Prostate cancer-derived exosomal PSM-E inhibits macrophage M2 polarization to suppress tumor invasion and metastasis through the RACK1/FAK/ERK pathway

doi:10.21203/rs.3.rs-4858983/v1

Download PDF

Research Article

Prostate cancer-derived exosomal PSM-E inhibits macrophage M2 polarization to suppress tumor invasion and metastasis through the RACK1/FAK/ERK pathway

https://doi.org/10.21203/rs.3.rs-4858983/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

It is well-established that understanding the mechanism of prostate cancer (PCa)-associated metastasis is paramount for improving its prognosis. Metastasis is known to involve the communication between tumor-associated macrophages (TAMs) and tumor cells. Exosomes are crucial in mediating this intercellular communication within the tumor microenvironment. Nonetheless, the role of exosomal proteins in PCa metastasis is not yet fully understood. Here, we investigated the mechanisms of prostate cancer-derived exosomal PSM-E on regulating macrophage M2 polarization to suppress tumor invasion and metastasis.

Methods

PSM-E levels in exosomes were detected by transmission electron microscopy and Western blotting analysis. The diagnostic value of urine-derived exosomal PSM-E in PCa were evaluated by LC-MS/MS, correlation analysis, and ROC curves analysis. The mechanisms underlying the inhibitory effect of exosomal PSM-E on the M2 polarization of macrophages was investigated by co-IP, IHC staining, and PCa tumorigenesis model.

Results

We revealed that exosomal PSM-E is upregulated in exosomes derived from the serum and urine of PCa patients. Clinically, an elevated exosomal PSME expression in urine is significantly correlated with an advanced pathological tumor stage and a high Gleason score. Our research also revealed that exosomal PSM-E inhibits prostate cancer cell proliferation, invasion, and metastasis by suppressing macrophage polarization in vitro and in vivo. Furthermore, we provided compelling evidence that exosomal PSM-E inhibits M2 polarization of macrophages by recruiting RACK1 and suppressing the ERK and FAK signaling pathways, consequently suppressing PCa invasion and metastasis. Furthermore, we found that the protease-associated domain of PSM-E and the fourth tryptophan-aspartate repeat of RACK1 are crucial for the interaction between PSM-E and RACK1.

Conclusions

Notably, exosomes carrying PSM-E from PCa urine could potentially serve as a biomarker for PCa, and targeting exosomal PSM-E may represent a strategy for preventing tumor progression in this patient population.

Prostate cancer

exosome

PSM-E

macrophage polarization

Prostate cancer (PCa) is a prevalent cancer and the second leading cause of cancer-related death among men, making it a growing global health concern [1,2]. While early-stage PCa is treatable, a third of cases progress to a more aggressive form associated with poor survival [1, 2]. Despite the advent of various therapeutic strategies, such as androgen deprivation therapy (ADT), tackling metastases remains challenging. Therefore, there is a pressing need for a deeper understanding of PCa metastasis to develop effective treatments [3]. While prostate-specific antigen (PSA) is frequently employed as a biomarker for PCa diagnosis and prognosis, clinically significant PCa cannot be consistently differentiated from prostatitis or benign prostatic hyperplasia (BPH) due to limited sensitivity and specificity of PSA detection, which leading to over diagnosis and overtreatment [4, 5]. Hence, there is an urgent need to identify more efficient biomarkers and develop more sensitive approaches for early diagnosis of PCa.

There is an increasing consensus suggesting that macrophages in a tumor microenvironment, tumor-associated macrophages (TAMs), are considered a significant contributor to PCa progression [6, 7]. Of note, Macrophages come in two distinct polarized forms: M1, which enhances immune responses and acts against tumors, and M2, which suppresses immune activities and promotes tumorigenesis [6–8]. Emerging evidence suggests that TAMs in tumors and nearby tissues, resembling M2 macrophages, exhibit pro-tumor characteristics, such as promoting PCa cell growth, invasion, and metastasis [6–8]. Furthermore, TAM infiltration is an independent risk factor for PCa recurrence, by reason of patients with fewer TAMs tend to have a better prognosis [9, 10]. In this respect, it has been reported that the prognosis of PCa patients with decreased TAM abundance is significantly better than those with higher abundance, indicating its relationship with the prognosis of PCa [9, 10]. Growing evidence suggests that tumor-released exosomes that are a kind of extracellular vesicles subpopulations with diameters ranging from 30 to 150 nm, can act as a carrier for intercellular signal exchange and are vital in shaping the tumor microenvironment [11–13]. These exosomes carry cargos like mRNA, proteins, and non-coding RNA, influencing the recipient cells and their microenvironment [11–13]. It has been established that tumor cells exosome-delivered proteins can regulate manipulate immune cells and establish a microenvironment conducive to tumor growth and metastasis [11–13]. It has been demonstrated by Urabe et al. that extracellular vesicles derived from metastatic prostate cancer can facilitate osteoclastogenesis by transferring CDCP molecules [14]. Lu et al. revealed that tumor cell exosomes could regulate monocyte M2 polarization through integration of α_vβ₆ integrin by cycling intracellularly in PCa progression [15]. However, the involvement of tumor-derived exosomes and TAMs in PCa progression warrants further investigation.

Prostate-specific membrane antigen (PSMA), primarily produced by prostate epithelial cells, is a type II transmembrane glycoprotein with functions related to N-acetylated α-linked acidic dipeptidase and folate hydrolase. It is significantly elevated in conditions like BPH, PCa, castration-resistant PCa, and metastatic PCa, and its expression levels correlate with tumor grade and stage [16, 17]. Notably, our group found that a novel identified alternatively spliced PSMA variant, namely PSM-E, is also specifically overexpressed in PCa and correlated strongly with PCa stage and grade [18]. Subsequently, our study has shown that PSM-E can inhibit the proliferation, migration, and invasiveness of PCa cells [18, 19]. Current evidences indicate that PSMA-containing exosomes hold promise as a target for early PCa diagnosis, treatment response assessment, and prognosis [20–22]. Recently, we proposed that PSM-E could suppress the production of inflammatory cytokines in the tumor microenvironment and inhibit monocyte chemotaxis [23]. Importantly, our findings further revealed PSM-E expression was negatively correlated with the infiltration of M2-like TAMs in PCa tissue [23]. PSM-E holds significant potential for early diagnosis and treatment response of PCa assessment owing to its high tissue and pathological specificity. Despite these discoveries, the mechanisms associating PSM-E with the tumor microenvironment in PCa progression remain unclear, warranting further research.

