBD, PF4, HbA1, C1R, and HBB were reduced by > 50% in comparison with those in the TFCs. We also observed APOE (a low-density lipoprotein that transport cholesterol from peripheral tissues to the liver for metabolism) in which group? was 1.7-fold higher than that in the TFC group and the AUC curve of APOE was 0.734 for PCa classification (Additional file
1: Fig. S2A–C). Although exosomal APOE were reported as potential biomarkers in several studies, we still need to be very cautious to verify if its exosome resource, as the molecular size of low-density lipoproteins is very close to exosomes, and APOE is also a high accumulative plasma protein [
26].Two exosomal-protein LRG1 and ITH3 and their combination showed a good potential as liquid biopsy markers to distinguish CRPC from PCa. Current data based on a cross-sectional study which recruits TFC, PCa, and CRPC patiets. For validation of the values of LRG1 and ITH3 or their combination as predictive markers for early prediction and monitoring of CRPC, a longitudinal cohort study with the observation of their levels during the whole natural history of CRPC progression need further investigation.
LRG1 is a member of the leucine-rich repeat sequence (LRR) protein family with eight repeat sequences. Previous studies have demonstrated that LRG1 is involved in the progression of tumors by promoting angiogenesis, including pancreatic cancer, lung cancer, bladder cancer, and colon cancer [
27‐
30]. The association of abnormal increase of plasma LRG1 with the degree of PCa malignancy was observed [
31]. Wang et al. showed that LRG1 is indispensable for promoting mouse ocular angiogenesis, and the lack of LRG1 was associated with significant pathological ocular angiogenesis through dysregulation of the TGF-β signaling pathway [
24]. In our study, LRG1 was enriched in CRPC, whose level was 1.7 times higher than that in PCa group in PRM validation, compared to a two-fold elevation in untargeted proteomics. However, whether the exosomal LRG1 derived from prostate cancer cells and the functional role of LRG1 protein in prostate cancer is far from known. IHC examination showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis. However, if LRG1 plays a role in prostate cancer distant metastasis and the machemism of LRG1 induced angiogenesis needs further study.
ITIH3 belongs to the α-trypsin inhibitor family and is enriched in the extracellular matrix and blood [
32]. One known function of this protein family is covalent binding to hyaluronic acid (HA) to stabilize the extracellular matrix (ECM). Several studies have suggested that ITIH3 exerts a tumor suppressor role in disease progression. For example, low expression of ITIH1 and ITIH3 resulted in a low number of lung metastases in a xenograft mouse model and increased the ability of cell attachment in vitro [
33]. In addition, Hamm et al. demonstrated that frequent loss of ITIH3 was observed in many solid tumors, such as lung cancer, gastric cancer, breast cancer, and ovarian cancer. Conversely, a significant increase in ITIH3 expression was observed in the plasma of patients with lung cancer [
34,
35]. In our study, we observed that the level of ITIH3 derived from CRPC patients was 2.04-fold higher than that in the PCa group. We speculated that ITIH3 distribution varied between the inside and outside of cells or even exosomes, which is likely due to ADT. Whether ITIH3 enrichment is specific to ADT requires future studies.
For untargeted metabolomics study, a total of 206 secondary spectrograms were obtained by mass spectrometry. In the comparison of PCa patients versus TFC, two elevated metabolites were observed in PCa samples relative to the TFC group, 2-(2-methylbutanoyl), and acetylglycine. Moreover, creatinine, dihydrothymine, and hydroxyoctanoic acid were higher in the TFC group, whose levels were 2–2.5 times than those in the PCa group. Interestingly, acetylglycine belongs to the amino acid pathway and may be involved in immunoregulation [
36,
37]. There was also some evidence that acetylglycine serves as a biomarker for disease diagnosis. Jonsson et al. reported that a significant increase in the level of acetylglycine in plasma was observed in glioma patients [
38]. Another example is the use of a combination of urinary acetylglycine and gamma-glutamylalanine to identify Vogt-Koyanagi-Harada disease, which is a multisystem disease of presumed autoimmune cause. Another metabolite, dihydrothymine, is an intermediate metabolite of thymine. Aberrant elevation of dihydrothymine may induce cytotoxicity. One interesting example illustrated that dihydropyrimidine dehydrogenase (DPYD) was induced by EMT-promoting transcription factors and generated dihydrothymine, which is necessary for the EMT process [
39]. However, different results were observed in our study, in which low levels of dihydrothymine were enriched in PCa samples. One potential explanation for the reduced dihydrothymine expression is that PCa may prefer to maintain high levels of dihydrothymine in internal tumor cells rather than to release them as exosomes.
Comparisons between CRPC and PCa showed that the cycloartocarpin and 2-methylglutaric acid content in the CRPC group were more than twofold those in the PCa group, while the levels of tridecanoic acid, undecanoic acid, and hydroxyoctanoic acid were abundant in the PCa group and 2–2.8-fold higher than those in the CRPC group. 2-Methylglutaric acid is an alpha, omega-dicarboxylic acid. Metribolone (R1881), also known as methyltrienolone, is a synthetic and orally active anabolic–androgenic steroid (AAS) that is widely used in scientific research as a ligand of interest in the androgen receptor (AR). Putluri et al. used 10 nM synthetic androgen (R1881) to treat VCaP prostate cancer cells for 24 h and found that 2-methylglutaric acid levels were significantly elevated in comparison with the levels in untreated controls [
40]. Thus, 2-methylglutaric acid could be a downstream metabolite dependent on AR activation. In our study, the 2-methylglutaric acid level was only increased in the CRPC group, which may suggest that relatively high levels of 2-methylglutaric acid are associated with ADT resistance.
Other differential metabolites included cycloartocarpin, tridecanoic acid, undecanoic acid, and hydroxyoctanoic acid. There is no strong evidence for the involvement of these metabolites in tumor progression and development, and they are unlikely to be the byproduct of CRPC. However, these metabolites exhibited excellent performance in distinguishing CRPC. ROC curve analysis was also performed for a series of metabolites, including cycloartocarpin, 2-methylglutaric acid, and hydroxyoctanoic acid, and the results showed that their AUC values were 0.87, 0.86, and 0.88, respectively. Subsequently, we constructed a combination diagnosis model using these four metabolites. Surprisingly, the AUC value of this model was 0.97, indicating an excellent ability to differentiate PCa from CRPC.
In summary, our current study applied an integrated proteomics and metabolomics analysis to describe the protein and metabolic profiles of plasma exosomes from CRPC and PCa patients as well as TFC control cohort. Several exosomal proteins and metabolites and their combinations showed potential values as CRPC markers that facilitate the discrimination of CRPC from PCa and TFC patients. Functional study of exosomal protein LRG1 confirmed its important role in PCa malignant progression.
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