Human Samples
Patients enrolled in MD-SELECT/EDRN trial were used to collect whole blood samples. The patients were classified into different Gleason grades (6, 7 and 9) according to their PSA levels and MRI scans results. We took 3 samples corresponding to each Gleason grade. All human investigations were carried out after the IRB approval by a local Human Investigations Committee and in accord with an assurance filed with and approved by the Department of Health and Human Services. Data has been anonymized to protect the privacy of the participants. Investigators obtained informed consent from each participant. The whole blood was centrifuged at 3000 rpm to collect the white layer containing Peripheral blood mononuclear cells (PBMCs). The PBMCs were later lysed in RIPA buffer to check eNOS and CSF1 expression using ELISA. The flash frozen prostate biopsies (n = 5) corresponding to Gleason grade 6 and 9 were taken from the Cancer Modeling Shared Source (CMSR) at the University of Miami. The frozen biopsies were used for doing BH4 estimation and Griess test. The CMSR also provided with the biopsy sections which were used for doing immunohistochemical staining for eNOS, CD206, CSF1 and CSF1R.
Cell culture
22R-v1 (CRL-2505), LNCaP (CRL-1740) and U937 cells (CRL-1593.2) were purchased from ATCC and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin and 2mM-L glutamine. TRAMPC2 cells (CRL-2731, purchased from ATCC) were maintained in DMEM medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin and100 mg/ml streptomycin.
Preparation of RNA and quantitative real-time PCR
Total RNA was extracted from cells using the TRIzol method and then reverse transcribed to complementary DNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA) according to the manufacturer’s protocol. The quantitative RT-PCR for indicated genes was performed in SYBR Universal PCR Master Mix (BIORAD, USA). Quantitation of mRNAs was performed using BIORAD™ Gene Expression Assays according to the manufacturer’s protocol. Samples were analyzed using the BIORAD sequence detection system. All PCRs were performed in triplicate, and the specificity of the reaction was determined by melting curve analysis at the dissociation stage. The relative quantitative method was used for the quantitative analysis. The calibrator was the average ΔCt from the untreated cells. The endogenous control was glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Western blotting
Cells were harvested and lysed in NP-40 buffer containing phenyl methyl sulfonyl fluoride and Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA). Protein expression was studied by exposing the membranes to antibodies against AR (Abcam, ab74272), AR-V7 (GeneTex, GTX33604), pERK (Cell signaling, 4370), ERK (Cell signaling, 9102S), pGSK-3beta (Cell signaling, 5558), p90RSK (Cell signaling, 11989), CD206 (Abcam, ab64693), VEGF (Abcam, ab46154) and GAPDH (Santa Cruz Biotechnology, SC47724). Immunoreactive bands were visualized using the Thermo Scientific Chemiluminescent Pico Kit.
Immunohistochemistry and fluorescence staining
For immunohistochemistry, tissue sections were stained with hematoxylin and eosin and analyzed by a genitourinary pathologist. For fluorescence staining, tissue slides were processed using antibody against F4/80 (cell signaling, 70076), iNOS (abcam, 178945) and pERK (Cell signaling, 4370) , followed by secondary antibodies tagged with Alexa Fluor® 488 or Alexa Fluor® 568 at room temperature for 30 minutes and 4,6-diamidino-2-phenylindole (Santa Cruz). All samples were assessed under a fluorescence microscope (Leica Microsystem, Wetzlar, Germany) at 60x magnification. Images were acquired using MetaMorph version 4.6 (Molecular Devices, Sunnyvale, CA, USA). Tumor xenograft tissues were fixed in 10% buffered formalin and embedded in paraffin. 5um thick sections were deparaffinized and rehydrated in sequential xylene and graded ethanol. Antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) in a microwave oven. Peroxidase and non-specific protein blocking were done as per the instructions using the Abcam ABC detection kit (ab64264) and incubated with the following primary antibody dilutions: anti-Androgen Receptor (Abcam, ab74272), anti-Ki67 (Abcam, ab15580), anti-F4/80 (Cell signaling, 70076), anti-ARV7 (GeneTex, GTX33604) and anti-CD206 (Abcam, ab64693) with 1:150 dilution while a dilution of 1:50 was used for anti-Prostate-Specific Antigen (Santa-Cruz Biotech, sc7316). They were subsequently incubated with biotinylated goat anti-polyvalent secondary antibody, followed by development using DAB substrate as per the instructions on the kit. All sections were lightly counterstained with hematoxylin and mounted with Cytoseal XYL. Images were taken using a brightfield microscope (Nikon E200) at 10X and 40X magnification and quantification was done using ImageJ software (NIH, USA).