This work focused on the impact of PSM-E in the tumor microenvironment on PCa progression and the underlying mechanisms. We provided hitherto undocumented evidence of PSM-E-containing exosomes in the extracellular environment of PCa patients, including serum and urine samples. Correlation analysis revealed a close association between the expression of urine exosomal PSM-E and the clinical stage and Gleason score in PCa patients. We also identified the ability of cellular exosomes to deliver PSM-E protein intercellularly, thereby transmitting its tumor-inhibiting and anti-metastatic effects. Furthermore, we revealed the involvement of PSM-E in macrophage polarization, suggesting that PCa-derived PSM-E-containing exosomes contribute to the regulation of M2 macrophage differentiation, a key factor in PCa progression. Mechanistically, PSM-E-laden exosomes from PCa cells were proved to suppress the FAK and ERK signal pathways by binding with RACK1. Taken together, our study illustrates that PSM-E-containing exosomes can hinder the progression of PCa by influencing macrophage polarization and metastasis, making it a potential prognostic marker for PCa progression and a target for anti-PCa therapy.

Participants, blood, and urine sampling

Serum and urine samples were collected from PCa patients undergoing surgery at the Department of Urology, the Affiliated Hospital of Sun Yat-sen University. Clinicopathological diagnoses were confirmed by at least two pathologists following the American Joint Committee on Cancer (AJCC) guidelines. Inclusion criteria required patients to have undergone prostate needle biopsy and received a histological diagnosis of PCa. Neither had undergone neoadjuvant androgen deprivation therapy nor had they ever been diagnosed with cancer. The control group consisted of males with total PSA levels below 4 ng/ml, showing no significant changes in ultrasound imaging or signs of prostatitis. The study involved 45 control cases, and 48 PCa cases. The first morning urine samples (10 ml) were collected from all participants, and peripheral venous blood was drawn into sodium citrate-free blood collection tubes to obtain serum. Both urine and blood samples were taken prior to any treatment. Prior to exosome was extracted from serum and urine samples of PCa patients by centrifugation of serum and urine at 3000 rpm for 10 min to remove cellular debris and deposits. This study followed ethical principles approved by the Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University.

Cell culture

Human PCa cell lines (PC3 and LNCaP) and human THP-1 were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA), and the human kidney cell line 293T was cultured in DMEM (Invitrogen), respectively. The culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen), 2 mM L-glutamine, 2 mM nonessential amino acids, 100 µg/ml streptomycin, and 100 units/ml penicillin (Invitrogen). Cells were maintained in an incubator under 37°C and a humidified atmosphere with 5% CO₂. THP-1 cells (1 × 10⁶ cells per well) were seeded in 60 mm dishs and settled for 24 h before being treating with 100 nM phorbol myristate acetate (PMA, Spring & Autumn, Nanjing, China), followed by culture with 20 ng/ml IL-4 for an additional 48 h to differentiate into M2 macrophages.

Plasmids and transfection

PSM-E plasmids with Flag epitope tags and RACK1 plasmids with HA epitope tags were amplified by RT-PCR and inserted into the pSin-EF2-puro vector. DNA sequencing was used to verify the plasmids. Small interfering RNA (siRNA) for PSM-E, purchased from Ruibo Biotechnology (Guangzhou, China). All PCR primers and siRNA sequence used in this work is shown in Supplementary Table S1. Plasmids and siRNA were transfected at specified concentrations using Lipofectamine 3000 reagents (Invitrogen) following the manufacturer's guidelines. Lentivirus containing PSM-E segments was harvested from 293T cells that transfected with overexpressing plasmids (pSin-EF2-puro vector), with psPAX2 and pMD2G. 48 hours after transfection, virus particles were harvested from the culture medium. PC3 and 293T cells were infected with lentivirus containing PSM-E ORF, and stable cell lines were selected and generated in DMEM supplemented with 5 µg/ml of puromycin.

Isolation and characterization of exosome

Three distinct cell groups were utilized in this study, including 293T cells stably overexpressing PSM-E-Flag (referred to as 293T-PSM-E), PC3 cells stably overexpressing PSM-E-Flag (referred to as PC3-PSM-E), and LNCap cells displaying high basal-level PSM-E protein. All these cell groups were grown to monolayer with 80–90% confluence in a medium supplemented with 10% of exosome-free FBS. Then, the conditioned medium was harvested and centrifuged twice (3000 × g for 15 minutes) to remove cells and centrifuged once (20,000 × g for 30 minutes) to remove cellular debris. Subsequently, the supernatants were further centrifuged at 100,000 × g for 70 minutes, and all these steps were meticulously performed at 4°C [24]. Then, the supernatants were concentrated ten-fold using an Amicon Ultra-15 centrifugal filter unit with a 3 kDa cutoff value. Exosomes derived from serum and urine were isolated using an Exosome Concentration Isolation Kit (Liaoning Rengen Biosciences Co., Ltd, Liaoning, China). These purified exosomes were resuspended in PBS. The protein content of these exosomes was quantified using the Bicinchoninic Acid protein assay kit (BCA, Thermo Scientific Pierce, Rockford, IL) with BSA as a reference, following the manufacturer's guidelines. As described previously, the exosomes were visualized through negative staining and analyzed via transmission electron microscopy (TEM) [25]. Finally, using Western blotting analysis, we characterized the exosomes loaded with PSM-E and excised the molecular weight-specific band from the gel for subsequent mass spectrometry analysis (MS).

Western blotting analysis

Cells were lysed using lysis buffer (composed of 50 mmol/L Tris-HCl with pH 7.4, 6% SDS, 5% mercaptoethanol, 10% glycerol, 1 mmol/L PMSF, and 0.1% bromophenol blue). The protein concentration of the lysates was quantified using the BCA assay kit with BSA as a reference, per the instructions. Subsequently, the lysed proteins were separated through SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Various primary antibodies were used, such as anti-CD63 (Santa Cruz Biotechnology, Dallas, Texas), anti-CD206 (Abcam, Cambridge, UK), anti-Flotillin-2 (Abcam), anti-Flag (Cell Signaling Technology, Beverly, MA), anti-RACK1 (Abcam), anti-HA (CST), anti-ERK (CST), anti-p-ERK (Cell Signaling Technology), anti-FAK (CST), anti-p-FAK, and anti-GAPDH (ABclonal Technology, Wuhan, China). Incubation with these antibodies was carried out overnight at 4°C, followed by a 1-hour incubation with anti-rabbit (ABclonal Technology) or anti-mouse (ABclonal Technology) secondary antibodies at room temperature. GAPDH was employed as a loading control for normalization in quantification. An enhanced ECL reagent (Thermo Fisher Scientific, Rockford, IL) was used to visualize the chemiluminescence bands.

