Exosomes derived from myeloid-derived suppressor cells facilitate castration-resistant prostate cancer progression via S100A9/circMID1/miR-506-3p/MID1

An androgen-independent human PCa cell line (PC3) and DU145 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained according to ATCC guidelines. We cultured PC3 cells in RPMI-1640 medium with 100 IU/mL penicillin, 10% fetal bovine serum (FBS), and 100 μg/mL of streptomycin, and maintained the cells in a humidified incubator with an atmosphere comprised of 5% CO₂ and 95% air at 37 °C.

We generated human MDSCs from human peripheral blood mononuclear cells (PBMCs) in vitro as previously described [6, 25]. Briefly, we isolated PBMCs from healthy donors by venipuncture, followed by differential density gradient separation (Ficoll HyPaque; Sigma-Aldrich, St. Louis, MO, USA). The PBMCs (1 × 10⁶ cells/mL) were cultured in T25 flasks with RPMI 1640 medium containing 10% heat-inactivated FBS with granulocyte–macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ, USA) at 10 ng/mL and IL-6 at 10 ng/mL for 1 week. In vitro-generated polymorphonuclear MDSCs (PMN-MDSCs) were characterized by flow cytometry for expressions of CD15, CD11b, and CD14. The PMN-MDSCs were CD15⁺ CD14^−/low CD11b⁺ (the sorting strategy is indicated by green boxes in Additional file 1: Fig S1A). We collected and used the cells for further experiments.

We cultured isolated MDSCs (2 × 10⁶ cells/mL) in RPMI-1640 medium containing 10% exosome-depleted FBS for 2 days. FBS was depleted from contaminating bovine exosomes by ultracentrifugation for at least half of a day at 100,000×g. We collected the culture medium using a 0.22 μm filter (BD Falcon; Corning, Corning, NY, USA), and extracted Exos via the ExoQuick Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) following standard procedures. Exos were resuspended in 200 μL of medium. Electron micrographs were observed by transmission electron microscopy (Tecnai-12; Philips, Amsterdam, Netherlands). The BCA protein assay kit was used to quantify the exosomes. Protein markers, CD9, CD63, Hap70, calnexin were determined by immunoblotting. The size and concentration of exosomes were directly measured using the NanoparticleTracking Analyzer (NTA, Malvern Panalytical, Malvern, UK). Identification of MDSC-exosomes was shown in Additional file 1: Fig. S1.

We performed circRNA microarray analysis using the Arraystar Human circRNA Array, version 2.0 (Arraystar, Rockville, MD, USA). We performed RNA extraction and microarray hybridization following standard procedures. We extracted total RNA from PC3 cells treated with phosphate-buffered saline (PBS) or MDSC-derived Exos (50 μg/mL) at 48-h using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) following standard procedures. We digested total RNA using RNAse R (Epicentre, Madison, WI, USA) to eliminate linear RNAs and enrich circRNAs. Then, the enriched circRNAs was amplified and transcribed into fluorescent cRNAs using the random priming method using the Arraystar Super RNA Labeling Kit (Arraystar), and hybridizing the cRNAs using the Arraystar Human circRNA Array, version 2 (8 × 15 K). Finally, the slides were washed and scanned using a Scanner G2505C (Agilent, Santa Clara, CA, USA) and the acquired array images were analyzed. Quantile normalization and subsequent data processing was performed using the R software limma package. CircRNAs differentially expressed with statistical significance between MDSC-Exo-PC3 and control-PBS-PC3 (fold-change (FC) ≥ 2 and p ≤ 0.05) were identified through Volcano Plot filtering. Hierarchical clustering was performed to show the distinguishable expression pattern of circRNAs among samples.

Patients (ages ranged from 49 to 78 years with a median age of 64 years) undergoing radical prostatectomy for localized PCa (n = 56) and benign prostatic hyperplasia (n = 31) undergoing transurethral resection of the prostate were collected from the Hangzhou Hospital of Traditional Chinese Medicine. All tissue specimens were pathologically confirmed as PCa by two experienced pathologists. The Ethics Committee of the Hangzhou Hospital of Traditional Chinese Medicine approved this study, and we obtained written consent from each participant prior to the study.

