Human subjects and fecal collection
This study was approved by IRB of Huashan hospital, Fudan University (Grant No.2020-531). All participants were informed of the aim of the study and signed the informed consent. PCa patients accepting surgical, hormonal, or any other kinds of therapies were collected at our urological department between Jan 2007 and December 2020. The diagnostic criteria of CRPC and HSPC were complied with EAU guidelines on PCa (19), and the recruitment criteria of the two cohorts of patients were described in our previous studies (3). Fresh feces were collected by patients themselves using a sterile “feces tube” with sealed screw cap. Samples were immediately sub-packaged and stored frozen at -80℃ until use.
Animals
This animal study was approved by IRB of Huashan hospital, Fudan University (Grant No. 2020-JS252). TRAMP mice were introduced from Jackson Laboratory (Bar Harbor, United States) and bred in Institute of Development Biology and Molecular Medicine of Fudan University. TRAMP male mice (age of 8 weeks) were randomly divided into four groups (n=10 for each group), singly housed, and maintained in a standard 12-hour light/dark condition with free access to rodent feed and water. Mice were treated with antibiotics containing 0.2 g/L ampicillin, neomycin, metronidazole, and 0.1 g/L vancomycin in drinking water for 1 week before human fecal microbiota transplantation (FMT) or SCFAs supplementation. For FMT, feces from five randomly selected either CRPC or HSPC patients were mixed with saline solution (20 mg ml-1) at equal weights, vortexed and centrifuged. Each time an aliquot of 200 μl of fecal suspension was administered to mice by oral gavage. For SCFAs supplementation experiment, a cocktail of 200 μl of SCFAs mixture (67.5 mM sodium acetate, and 40 mM sodium butyrate) (20) or saline solution was administered to mice by oral gavage. FMT or SCFAs were administrated twice a week and maintained for 16 weeks, then mice were anesthetized and sacrificed to collect feces and prostate.
16s rRNA sequencing and analysis
The fecal DNA of mice accepting FMT were extracted using the DNeasy PowerSoil Kit (QIAGEN, Inc., Netherlands) following manufacturer’s instruction. Regions V3–V4 of the 16S rRNA gene were amplified using the forward primer 5′-ACTCCTACGGGAGGCAGCA-3′, and the reverse primer 5′- GGACTACHVGGGTWTCTAAT-3′. The PCR program was set as follow: 98°C 10min, 25 cycles of 98°C 15 s, 55°C 30 s, 72°C 30 s, 72°C 5 min. Equimolar amplicon pool was obtained, and paired-end 2×300 bp sequencing was performed at Illlumina MiSeq platform (Shanghai Personal Biotechnology Co., Ltd., China). Low-quality reads and adapter contamination were discarded. High-quality reads were aligned using FLASH, and operational taxonomic units (OTUs) was delineated using UCLUST at 97% cutoff. LEfSe analysis was used to detect differential microbiotas (21).
SCFAs analysis
The SCFAs content of mice feces were quantified with an Agilent 7890A Gas chromatograph coupled to Pegasus GC-TOFMS system (LECO Corporation, St. Joseph) . An amount of 100 mg fece of each sample was added to 50μl 15% phosphoric acid, 100μl 125μg/ml internal standard (isohexanoic acid), and 400 μl ether. Samples were vortex mixed for 1 min, and centrifuged at 14000g 4°C for 10min. A 200μl aliquot of supernatant was finally transferred to test. The GC-GC-TOFMS separation was performed using an Agilent HP-INNOWAX capillary column (30m × 0.25mm ID × 0.25μm, Waters, Ireland). The parameters were set as follow: helium gas flow=1 ml/min; column temperature= 70°C for 1min, raised to 170°C at 10°C/min, raised to 240°C at 25°C/min and maintained for 2 min; inlet, interface, and ionization source temperatures= 250°C, 230°C, and 250°C; electron impact ionization= 70 eV; injection volume= 1μl; m/z scan range= 40-550. SCFAs were analyzed in full scan mode (22). ChromaTOF® software (LECO Corporation) was used to collect and process data. Identification of SCFAs was carried out by referring to NIST Standard Reference Data .