LC-MS/MS analysis of PSM-E

The study utilized the Waters Series ACQUITY Premier system (Waters Corporation, Milford) and a Xevo TQ-S LC/MS mass spectrometer (Waters Corporation, MA). Liquid chromatography separations were carried out on a ZORBAX Eclipse XDB-C18 (Agilent, Waldbronn, Germany) at room temperature. The mobile phase comprised solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in methanol). A linear gradient with a flow rate of 0.3 mL/min was applied in the following manner: 10% B (0 min), 10% B (1 min), 90% B (4 min), 90% B (8 min), and 10% B (9 min). An electrospray ion source was used to interface the mass spectrometer with the positive MRM mode. The injection volume was 10 µl. The electrospray capillary voltage was optimized to 70 V, and the nebulizer pressure was set to 35 psi. The data were collected using the Waters MassLynx Mass Spectrometry Software (version 4.1).

Wound healing assay

Cells (1 × 10⁶ cells/well) were seeded in a six-well plate, ensuring they reached 90% confluence at the time of scratch wounding. To inhibit cell mitosis, cells were pretreated with mitomycin C at 10 µg/ml (Sigma-Aldrich) for 2 hours. The non-adherent cells were eliminated by washing them with a culture medium after three straight scratch wounds were created in each well using a pipette tip (10 µl). Purified exosomes derived from 293T-PSM-E cells or 293T-Vector cells were added to the wells and incubated for up to 72 hours. Using a bright-field microscope, the wound width was measured immediately after wounding (at 0 hours) and at 24 hours.

Invasion assays

The invasion assays were conducted using cell culture inserts with 8.0 µm pore size (Falcon, Corning, Tewksbury MA), coated with 10% Matrigel or not (BD Biosciences). In brief, the upper chamber was seeded with PC3, and the lower chamber was incubated overnight with 500 µl of fresh medium. The medium was then replaced with fresh medium, and the cells were incubated with medium alone or stimulated with purified exosomes derived from 293T-PSM-E cells or 293T-Vector cells for 72 hours. Those cells in the upper chamber that were non-migratory were removed, and the remaining cells were gently washed with 1×PBS. A 0.5% crystal violet solution was used to stain these adhered cells that had migrated through the filter for 10 minutes. Images were captured using light microscopy.

RNA extraction and quantitative real-time PCR

Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The first-strand cDNA was synthesized using HiScript II Q Select RT SuperMix for qPCR (Vazyme, Jing Nan, China), and real-time PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme,) or AceQ qPCR Probe Master Mix (Vazyme) according to the manufacturer's protocols. The primer groups used for real-time PCR are provided in Supplementary Table 1. Three independent experiments were conducted, and the data were expressed as ratios relative to GAPDH and presented as the mean ± S.D. values.

Co-immunoprecipitation

Cells transfected with the specified plasmids for 48 hours were harvested and collected with lysis buffer (1 mmol/L EDTA, 25 mmol/L HEPES, 2% glycerol, 150 mmol/L NaCl, 1% NP-40, and cocktail (Roche, Basel, Switzerland). Mixed lysates were kept on ice for 30 minutes, followed by 10 minutes of centrifugation at 12,000 rpm. For pre-cleaning, the supernatant was incubated with 20 µl of agarose beads (Calbiochem) for 1 hour with rotation at 4°C. After centrifuging at 2,000 rpm for 1 minute, the lysates were then incubated overnight at 4°C with the indicated antibodies or IgG antibody. Then, the precipitates were washed three times with IP wash buffer (150 mmol/L NaCl, 20 mmol/L HEPES, 2% glycerol, 1 mmol/L EDTA, 0.1% NP-40, and 1 mmol/L EGTA). After completely removing the liquid, the bound proteins were resolved in 1× sample buffer, subjected to SDS-PAGE, and detected by Western blotting analysis.

In vivo tumorigenesis model

C57BL/6 mice aged 5–6 weeks were obtained from Guangzhou Forevergen Medical Laboratory Animal Center and housed in specific pathogen-free (SPF) barrier facilities on a 12-hour light/dark cycle. On day zero, RM-1 cells (1 × 10⁵) were subcutaneously injected into the right dorsum. Before treatment, mice were randomly divided into two treatment groups and two control groups in a blinded fashion, with each group consisting of five mice. On day 7, when tumors had formed and were measurable, two treatment groups of mice received intraperitoneal (i.p.) injections of PC3-Vector-Exosomes (10 µg/kg body weight) and LNCaP-siPSM-E-Exosomes (10 µg/kg body weight) every three days. In contrast, the vehicle-control animals received i.p. doses of 200 µl PC3-Vector-Exosomes or LNCaP-siNC-Exosomes solution per mouse daily. Every four days, the diameter of each mouse tumor was measured with a digital caliper by measuring perpendicular diameters in a blinded manner. And tumor volume was calculated using the formula: tumor volume (mm³) = (length [mm]) × (width [mm])² × 0.5. The weight of each mouse tumor was also recorded. We euthanized all animals, and dissected and weighed the tumors at the end of the experiment. All procedures involving animals were approved by the Ethics Committee of Guangzhou Forevergen Medical Laboratory Animal Center under approval No. IACUC-AEWC-F2305037.

Immunohistochemical staining (IHC)

IHC analysis was performed in accordance with the protocol previously described [26]. In brief, Paraffin sections of mouse prostate cancer tissues were prepared and firstly deparaffinized and hydrated. Microwave method used to retrieve antigens in the, followed by incubation with 0.3% hydrogen peroxide solution to block endogenous peroxidase activity. An anti-CD206 antibody (CST) was used to detect CD206 level in mouse tumors tissue. An antibody against Ki67 (Servicebio; Wuhan, China) were used to detect Ki67 level in mouse tumors tissue. The sections were developed using 3,3’-diaminobenzidine tetrahydrochloride and peroxidase after incubation with primary and secondary antibodies. After counterstaining with hematoxylin, the sections were mounted in a nonaqueous mounting medium. Images were visualized using a Leica DM2500 microscope system (Leica, Germany). Image J Version 1.5i software (Media Cyernetics, Inc, USA) was used to evaluate the percentage of the staining positively area.

Statistical analysis

This study was carried out using the GraphPad Prism software (version 9.5) for all statistical analyses. Data conforming to normal distribution were presented as means ± standard deviations (S.D.) from three independent experiments. Comparisons between the two groups were made using independent Student's t-tests. The one-way ANOVA was used for multiple comparisons of continuous variables, followed by the Tukey-Kramer test. Data that non-normally distributed were expressed with the median and interquartile range, and the Kruskal-Wallis and Dunn's tests were used for multiple comparisons. Correlations were determined through Spearman rank correlation analysis. Additionally, receiver-operating characteristic (ROC) analysis was carried out for the levels of PSM-E, tPSA, and f/t PSA in the context of PCa. Plotting true-positive fractions (sensitivity) against false-positive fractions (specificity) can be accomplished by changing the cutoff value used in the analysis. Diagnostic test accuracy is measured by the areas under ROC curves. Significant differences were denoted in charts as follows: *p < 0.05, **p < 0.01, and ***p < 0.001. p values less than 0.05 indicated a statistically significant difference, while "ns" indicated no significance. All statistical analyses were based on at least three biological replicates.