We extracted RNA and miRNAs from the cultured cells and tissues using TRIzol reagent (Invitrogen, Karlsruhe, Germany) and an mirVana miRNA isolation kit (Ambion, Austin, TX, USA) following standard instructions. We then synthesized cDNAs using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). We performed qPCR analyses using an ABI PRISM 7500 Sequence Detection System (Life Technologies, Grand Island, NY, USA). We used U6 snRNA as an endogenous control of miRNAs, and normalized circRNAs levels to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We calculated relative gene expression levels using the 2^−ΔΔCt method. The primers used for RT-PCR are included in Table 1.

Small interfering RNAs (siRNAs) targeting human circMID1 (si-circMID1), MID1 (si-MID1), and nonspecific negative control oligos (si-control), miR-506-3p-mimics, and the corresponding negative control mimic (miR-NC), anti-miR-506-3p and anti-miR-ctl plasmid-mediated overexpressing circMID1 (over-circMID1) or MID1 (over-MID1) vector, and siRNA targeting S100A9 (si-S100A9) were obtained from Gene Pharma (Shanghai, China). The lentivirus targeting circMID1 (sh-circMID1) was purchased from GeneChem (Shanghai, China). We performed transfections using Lipofectamine 3000 (Invitrogen) following the standard instructions. Overexpression or knockdown effects were measured by RT-qPCR using RNA extracted 2 days after transfection.

For treatments, we preincubated PC3 cells with si-control, si-circMID1, or si-MID1, and anti-miR-ctl, or anti-miR- 506-3p, and miR-NC, or miR-506-3p, over-circMID1 or over-MID1 for 30 min and then treated them with or without MDSC-derived Exos (50 μg/mL) for the indicated times.

We assessed cell proliferation using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China). In brief, we seeded cells (3 × 10⁴) into plates with 96 wells and incubated them at 37 °C for 1 day before transfection. We added CCK-8 solution (10 μL) to every well 2 days after transfection. At the indicated time points, we measured absorbance at a wavelength of 450 nm for 5 days using a Spectra Max 250 spectrophotometer (Molecular Devices, San Jose, CA, USA). The experiments were performed in triplicate.

We performed Transwell assays for PC3 and DU145 cell invasion and migration in 8.0 µm Boyden chambers with 24 wells. For the invasion assays, the chambers were precoated with Matrigel, and cells (3 × 10⁴) were seeded in serum-free medium into the upper level of the chamber. We added full culture medium to the lower chamber as a chemoattractant. After 1 day of incubating at 37 °C with 5% CO₂, the membranes were fixed with methanol and stained with 0.1% Crystal Violet. We randomly selected five visual fields (200×) and counted the number of the invading cells using a light microscope. We also carried out migration assays without precoating the chambers.

Exosomes (20 mg) derived from MDSCs were labeled with CFSE dye, which was diluted at a ratio of 1:1000. After 15 min incubation at 37 °C, the exosomes were harvested and washed with PBS by centrifugation (100,000×g, 70 min). CFSE-labeled exosomes or unlabeled exosomes were added to PC3 and DU145 cells and incubated for 48 h at 37 °C, and the cellular uptake was observed using a confocal fluorescence microscope. The sample was treated in the same manner and used as a control for any free CFSE dye left in the solution. Immunofluorescence microscopy was done as previously described [26]. Primary antibody was mouse IgG anti-alpha-tubulin DM1A (1:2000) (Sigma). Secondary antibody was donkey anti-mouse IgG AlexaFluor 594 (1:500) (Molecular Probes).

The constructs containing wild-type (WT) or mutant (MUT) cirMID1 sequences or the MID1 3'-UTR containing the WT or mutated miR-506-3p binding site were subcloned into the luciferase gene by using a pmir-GLO vector (Promega, Madison, WI, USA). We co-transfected HEK293 or PCa cells with 100 ng of luciferase reporter vectors combined with miRNAs mimics/NC or a miR-506-3p mimics/NC (200 pmol) using Lipofectamine 2000 (Invitrogen and Thermo Fisher Scientific). After 2 days of transfection, we detected firefly and Renilla luciferase activities using a dual luciferase reporter assay system (Promega), and expressed the specific activity as the relative activity of firefly to Renilla luciferase. The experiments were conducted in triplicate.