Prostate Histopathology
Mice prostate were sectioned and fixed in 4% paraformaldehyde, placed in 70% ethanol and dehydrated, embedded in paraffin, and stained using hematoxylin and eosin. Histopathological diagnosis were blindly given by two independent skilled uropathologists at our hospital according to a modified grading system of prostate histopathology for TRAMP mice (23). The histologic features were classified as: (I) normal tissue (NT); (II) low-grade PIN (LGPIN); (III) high-grade PIN (HGPIN); (IV) well-differentiated adenocarcinoma (WD-Adeno); and (V) poorly-differentiated adenocarcinoma (PD-Adeno). Quantitative analysis was conducted as previously described with minor modification, 10 random fields were captured at 10magnification, with the most advanced histological feature in each field was included in calculation. The numbers and percentages of different subtypes of lesions were established and compared among groups.
Cell culture
Human PCa cell lines (DU145, PC3) and murine macrophage cell line RAW 264.7 were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All the cell lines were cultured at 37°C in 5% CO2 using RPMI 1640 medium (Gibco, USA) containing 10% FBS (Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin.
SCFAs treatment, wound-healing and matrigel invasion assay
Different doses of SCFAs mixture (0.02mM, 0.2mM, and 2mM, with a ratio of sodium acetate to sodium butyrate at 1:1) were used to treat PCa cells. For the wound-healing assay, PCa cells (1×105) were seeded to confluence in 12-well plates. A monolayer was scratched, and the cells were cultured in complete medium supplemented with/without SCFAs. Cells were photographed at 0 h, 12 h and 24 h, and the closure area of wound was calculated. For the matrigel invasion assay, PCa cells (1×105) with serum-free medium were placed in the upper chambers of 24-well transwell plates coated with matrigel, supplemented with/without SCFAs, while the bottom chambers were filled with complete medium. After 48 h, cells attached to the upper surface of the membrane were removed, whereas cells reached to the underside of the chamber were fixed, stained, and counted. To determine the impact of macrophages on PCa cells, PCa/macrophage cells were co-cultured in 24-well transwell plates for 48 h, supplemented with/without SCFAs, the CM (condition medium) or control media were collected, diluted with 10% FBS (1:1), and placed into the lower chambers of 24-well transwell plates. PCa cells with serum-free medium were placed in the upper chambers to perform matrigel invasion assay. Each experiment was repeated three times.
Transmission electron microscopy
PCa cells treated with/without SCFAs were fixed with 2.5% glutaraldehyde for 4 h, post fixed in 1% OsO4 for 1 h, rinsed three times with 0.1 M phosphate buffer, and dehydrated in a graded series of ethanol. Then, cells were consecutively infiltrated with acetone and epoxy resin (2:1), acetone and epoxy resin (1:1), and epoxy resin for 24 h. Finally, cells were embedded in epoxy resin for 48 h at 60°C. Sections were made at a thickness of 80 to 100 nm, stained with aqueous uranyl acetate and lead citrate, and then analyzed with transmission electron microscope (FEI Tecnai G2 20 TWIN ). For each group, 20 independent sectioned cells were examined and the number of autophagosomes per cross-sectioned cell was calculated (24).
RNA-seq transcriptome analysis
Total RNA of SCFAs-challenged DU145 cells were extracted using TRIzol® Reagent (Invitrogen). Untreated DU145 cells were used as control. The cDNA library was prepared with 1 μg of total RNA using the TrueSeq RNA Library Preparation Kit (Illumina, USA). The mRNAs was separated using Oligo (dT) magnetic beads, and cDNA library was amplified and sequenced using an Illumina PE 250 platform. Raw reads were filtered using Trimmomatic software (Version 0.32) to remove adapter sequences and low-quality reads (base quality <20, read length <75 bp). The high-quality clean reads were mapped to reference genome using HISAT2 software (Version 2.2.1), and processed to gain FPKM value using HTseq software (Version 0.9.1) to ascertain differential genes between groups. Functional annotations were conducted by comparing reads to databases of Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg) (25).