Clinical characteristics of the study samples

A summary of the participants' baseline characteristics is shown in Table 1, which encompassing age, Gleason score, TNM stage, lymph node metastasis, and PSA serum levels. Serum and urine samples were collected from 93 participants, including control (n = 45), and PCa (n = 48) cases.

Table 1

Associations between urine-derived exosomal PSM-E expression and clinicopathological characteristics of PCa patients
Characteristic	Cases (%) (n = 48)	PSM-E expression (%)		p
Characteristic	Cases (%) (n = 48)	high	low	p
Age
< 66	16 (33.3)	8	8	0.838
≥ 66	32 (66.7)	17	15	0.838
Gleason score
≤ 6	6 (12.5)	2	4	0.028 *
3 + 4	12(25)	5	7
4 + 3	11(22.9)	8	3
≥ 8	19 (39.6)	10	9
TNM stage
I/II	17 (35)	5	12	0.003 **
III/IV	31 (65)	20	11	0.003 **
Lymph node metastasis
Yes	6 (12.5)	2	4	0.383
No	42 (87.5)	20	22	0.383
fPSA
< 4 ng/ml	40 (83.3)	19	21	0.113
≥ 4 ng/ml	8 (16.7)	6	2	0.113
p value < 0.05 was considered significant. p < 0.05, p < 0.01, and **p < 0.001

PSM-E is encapsulated within serum and urine-derived exosomes from PCa patients

Exosomes were isolated from serum or urine samples obtained from the participants using the corresponding commercial kits. Subsequently, exosomes purified from 5 samples underwent transmission electron microscopy and Western blotting analysis to detect PSM-E protein levels. The electron micrographs of exosomes revealed vesicle sizes primarily around 100 nm (Fig. 1A, B). Notably, PSM-E was enriched in serum-derived exosomes from BPH and PCa patients, and abundant in those isolated from urine samples of BPH and PCa patients (Fig. 1C, D). Importantly, PSM-E levels in PCa patients were significantly higher than those in control group. In contrast, exosomes from the control group did not contain detectable PSM-E, with CD63 and flotillin-2 as exosomal control markers. In summary, these findings demonstrate that PSM-E is found in both the serum and urine of BPH and PCa patients.

Urine-derived exosomal PSM-E yielded a good diagnostic performance for PCa

In an attempt to investigate the value of urine-derived exosomal PSM-E in PCa, LC-MS/MS analysis was used to detect the protein level of PSM-E in the exosomes of each group by recognizing the specific peptide FLAAYACTGCLAER of PSM-E. As shown in Fig. 1E, urine-derived exosomal PSM-E concentration was significantly elevated in the PCa patients compared to the control group (p < 0.001). Exosomal PSM-E expression exhibited significantly higher levels in patients with PCa than those with BPH (p < 0.01). Remarkably, high level of exosomal PSM-E positively correlated with a high Gleason score (≥ 8, p < 0.05) and advanced pathological tumor stage (III/IV TNM stage p < 0.01) (Fig. 1F, G,).

To analyze the diagnostic specificity and sensitivity of exosomal PSM-E for PCa compared with those of tPSA, ROC curves for the detection of PCa were applied. As shown in Fig. 1H, I, in all PCa subjects, the values of AUC were 0.8904 (95% CI, 0.8311 to 0.9497) for exosomal PSM-E, 0.5295(0.3812 to 0.6779) for tPSA and 0.7189 (95% CI, 0.5890 to 0.8487) for f/t PSA. These differences between exosomal PSM-E and f/t PSA were statistically significant in all PCa subjects (p < 0.0001 and p < 0.001). Using a cutoff value of 42.08 ng/ml for exosomal PSM-E, exosomal PSM-E level significantly differentiated PCa patients from control cases among consecutive patients with 75% sensitivity and 80% specificity for diagnosing PCa. These results suggest that exosomal PSM-E exhibited good performance in distinguishing PCa and outperformed other traditional clinical biomarkers tPSA or f/t PSA.

PSM-E can be exported from cells by exosomes

We next sought to investigate whether PSM-E is present in cell-derived exosomes. Specifically, PSM-E was overexpressed in 293T and PC3 cells, which had low basal levels of PSM-E (Supplementary Figure S1A), through transfection with the PSM-E-Flag construct. Subsequently, culture medium supernatants were collected and processed through a series of steps, which included two rounds of centrifugation at 4°C so as to remove cells and cell debris. The resulting supernatants were further passed through 0.22-µm filters. After this, using a centrifugal filter unit (Amicon Ultra-15) with a 3 kDa cutoff value, the supernatants were concentrated ten-fold. As depicted in Fig. 2A and 2B, our findings demonstrated the presence of PSM-E-Flag protein both in the cytoplasm and the in culture medium supernatants. Additionally, Western blotting analysis, utilizing a PSM-E-specific antibody, confirmed the presence of PSM-E protein in the supernatant derived from LNCaP cells with high basal levels of endogenous PSM-E (Fig. 2C). Meanwhile, we constructed siRNAs targeting the back-splice region of PSM-E (PSM-E siRNA) to depleted basal-level PSM-E expression in LNCaP cells (Supplementary Figure S1B).

Since a putative signal peptide is not present in the amino acid sequence of PSM-E, we hypothesized that PSM-E is exported via an exosome-mediated mechanism. To validate this hypothesis, we isolated exosomes from the culture media of various cell lines, including 293T cells transfected with PSM-E (293T-PSM-E), 293T cells transfected with control vector (293T-Vector), PSM-E-transfected PC3 cells (PC3-PSM-E), control vector-transfected PC3 cells (PC3-Vector), LNCaP cells, PSM-E siRNA transfected LNCaP cells (LNCaP-siRNA), and a non-targeting control siRNA transfected LNCaP cells (LNCaP-siNC). As shown in Fig. 2D-F, electron microscopy revealed the typical double-layer membrane structure of exosomes with an approximate diameter of 100 nm. Notably, PSM-E was also abundant in those exosomes derived from 293T-PSM-E, PC3-PSM-E and LNCaP-siNC cells. In contrast, exosomes obtained from 293T-Vector, and PC3-Vector did not exhibit detectable levels of PSM-E, and transfection of PSM-E-siRNA efficiently depleted PSM-E expression level in these exosomes (Fig. 2G-I). These results were validated using CD63 and flotillin-2 as exosomal control markers (Fig. 2G-I). The presence of PSM-E in the exosome composition was further confirmed using mass spectrometry (Fig. 2J). Collectively, these data demonstrate that exosome secretion of PSM-E is not limited to LNCaP cells, which express high levels of endogenous PSM-E, but also occurs in 293T and PC3 cells transiently expressing exogenous PSM-E.