We performed RIP assays using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) following the standard protocol. PC3 cells (2 × 10⁷) were lysed in RIP lysis buffer and the cell lysates incubated, dividing them into two equivalent parts, with either isotype-matched anti-IgG antibody (Millipore) or 5 μg human anti-Argonaute2 (AGO2) antibody (Millipore) with rotation at 4 °C overnight. We added magnetic beads and continued incubation at 4 °C for 1 h. We immunoprecipitated the samples with proteinase K at 55 °C for 1 h, then treated the complexes of RNA with TRIzol reagent (Life Technologies) for further purification. We utilized purified RNA to detect circMID1 and miR-506-3p expression levels using qRT-PCR.

For western blots, in brief, we collected cells from various treatment groups and lysed them in RIPA buffer (Beyotime Biotechnology, Nantong, China) including 1% protease inhibitor (Cell Signaling Technology). The protein concentration was measured using the BCA Protein Assay kit (Beyotime Biotechnology), and total protein (50 μg) was separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; 10%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk in Tris-buffered saline-Tween (TBST) buffer under room temperature for 1 h, we incubated the membranes with primary antibody against S100A9, DHX9, QKI, MID1 (1:1000; Abcam, Cambridge, MA, USA), for 1 day at 4 °C, followed by incubation with HRP-conjugated secondary antibody (1:5000; Rockland, Limerick, PA, USA) at room temperature for 1 h. Anti-GAPDH antibody (1:1000; Affinity, Cambridge, MA, USA), β-actin or Hsp70 (1:1000; Abcam) measured GAPDH, β-actin or Hsp70 as protein loading controls. We visualized the blots using a chemiluminescent reaction system (ECL; Beyotime, Shanghai, China).

All male BALB/c (nu/nu) mice were purchased from the Laboratory Animal Center of Zhejiang University. Animals were maintained in standard conditions with water and food provided ad libitum. The Institutional Animal Care and Use Committee at of the Second Affiliated Hospital in Zhejiang University approved the animal studies. For xenograft animal assays, 4 ~ 6-week-old BALB/c nude mice were injected subcutaneously with a total of 5 × 10⁶ cells into the right flank (five mice per group). We measured the tumor size every 5 days for 1 month, and the volume was determined using the formula: volume = (length × width²) × 0.5. After 1 month, we euthanized the mice by carbon dioxide asphyxiation and removed the tumors and weighed them. Finally, the tumor tissues were immediately flash-frozen in liquid nitrogen and stored at − 80 °C until analysis.

The tumors were resected and fixed in 4% paraformaldehyde, embedded with paraffin, and cut into 5 μm sections. The slices were dewaxed with xylene and dehydrated in graded ethanol, followed by blocking endogenous peroxidase with 3% hydrogen peroxide. After antigen retrieval, the sections were incubated with primary antibodies against MMP-2 (1:1000; Abcam, Cambridge, UK) or Ki67 (1:1000; Abcam) overnight at 4 °C. Then, the slides were incubated with horseradish peroxidase-labeled secondary antibody (1:1000, Abcam) for 1 h. The slides were developed with a diaminobenzidine (DAB) detection kit (Dako Cytomation, Glustrop, Denmark). After being counterstained with hematoxylin, the samples were observed using a light microscope (Olympus, Tokyo, Japan).