Macrophage recruitment assay
PCa cells were cultured with/without SCFAs for 48 h, the CM or control media were collected, diluted with 10% FBS, and placed into the lower chambers of 24-well transwell plates. RAW264.7 cells (1×105) with serum-free medium were placed in the upper chambers without matrigel to perform macrophage migration assay (26). After 48 h, the migrated cells were collected, stained, and counted. Each experiment was repeated three times.
Western blot
Mice prostate tissues or human PCa cell lines were extracted using RIPA lysis buffer containing protease inhibitors (Sigma-Aldrich), and concentrated using BCA Protein Assay kit (Vigorous Biotechnology, China). Equal proteins were resolved using 10% SDS-PAGE before being transferred onto nitrocellulose membranes (Millipore, USA). Membranes were blocked with 5% BSA in TBST buffer and incubated with anti-LC3 (1: 800, 12741S, Cell Signaling), anti-TLR3 (1:800, 17766-1-AP, Proteintech), anti-total-NF-κB (1:1000, 4764S, Cell Signaling, USA), anti-Phospho-NF-κB (1:1000, 3033S, Cell Signaling), anti-ERK (1:1000, 11257-1-AP, Proteintech), anti-Phospho-ERK (1:1000, 28733-1-AP, Proteintech), anti-MEK (1:1000, 4694S, Cell Signaling), anti-Phospho-MEK (1:1000, 3958S, Cell Signaling), anti-MAPK (1:1000, 4695S, Cell Signaling), anti-Phospho-MAPK (1:1500, 4370S, Cell Signaling) at 4°C overnight and HRP-coupled secondary antibodies at room temperature for 1h. GAPDH and β-ACTB were used as internal controls.
Immunohistochemistry
Immunohistochemistry was performed on prostate sections of TRAMP mice and PCa patients, respectively. Primary antibodies against F4/80 (1:500, 28463-1-AP, Proteintech), CD163 (1:500, 16646-1-AP, Proteintech), iNOS (1:200, 18985-1-AP, Proteintech), CCL20 (1:200, PA5-114709, Invitrogen), and CCR6 (1:400, 66801-1-Ig, Proteintech) were used. After washing 3 times with PBS, the sections were incubated with HRP-coupled secondary antibodies for 30 min at room temperature. DAB was added for 3-4 min, then slides were dehydrated with increasing ethanol concentrations and xylene, and sealed. The scores of Mφ markers (F4/80, CD163, iNOS) were calculated by counting the positively stained cells per area from 10 random fields at 40magnification. The scores of CCL20 and CCR6 were classified into three groups according to the staining intensity (score 1 to 3) from 10 random fields at 40 magnification.
Quantitative RT-PCR and Elisa
Total RNA extraction of mice prostate tissues or human PCa cell lines were carried out using TRIzol® reagent (Invitrogen). The cDNA was obtained using a HiScript® 1st Strand cDNA Synthesis Kit (Vazyme, China). qRT-PCR was carried out using a AceQ qPCR SYBR Green Master Mix (Vazyme) on ABI Prism® 7900HT sequence detection system (Thermo Fisher Scientific, USA). Expression fold changes were determined using the 2−ΔΔCT method. The qRT-PCR primers used were shown in Table S1. Also, the ELISA kit (R & D Systems) was used to detect the levels of IL6, CCL20, TNF, MMP3 and MMP9 in culture supernatant. Each experiment was repeated three times.
Autophagy inhibition and RNA interference
PCa cells were co-treated with 3-MA (5 mM, Sigma-aldrich), or transfected with siRNAs targeting TLR3 (sc-36685, Santa Cruz) or CCL20 (sc-43935, Santa Cruz) for 48 h before performing wound-healing, matrigel invasion, and macrophage recruitment assay. Cell transfection were conducted via Lipofectamine® 3000 (Invitrogen) according to provided protocols.
Statistical Analysis
GraphPad Prism Version 8.0 was used for data analysis. Two-tailed Student’s t-test was used to evaluate statistical significance between two groups. One-way ANOVA was used to evaluate statistical significance among three or more groups. Mann-Whitney U-test was used to analyze nonparametric data. Data were expressed as mean ± standard deviation (SD) or as medians with inter-quartile ranges. P ≤ 0.05 indicated statistically significant difference.