Exosomal PSM-E can be transferred to recipient cells

Exosomes are protein-containing extracellular vesicles known to play a major role in protein transport among cells. In light of the discovery that PSM-E is contained in exosomes and can be exported from cells expressing it, we investigated whether PSM-E can be transferred to recipient cells. To this end, we tested such a possibility by employed a co-culture system. After transfecting PC3 and 293T cells with a Flag epitope-tagged PSM-E plasmid, these cells were co-cultured with M0 macrophages in the transwell plates (Fig. 3A). Our data demonstrated that the Flag epitope-tagged PSM-E from the upper transwells was delivered into the recipient M0-type THP-1 cells, which were seeded in the lower wells (Fig. 3A), confirming that cells can indeed secrete extracellular PSM-E that is taken up by M0 macrophages.

Next, we tested whether recipient cells could take up exosomal PSM-E when treated with purified exosomes derived from PSM-E-expressing cells. We differentiated the human monocyte cell line THP-1 into M0-like macrophages using PMA, characterized by their adherent morphology and elevated expression of the macrophage marker CD68 (Supplementary Figure S2A). Subsequently, these M0 macrophages were incubated with exosomes purified from different sources, including PC3-Vector, PC3-PSM-E, LNCaP-siNC, and LNCaP-siPSM-E. As shown in Fig. 3B, an increased abundance of PSM-E in the recipient M0 macrophages treated with exosomes from PC3-PSM-E (PC3-Vector-Exos and PC3-PSM-E-Exos, 80 µg/ml). Additionally, the identification of PSM-E proteins in the lysates of recipient M0 macrophages treated with exosomes from LNCaP-siNC and LNCaP-siPSM-E further confirmed the transfer of PSM-E (Fig. 3C). A fluorescent dye PKH67 was added to the culture medium of M0 macrophages to identify these exosomes derived from cells. After 12 hours, the THP-1 cells exhibited efficient uptake of the cell-secreted exosomes, as indicated by the presence of green fluorescence staining in M0 macrophages (Fig. 3D).

Furthermore, to validate that exosomal transfer of PSM-E was responsible for the increase in PSM-E protein levels in recipient cells, PSM-E levels was measured in the recipient cells treated with various concentrations of PSM-E-containing exosomes. As shown in Fig. 3E, F, the finding demonstrated that the levels of PSM-E proteins in the lysates of recipient M0 macrophages and PC3 cells treated with PSM-E-laden exosomes derived from 293T-PSM-E cells (at 40 µg or 80 µg) were dose-dependent, as confirmed by immunoblotting. Importantly, when an extracellular vesicle secretion inhibitor, GW4869, was added to the cells 24 hours prior, it blocked exosome production and PSM-E delivery into the recipient M0-THP-1 cells, indicating that cells primarily secrete extracellular PSM-E in an exosome-dependent manner (Fig. 3G). Overall, these results demonstrate that prostate cancer cells can secrete PSM-E-containing exosomes efficiently taken up by recipient cells.

Exosomal PSM-E inhibits PCa cell migration, invasion, and the polarization of M0 macrophage to the M2 phenotype

Our previous studies revealed that PSM-E could suppress the proliferation, migration, and invasiveness of prostate cancer cells^18,19. To explore the effects of exosomal PSM-E on tumor migration and invasion, we conducted wound healing assays and transwell invasion experiments on recipient PC3 cells treated with exosomes derived from 293T-PSM-E and 293T-Vector cells at different concentrations (40 µg or 80 µg/ml). The results indicated that 293T-PSM-E-Exosomes inhibited PC3 cell migration and invasion compared to 293T-Vector-Exosomes (Fig. 4A, B). These results suggest that exosomal PSM-E can also suppress the invasive and metastatic abilities of PC3 cells.

Our previous findings revealed that PSM-E was specifically overexpressed in PCa and its expression is negatively correlated with the expression of CD206 (a specific marker of M2-type macrophages), which suppressing the secretion of inflammatory cytokines in the PCa microenvironment and inhibiting the chemotaxis of monocytes ²³. In this study, we focused on investigating PSM-E's potential impact on the tumor microenvironment in the context of PCa progression ²³. Additionally, M0 macrophages were treated with IL-4 to induce their differentiation into M2-like macrophages (Supplementary Figure S2B). Real-time RT-PCR analysis revealed a significant increase in the levels of M2 markers, including CCL17, CCL18, and CCL22, in M2 macrophages (Supplementary Figure S2C). Subsequently, we examined the effects of 293T cell-derived exosomal PSM-E on the polarization of M0 macrophages towards the M2 phenotype. The expression levels of M2 markers (CCL17, CCL18, and CCL22) markedly decreased in M0 macrophages co-treated with 293T-PSM-E-Exos and IL-4, compared to those in the 293T-vector-Exos and IL-4 treatment groups (Fig. 4D), suggesting that the proportion of M2-type macrophages in THP-1 cells was reduced following treatment with PSM-E exosomes. In contrast, there was no change in the expression of M1 macrophage markers (iNOS and IL-1β) after similar treatment with 293T cell-derived exosomal PSM-E (Fig. 4E). As shown in Fig. 4F, our results demonstrated that the levels of CD206 proteins in the lysates of recipient THP-1 macrophages (THP-1 treated with PMA and IL-4) exposed to PSM-E-laden exosomes derived from 293T-PSM-E-cells (40 µg, 80 µg) exhibited a dose-dependent decrease, as determined by immunoblotting. In summary, the above findings confirm that cell-derived exosomal PSM-E can suppress the M2 polarization of macrophages.

PMS-E colocalizes and interacts with RACK1

We further sought to explore the mechanisms underlying the inhibitory effect of PCa-derived exosomal PSM-E on the M2 polarization of macrophages. To this end, using the protein G-conjugated IgG antibody or anti-Flag monoclonal antibody, lysates from PC3-PSM-E-Flag cells were immunoprecipitated (Fig. 5A), followed by a pull-down experiment, and subsequently analyzed the immunoprecipitates by LC-MS/MS (Fig. 5A). We screened the putative interacting proteins with PSM-E obtained from IP-MS analysis based on the unique peptides (unique peptides ≥ 2) and peptide spectrum scores ranking (Fig. 5A). Notably, RACK1 was picked out as a potential candidate protein, and the mass spectrometer of the RACK1 peptide segment was depicted in Fig. 5B. To validate that PSM-E interacts with RACK1, we performed co-immunoprecipitation (co-IP) assays after co-transfection of PSM-E-Flag and RACK1-HA expression plasmids in 293T cells. Significantly, as shown in Fig. 5C and 5D, our results showed that RACK1-HA was precipitated by the Flag antibody, meanwhile, PSM-E-Flag was precipitated by the HA antibody, revealing the formation of a complex between PSM-E-Flag and RACK1-HA. Furthermore, the interaction between Flag-tagged PSM-E and RACK1 was investigated using an IP assay using endogenous RACK1 as a precipitate, which resulted in the successful pull-down of endogenous RACK1 together with Flag-tagged PSM-E (Fig. 5E).