To determine the role and molecular mechanisms underlying MDSC-Exo in PCa progression, PC3 cells were co-cultured with MDSC-Exo or control-PBS and then PC3 cells were collected for their circRNA profiles analysis by using circRNA microarrays. We detected a total of 4248 circRNAs. Among them, 90 circRNAs were differentially expressed (fold-changes > 2.0; P < 0.05 and FDR < 0.05) between MDSC-Exo-PC3 cells and control-PBS-PC3 cells (Fig. 1A). Of the 90 circRNAs, 12 circRNAs were upregulated and 78 were downregulated in MDSC-Exo-PC3 cells compared with controls. To further evaluate the microarray data, we selected the four most upregulated and four most downregulated circRNAs to confirm their expressions in PC3 cells with different treatments by using qRT-PCR (Fig. 1B, C). The hsa_circ_0007718 was the top upregulated circRNA in MDSC-Exo-PC3 cells. By browsing the hg19/GRCh37 reference genome, hsa_circ_0007718, located at chrX:10491131–10535643, was assumed to derive from the MID1 gene (midline 1), which is located on chromosome Xp22.2. In this manner, we termed hsa_circ_0007718 as “circMID1.”

To confirm that circMID1 was differentially expressed in PCa, we examined prostatectomy specimens of PCa patients and benign prostatic hyperplasia (BPH) using qRT-PCR. We discovered that the circMID1 expression was significantly higher in PCa compared to BPH tissues (p < 0.01, Fig. 1D). The circMID1 expression was also higher in CRPC patients compared to HSPC patients (p = 0.008, Fig. 1E). In summary, the results suggested that circMID1 upregulation might be relevant to PCa progression.

Because circMID1 was upregulated in MDSC-Exo-PC3 cells, we utilized RNA interference to knockdown circMID1 expression and validate its biological mechanisms during MDSC-Exo-regulated CRPC propagation. The qRT-PCR analyses showed that circMID1 expression in the PC3 cell lines was successfully inhibited by siRNAs (Fig. 2A). In vitro investigations showed that MDSC-Exo promoted the PC3 cell migration, proliferation, and invasion (Fig. 2B–D). However, CCK-8 assays detected that circMID1 downregulation significantly suppressed PC3 cell proliferation induced by MDSC-Exo (Fig. 2B). Next, we assessed the circMID1 role in prostate cancer migration and invasion using Transwell assays. The circMID1 knockdown inhibited PC3 cell invasion and migration induced by MDSC-Exo (Fig. 2C, D).

We next investigated the underlying mechanism of the circMID1 increase in PC3 cells. The proinflammatory protein, S100A9, was previously shown to be abundant in MDSC-shed exosomes and to have a chemotactic function for MDSCs [27]. As shown in Fig. 3A, abundant S100A9 protein was detected in MDSC-Exo. Then, S100A9 expression was knocked down in MDSCs using siRNA. MDSC-Exo^si-S100A9, which had a lower expression of S100A9 in exosomes, was isolated and purified (Fig. 3B). The knockdown of S100A9 significantly reduced circMID1 expression levels in MDSC-Exo-treated PC3 and DU145 cells (Fig. 3C). As RNA binding proteins (RBPs) are necessary for circRNA [28], we then examined whether RBPs DHX9 and QKI, which regulate the biogenesis of a number of circRNAs [29, 30], were responsible for circMID1 biogenesis. The knockdown of S100A9 significantly increased DHX9 protein expression levels in MDSC-Exo-treated PC3 and DU145 cells (Fig. 3D). DHX9 is a well-known RNA helicase that can interact with inverted Alu repeats (IARs) to decrease the expression of a subset of circRNAs [29]. Abundant IARs are found within the MID gene locus, some of which are located around exons 7 to 8 (Fig. 3E). The results confirmed that circMID1 abundance increased after silencing DHX9 (Fig. 3F, G), suggesting that DHX9 could be a potential regulator.

Additionally, to study the internalization of MDSC-derived exosome S100A9 by PCa cells, MDSC-Exo was labeled with carboxyfluorescin diacetate succinimidyl ester (CFSE) fluorescent dye. The uptake of MDSC-Exo S100A9 was observed using a confocal microscope. We found that CFSE-labeled exosomes were localized in the cytoplasm, the uptake of CFSE exosomes by PCa cells was evident at 48 h, suggesting that the number of CFSE exosomes absorbed by PC3 and DU145 cells was observed, but not in the control cells treated with free CFSE dye or incubated unlabeled exosomes (Fig. 3H). The S100A9 levels in PC3 cells after 48 h incubation with CFSE exosomes was then detected (Fig. 3I), indicating that S100A9 was highly expressed in MDSC-Exo-treated PC3 and DU145 cells.