To identify the specific domains of PSM-E responsible for its interaction with RACK1, we generated two deletion mutants that removed the protease-associated domain (PA) and the peptidase M28 domain (M28) of PSM-E. Co-IP assays revealed that the protease-associated domain of PSM-E was crucial for its interaction with RACK1 (Fig. 5F). It is well-established that RACK1 belongs to the tryptophan- aspartate repeat (WD-repeat) proteins family which share significant homology with the β subunit of G-proteins. We generated five serial WD domain deletion constructs, including full-length (FL), WD2-7, WD3-7, WD4-7, and WD5-7 (Fig. 5G). Co-IP assays using an anti-HA antibody and subsequent immunoblotting with an anti-Flag antibody showed that the fourth WD domain of RACK1 was essential for its interaction with PSM-E (Fig. 5G). Taken together, these results indicate that the protease-associated domain of PSM-E and the fourth WD domain of RACK1 are crucial for the interaction between PSM-E and RACK1.

Exosomal PSM-E PCa growth and correlates with low infiltration of M2 macrophage in vivo

To assess the anti-PCa activity of exosomal PSM-E in vivo, we established a subcutaneous PCa tumorigenesis model in immunocompetent C57BL/6J mice. In this model, we subcutaneously injected RM-1 cells into C57BL/6J mice to develop solid tumors until the tumors reached a volume of approximately 50 mm³ (Supplementary Figure S3A). We then monitored the tumor growth after intraperitoneal injections of exosomes derived from PC3 cells with exogenously overexpressed PSM-E (PC3-Vector-Exos, PC3-PSM-E-Exos) or LNCaP cells with high basal levels of endogenous PSM-E (LNCaP-siNC-Exos and LNCaP-siPSM-E -Exos) every three days for four weeks (Supplementary Figure S3A). As shown in Supplementary Fig. 6A and Figure S3B, the volume of the homograft PCa tumors was significantly reduced by 43.6in mice treated with PC3-PSM-E-Exos. The weight of the homograft tumors at the experimental endpoint was also notably reduced by 49.12 ± 3.81% in C57BL/6 mice treated with PC3-PSM-E-Exos (Fig. 6B). Conversely, compared to the LNCaP-siNC-Exos group, the volume and weight of tumors derived from the LNCaP-siPSM-E-Exos group were significantly increased (Fig. 6C, D and Figure S3C). IHC analysis of mouse homograft tumors revealed a significantly reduced expression of CD206⁺ (a specific marker of M2-type tumor-associated macrophages) in the PC3-PSM-E-Exos treatment group, while a higher expression of CD206⁺ was observed in the LNCaP-siPSM-E-Exos treatment group (Fig. 6E). Furthermore, the results revealed higher expression of the proliferation marker Ki67 in the tumors derived from the LNCaP-siPSM-E-Exos group compared to those in the PC3-PSM-E-Exos group (Fig. 6E). These findings suggest that exosomal PSM-E can suppress tumor growth and correlate with a lower infiltration of M2 macrophages in vivo.

Exosomal PMS-E inhibits M2 polarization of macrophages via suppression of the ERK and FAK signaling pathways

Numerous studies have indicated that the activation of the ERK signaling pathway in macrophages is involved in the M2 polarization of macrophages [27]. Moreover, RACK1 has been reported to regulate the ERK and FAK pathway in various cellular activities [28]. To gain a deeper understanding of the RACK1 signaling cascades that mediate the inhibitory effect of exosomal PMS-E on M2 macrophage polarization, we examined changes in the phosphorylation status of RACK1 downstream substrates, such as ERK and FAK, using Western blotting. As shown in Fig. 7A and 7B, exosomal PSM-E led to a significant decrease in the levels of phosphorylated FAK and ERK (p-FAK and p-ERK) without affecting the expression of total ERK and FAK in both PC3 and M0 macrophages (THP-1 macrophage cells induced by PMA) in a dose-dependent manner. In contrast, the attenuation of PSM-E expression in LNCaP cells through transfection with PSM-E siRNA (siPSM-E) reversed the activation of the ERK/FAK pathway (Fig. 7C). Notably, M2-like macrophages treated with FR180204 (an ERK inhibitor) for 12 hours exhibited a significant decrease in phosphorylated ERK and FAK levels without affecting the expression of total ERK and FAK (Fig. 7D). As expected, CD206 expression decreased in M2 macrophages treated with 293T-PSM-E-Exos (Fig. 7E). In summary, these results suggest that exosomal PSM-E could inhibit M2 macrophage polarization via suppressing the RACK1-ERK/FAK signaling pathway.

It is now understood that metastasis is the leading cause of PCa-related mortality and results from complex interactions between the tumor microenvironment and tumor cells within the prostate [29], which drives tumor progression and distant spread. Herein, we demonstrated the presence of PSM-E-enriched exosomes in the urine of PCa patients, which significantly correlated with PCa tumor grade. Importantly, we uncovered that PSM-E-enriched exosomes could hinder PCa progression by facilitating communication between PCa cells and TAMs in the metastatic microenvironment. We also elucidated the underlying mechanism of PSM-E-triggered TAM polarization, revealing that RACK1, an integral component of PSM-E, suppresses the ERK and FAK signaling pathways during M2 polarization. The involvement of exosomal PSM-E in the interplay between TAMs and tumor cells provides insights into the molecular mechanisms of PCa proliferation and metastasis and suggests that it may serve as a novel marker for liquid biopsy in PCa.

It is well-established that a large proportion of PCa cases diagnosed through PSA screening are not clinically significant due to a high false-positive rate. Consequently, there is an urgent need for development of novel biomarkers to enhance the accuracy of PCa diagnosis. Current evidence suggests that exosomes can transfer proteins, RNAs, DNA, lipids, and metabolites, making them promising surrogates for tissue biopsy [30, 31]. Thus, exosome isolated from body fluids, such as serum, saliva, and urine, from cancer patients, have emerged as important alternative biomarkers for tumor early diagnosis and prognosis prediction, as they carry host oncogenes and tumor suppressor genes [30–32]. Recently, the exosomes in blood, saliva or urine from patients suffering from different cancers have been frequently reported to be liquid biopsy sources for cancer detection [30, 31]. Our current study demonstrated for the first time that the presence of PSM-E-containing exosomes in the extracellular environment, including serum and urine from patients with PCa. We found that the PCa group had significantly higher urine-derived exosomal PSM-E concentrations than the control group, also with markedly higher levels of exosomal PSM-E compared to those with BPH. Furthermore, a positive correlation was found between high levels of exosomal PSM-E and high Gleason scores, advanced pathological tumor stages. Notably, urine-derived exosomal PSM-E yielded an AUC of 0.8904 in diagnosing PCa. These findings suggest that urine-derived exosomal PSM-E represents a novel, non-invasive indicator for early PCa diagnosis and progression prediction, contributing to the development of non-invasive liquid biopsy techniques in the future.

Emerging evidence has highlighted the role of TAM recruitment and M2 polarization in tumor growth, invasion, and metastasis [33]. Macrophages in the tumor microenvironment typically display an M2-like phenotype, promoting the growth, metastasis, and invasion of prostate cancer cells [34]. Notably, tumor-derived exosomes, as a primary source of environmental signals, have been shown to induce macrophages to adopt an M2-like phenotype [33, 34]. Thus, the cargo within exosomes regulates highly efficient communication between tumor cells and macrophages. Our study demonstrated the PSM-E-containing exosomes was found in the tumour extracellular environment, and unveiled their capacity to transport PSM-E between cells, thereby conveying its anti-tumor impact. Our findings showed that PSM-E-containing exosomes could suppress PCa cell invasion and metastasis in vitro. Intriguingly, in a prostate cancer mouse model, exosomal PSM-E significantly inhibited tumor growth. Furthermore, our study demonstrated that high PSM-E expression and M2-type TAMs exhibited opposing trends in both cellular and animal models, consistent with our previous findings in clinical PCa tissues [23]. Based on these findings, it is highly conceivable that PSM-E-containing exosomes inhibit M2 macrophage polarization, attenuating the pro-tumor effect and suggesting PSM-E as a promising therapeutic target for PCa progression.

At the molecular level, we identified RACK1 as the binding partner of PSM-E. It has been established that PSM-E contains a protease-associated domain (150–248), peptidase-28 domain (355–546), and TfR domain (639–703). We thus deleted the protease-associated domain and peptidase-28 domain of PSM-E and found that the protease associated domain of PSM-E is indispensable for its interaction with RACK1. Meanwhile, RACK1 is a 36-kDa protein with seven tryptophan-aspartate repeat (WD-repeat)domains that mediate protein-protein interactions [28]. We further examined which portions of RACK1 were critical for binding to PSM-E, and the co-IP experiments indicated that the fourth WD domain was crucial for their interaction. This domain is also required for membrane localization, indicating that PSM-E may exert its function by interacting with and influencing RACK1. RACK1 is a highly conserved WD40 repeat scaffold protein known to bind to many proteins, thereby impacting multiple signal transduction pathways through compartmentalization [28]. RACK1 is able to integrate inputs from different signaling pathways because WD40 repeats interact with different signaling molecules simultaneously, including ERK signaling [28] and FAK signaling [35].

Our findings provided evidence of how PCa cell-derived exosomal PSM-E suppressed tumor metastasis by modulating the RACK1/FAK/ERK signaling pathway. We also revealed that serum and urine exosomal PSM-E could serve as a marker for metastatic disease and prognostic prediction in PCa patients. Our study uncovered the crucial mechanism of exosomal PSM-E-mediated intercellular communication from PCa cells to the tumor microenvironment, endowing common PCa cells with metastatic features, paving the way for a potential non-invasive diagnostic approach for PCa.

Data sharing statement

All data available upon request to the corresponding authors.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the National Natural Science Foundation of China (82071352, 82341094, 82171675, 81672556), Guangdong Basic and Applied Basic Research Foundation (2022A1515011156, 2021A1515010597), Guangdong Marine Economy Development Special Project (NO. GDNRC [2022]35).

Author Contribution

Conception and design: KC, YC, HG and XZ; Experiments and analysis: XQ, RN, YT, YH, WR, WZ, HW, JZ, MX and XZ; Drafting the manuscript: XQ, RN, YT, and YH. Supervision: KC, YC, and XZ. All authors contributed to the revision of the manuscript and approved the final version.