To investigate the potential miRNAs associated with circMID1, the possible binding partners of circMID1 were predicted by bioinformatics analyses using two databases in the public domain: STARBASE version 3.0 and CIRCBASE. Bioinformatics data demonstrated that circMID1 interacted with several miRNAs including miR-27a-3p, miR-513b-5p, miR-888-5p, miR-320, miR-22-3p, miR-506-3p, miR-124-3p and so on. The luciferase reporter vector including the circMID1 sequence was constructed, which was transfected with various miRNA mimics into HEK293 cells. The results showed that miR-506-3p exclusively and significantly reduced fluorescein intensity (Fig. 4A). circMID1 had a binding site for miR-506-3p (Fig. 4B), suggesting that circMID1 had a stable interaction with miR-506-3p. Therefore, we conducted dual-luciferase reporter assays to determine whether miR-506-3p functioned as a circMID1 target in PCa cells. The luciferase activity was markedly reduced as cells were co-transfected with luciferase reporters, circMID1-WT and miR-506-3p mimics, meanwhile, co-transfection with mutated luciferase reporter circMID1-MUT and miR-506-3p mimics did not significantly affect luciferase activity in PC3 and DU145 cells (Fig. 4C). Previous results suggested that Argonaute 2 (Ago2) protein binds with miRNAs and circRNAs to form an RNA-induced silencing complex. We therefore performed RNA immunoprecipitation (RIP) assays to pull down RNA transcripts bound to Ago2 in PC3 cells. We found that miR-506-5p and circMID1 were pulled down efficiently by anti-Ago2, but not by the nonspecific anti-IgG antibody (Fig. 4D). Subsequently, we verified the circMID1 effect on PCa cell miR-506-3p expression. Knockdown results showed that si-circMID1 upregulated miR-506-3p expression in PC3 and DU145 cells without or with MDCS-Exo treatment (Fig. 4E). To further validate the direct binding between circMID1 and miR-506-3p, we performed MS2bp-MS2bs based RIP assay in PC3 cells. As shown in Fig. 4F, miR-506-3p was significantly enriched in RNAs retrieved from MS2bs-circMID1 compared with that from MS2bs-circMID1mt or control MS2bs-Rluc, indicating the specific interaction between circMID1 and miR-506-3p. Moreover, the miR-506-3p expression levels in the PCa samples were significantly lower than that of BPH tissues (Fig. 4G), and miR-506-5p was negatively associated with circMID1 in PCa tissues (Fig. 4H). The results agreed with the observed circMID1 overexpression in PCa and demonstrated that circMID1 was a miR-506-3p sponge in PCa.

To detect if circMID1 acted as a ceRNA to liberate the MID1 expression and sequester miR-506-3p, we then characterized MID1 expression in PCa tissues. The data verified that MID1 was upregulated in PCa tissues (Fig. 5A). Pearson’s correlation illustrated that MID1 was negatively correlated with miR-506-3p and positively correlated with circMID1 (Fig. 5B, C). In addition, we employed TargetScan to indicate the miR-506-3p putative target genes, which showed that MID1 was predicted (Fig. 5D). To verify this finding, we performed luciferase reporter assays, which showed that luciferase activity decreased significantly when being co-transfected with miR-506-3p mimics and a luciferase reporter containing WT MID1 3′-UTR, but not with a mutant luciferase reporter in PC3 and DU145 cells (Fig. 5E). Western blotting and qRT-PCR verified that MID1 protein and mRNA expression levels were decreased by miR-506-3p mimics and increased with miR-506-3p depletion by its antagomir in PC3 and DU145 cells (Fig. 5F, G). Together, these results suggested that MID1 was a direct miR-506-3p target, which might also sequester miR-506-3p.