Acknowledgements

Not applicable

Siegel RL, Miller KD, Fuchs HE and Jemal A. Cancer statistics, CA: a cancer journal for clinicians. 2022; 72: 7-33.
Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A and Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians. 2021; 71: 209-249.
Wyatt AW and Gleave ME. Targeting the adaptive molecular landscape of castration-resistant prostate cancer. EMBO Mol Med. 2015; 7(7): 878-894.
Prensner JR, Rubin MA, Wei JT, and Chinnaiyan AM. Beyond PSA: the next generation of prostate cancer biomarkers. Sci Transl Med. 2012; 4(127): 127rv123.
Lilja H, Ulmert D and Vickers AJ. Prostate-specific antigen and prostate cancer: prediction, detection and monitoring. Nat Rev Cancer. 2008; 8(4): 268-278.
Turley SJ, Cremasco V and Astarita JL. Immunological hallmarks of stromal cells in the tumour microenvironment. Nat Rev Immunol. 2015; 15(11): 669-682.
Cassetta L and Pollard JW. Tumor-associated macrophages. Curr Biol. 2020; 30 (6): R246-R248.
Pan Y, Yu YD, Wang, XJ and Zhang T. Tumor-Associated Macrophages in Tumor Immunity. Front immunol. 2020; 11: 583084.
Han CL, Deng YX, Xu, WC, Liu Z, Wang T, Wang SG, Liu JH and Liu XM. The Roles of Tumor-Associated Macrophages in Prostate Cancer. J Oncol. 2022; 7: 8580043.
Shi F, Sun MH, Zhou Z, Wu L, Zhu Z, Xia SJ, Han BM, Zhao YY, Jing YF and Cui D. Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway. Int J Biol Sci. 2022; 18(16): 5994-6007.
Kalluri R and LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020; 367 (6478): eaau6977.
van Niel G, Carter D, Clayton A, Lambert DW, Raposo G and Vader P. Challenges and directions in studying cell-cell communication by extracellular vesicles. Nat Rev Mol Cell Biol. 2022; 23(5): 369-382.
Wang XY, Huang J, Chen WJ, Li GP, Li ZH and Lei JY. The updated role of exosomal proteins in the diagnosis, prognosis, and treatment of cancer. Exp Mol Med. 2022; 54(9): 1390-1400.
Urabe F, Kosaka N, Yamamoto Y, Ito K, Otsuka K, Soekmadji C, Egawa S, Kimura T and Ochiya T. Metastatic prostate cancer-derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein. J Extracell Vesicles. 2023; 12(3): e12312.
Lu H, Bowler N, Harshyne LA, Craig Hooper D, Krishn SR, Kurtoglu S, Fedele C, Liu Q, Tang HY, Kossenkov AV, et al. Exosomal αvβ6 integrin is required for monocyte M2 polarization in prostate cancer. Matrix Biology. 2018; 70: 20-35.
Plichta KA, Graves SA and Buatti JM. Prostate-Specific Membrane Antigen (PSMA) Theranostics for Treatment of Oligometastatic Prostate Cancer. Int J Mol Sci. 2021; 22(22): 12095.
Silver DA, Pellicer I, Fair WR, Heston WD and Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997; 3(1): 81-5.
Cao KY, Mao XP, Wang DH, Xu L, Yuan GQ, Dai SQ, Zheng BJ and Qiu SP. High expression of PSM-E correlated with tumor grade in prostate cancer: a new alternatively spliced variant of prostate-specific membrane antigen. The Prostate. 2007; 67: 1791-1800.
Cao KY, Xu L, Zhang DM, Zhang XM, Zhang T, He X, Wang Z, Feng FS, Qiu SP and Shen GX. New alternatively spliced variant of prostate-specific membrane antigen PSM-E suppresses the proliferation, migration and invasiveness of prostate cancer cells. Int J Oncol. 2012; 40(6): 1977-1985.
Negahdary M, Sattarahmady N, and Heli H. Advances in prostate specific antigen biosensors-impact of nanotechnology. Clin Chim Acta. 2020; 50: 43-55.
Li P, Yu XY, Han WJ, Kong Y, Bao WY, Zhang JQ, Zhang WC and Gu YQ. Ultrasensitive and Reversible Nanoplatform of Urinary Exosomes for Prostate Cancer Diagnosis. ACS Sens. 2019; 4(5): 1433-1441.
Wang CB, Chen SH, Zhao L, Jin X, Chen X, Ji J, Mo ZN and Wang FB. Urine-derived exosomal PSMA is a promising diagnostic biomarker for the detection of prostate cancer on initial biopsy. ClinTransl Oncol. 2023; 25(3):758-767.
Niu RX, Yuan L, Tan CH, et al. Inhibitory effect of PSM-E on secretion of inflammatory cytokines and chemotaxis of monocytes. J Trop Med. 2021;4:1672-3619.
Xu Y, Yao Y, Yu L, et al. Clathrin light A-enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis. J Extracell Vesicles. 2023;12(8): e12359.
Wei Y, Li M, Cui S, Wang D, Zhang CY, Zen K and Li L. Shikonin Inhibits the Proliferation of Human Breast Cancer Cells by Reducing Tumor-Derived Exosomes. Molecules (Basel, Switzerland). 2016; 21(6):777.
Jin H, Xu M, Padakanti PK, Liu Y, Lapi S and Tu Z. Preclinical evaluation of the novel monoclonal antibody H6-11 for prostate cancer imaging. Mol Pharm. 2013; 10(10): 3655-3664.
He L, Jhong JH, Chen Q, Huang KY, et al. Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors. Cell Rep. 2021; 37(5): 109955.
Vomastek T, Iwanicki MP, Schaeffer HJ, Tarcsafalvi A, Parsons JT and Weber RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility. Mol Cell Biol. 2007; 27(23): 8296-8305.
He Y, Xu W, Xiao YT, Huang H, Gu D and Ren S. Targeting signaling pathways in prostate cancer: mechanisms and clinical trials. Signal Transduct Target Ther. 2022; 7(1): 198.
Wang X, Tian L, Lu J and Ng IO. Exosomes and cancer-Diagnostic and prognostic biomarkers and therapeutic vehicle. Oncogenesis. 2022; 11(1): 54.
Bahmad HF, Jalloul M, Azar J, Moubarak MM, Samad TA, Mukherji D, Al-Sayegh M, and Abou-Kheir W. Tumor Microenvironment in Prostate Cancer: Toward Identification of Novel Molecular Biomarkers for Diagnosis, Prognosis, and Therapy Development. Front Genet. 2021; 12: 652747.
Yu D, Li Y, Wang M, Gu J, Xu W, Cai H, Fang X and Zhang X. Exosomes as a new frontier of cancer liquid biopsy. Mol Cancer. 2022; 21(1): 56.
Boutilier AJ and Elsawa SF. Macrophage Polarization States in the Tumor Microenvironment. Int J Mol Sci. 2021; 22(13): 6995.
Izumi K and Mizokami A. Suppressive Role of Androgen/Androgen Receptor Signaling via Chemokines on Prostate Cancer Cells. J Clin Med. 2019; 8(3): 354.
Kiely P, Baillie G, Houslay M and O'Connor R. RACK1 is an essential scaffolding protein for regulation of FAK phosphorylation by IGF-I and integrins, and for tumour cell proliferation. Cancer Res. 2007; 67: 4153.

No competing interests reported.

Download PDF

Reviews received at journal
14 Sep, 2024
Reviewers agreed at journal
14 Sep, 2024
Reviews received at journal
02 Sep, 2024
Reviewers agreed at journal
23 Aug, 2024
Reviewers agreed at journal
22 Aug, 2024
Reviewers invited by journal
22 Aug, 2024
Editor assigned by journal
05 Aug, 2024
Submission checks completed at journal
05 Aug, 2024
First submitted to journal
04 Aug, 2024

You are reading this latest preprint version

Prostate cancer-derived exosomal PSM-E inhibits macrophage M2 polarization to suppress tumor invasion and metastasis through the RACK1/FAK/ERK pathway

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Participants, blood, and urine sampling

Cell culture

Plasmids and transfection

Isolation and characterization of exosome

Western blotting analysis

LC-MS/MS analysis of PSM-E

Wound healing assay

Invasion assays

RNA extraction and quantitative real-time PCR

Co-immunoprecipitation

Immunohistochemical staining (IHC)

Statistical analysis

Results

Clinical characteristics of the study samples

PSM-E is encapsulated within serum and urine-derived exosomes from PCa patients

Urine-derived exosomal PSM-E yielded a good diagnostic performance for PCa

PSM-E can be exported from cells by exosomes

Exosomal PSM-E can be transferred to recipient cells

PMS-E colocalizes and interacts with RACK1

Discussion

Conclusions

Declarations

Data sharing statement

Ethics approval and consent to participate

Funding

Author Contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

Status:

Version 1