We then characterized circMID1 expression after MID1 knockdown and found circMID1 repression (Fig. 5H). To validate if MID1 acted as a ceRNA, we co-transfected si-MID1 and miR-506-3p inhibitors and discovered that circMID1 repression was reversed in PC3 and DU145 cells (Fig. 5H). We then detected MID1 mRNA and protein expressions after circMID1 knockdown and observed reduced expression of both. The expression was reversed after co-transfection with miR-506-3p inhibitors in PC3 and DU145 cells (Fig. 5I, J). These data suggested that MID1 and circMID1 functioned as ceRNAs by harboring miR-506-3p.

To determine the effects of the circMID1/miR-506-3p/MID1 pathway on MDSC-Exo-regulated CRPC propagation, the circRNA-miRNA-mRNA axis was blocked or overexpressed through knockdown in PC3 cells treated with MDSC-Exo, and then analyzed by conducting functional assays. Data demonstrated that circMID1 knockdown or miR-506-3p overexpression or MID1 knockdown inhibited the proliferation, invasion, and migration of MDSC-Exo-treated PC3 cells, while miR-506-3p inhibition or MID1 overexpression rescued the circMID1-deficient or miR-506-3p-overexpressed cell proliferation, invasion, and migration defects after treatment with MDSC-Exo (Fig. 6A, B). In addition, we found that knockdown of S100A9 significantly increased the miR-506-3p levels and reduced MID1 expression levels in MDSC-Exo-treated PC3 cells (Fig. 6C). In MID1-overexpressed PC3 cells, the circMID1 expression levels were increased and the miR-506-3p levels reduced (Fig. 6D). The knockdown of S100A9 inhibited MDSC-Exo-treated PC3 cell proliferation, migration, and invasion, while circMID1 overexpression or miR-506-3p inhibition or MID1 overexpression rescued the inhibition effect of S100A9 knockdown (Fig. 6E, F). Moreover, we also found that in MDSC-Exo-treated DU145 cells the circMID1 and MID1 expression levels were increased and the miR-506-3p levels were reduced, while knockdown of S100A9 significantly increased the miR-506-3p levels and reduced circMID1 and MID1 expression levels (Additional file 2: Fig. S2A). The circMID1 knockdown or miR-506-3p overexpression or MID1 knockdown inhibited the proliferation, invasion, and migration of MDSC-Exo-treated DU145 cells (Additional file 2: Fig. S2B and S2D). The knockdown of S100A9 inhibited MDSC-Exo-treated DU145 cell proliferation, migration, and invasion, while circMID1 overexpression or miR-506-3p inhibition or MID1 overexpression rescued the inhibition effect of S100A9 knockdown (Additional file 2: Fig. S2C and S2E). Thus, the results showed that MDSC-Exo promoted prostate cancer cell migration, proliferation, and invasion through S100A9/circMID1/miR-506-3p/MID1 signaling.

Based on the previous results, the role of MDSC-Exo and circMID1 in PC3 tumor growth in vivo was evaluated by xenograft nude mice tumor formation assays. Nude mice were injected with control or MDSC-Exo-treated PC3 cells, or MDSC-Exo-PC3 cells transfected with si-control or si-circMID1, and tumor growth was measured. We found that MDSC-Exo promoted tumor growth, whereas knockdown of circMID1 caused a significant decrease in tumor growth (Fig. 7A–C). MDSC-Exo-upregulated circMID1 and MID1 expressions were inhibited by si-circMID1 in tumor tissues, while downregulated miR-506-3p expression was increased by si-circMID1 (Fig. 7D). Furthermore, immunohistochemical staining was carried out to determine the expression of the proliferation marker Ki67 and the invasion-associated factor MMP-2. The expression of Ki67 and MMP-2 in the Exo group was significantly increased compared to that in the control group; in comparison with the Exo group, the expression of Ki67 and MMP-2 in the Exo-sh-circMID1 group was significantly decreased (Fig. 7E, F). Together, these results indicated that silencing circMID1 inhibited the promotional effects of MDSC-Exo on prostate cancer growth via regulating signaling of miR-506-3p/MID1.