For fluorescence staining, post-antigen retrieval, the slides were permeabilized using 0.4% triton-X with 5%BSA. After permeabilization, sections were put in a blocking solution containing 5%BSA in PBST for 30 minutes at room temperature. Afterwards, incubation in primary antibodies were done overnight. Next day, slides were washed and incubated with standard fluorescent tagged secondary antibodies for an hour at room temperature. Following washing, antifade containing 4′,6-diamidino-2-phenylindole (DAPI) was used for mounting. Sections were imaged using a confocal microscope (Leica Microsystem, Wetzlar, Germany).
MTT assay
22RV1 and LNCAP cells were seeded in 96-wells plate in quadruplets and were treated with varying doses of GSNO or GW2580 (CSF1R inhibitor) or with a combination of GSNO and GW2580. MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) assay reagents were added, and the absorbance was measured at 562nm after 0, 3, 5, 7, and 9 days.
Animals
The animal protocol was approved by the Institutional Animal Care and Use Committee of University of Miami Miller School of Medicine, Miami, FL. SCID and C57BL6 (6 weeks old) mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Castration experiments were performed in all the C57BL6 mice. For castration, mice were anesthetized using Isoflurane (Abbott Laboratories). The perineal region was cleaned three times with ethanol and a betadine scrub (VWR, AJ159778), and sterile dissecting shears were used to make a 4–5 mm incision in this region. Using two sterile forceps, the testes were located, and a ligature was made around the testicular vessels and the tunica albuginea that encases the testes. The testes were amputated with dissecting shears and the scrotum was sutured closed with 6-0 Ethicon black monofilament nylon (Ethicon Inc., 1665). A local triple antibiotic was applied over the region of the wound to facilitate healing. C57BL6 mice were grouped into control and experimental groups. All the mice were grafted with 1 million TRAMPC2 cells subcutaneously. The experimental group received 10mg/kg/day of GSNO, or 40mg/kg/day or 10mg/kg/day GSNO+40mg/kg/day GW2580 treatment intraperitoneally (IP) for 2 weeks, while the control group received PBS IP. After treatment, animals were survived for an additional two weeks before humanely sacrificing them. At the end time point, blood was collected via cardiac puncture and tumor grafts, lungs, and spleen were harvested for further analysis. Tumor volume (V) was measured regularly until the mice were sacrificed by measuring the length (L) and width (W) of the tumor with calipers by using the formula: V= 1/2(length × width2).
Flow cytometry analysis for immune markers
For making single cell suspension from TRAMPC2 tumors, a piece of tumor was taken and minced with scissors or blade. These minced tumors were digested using digestion buffer containing collagenase by agitation at 37oC for 1.5 hrs and vortexing for a min, every 30 min. It was followed by adding RBC lysis buffer and 0.25%trypsin-EDTA and incubating again @ 37oC for 15 mins. Lastly, 5ml of chilled FACS buffer (PBS+2% FBS) was added and the digestion mixture was then filtered with 40um cell strainer. The cells were allowed to spin down at 450g for 5min and the collected pellet was resuspended in 2 ml FACS buffer. These isolated single cells were incubated with different antibodies following standard staining protocols for intra-cellular and membrane bound antibodies. The details of the antibodies are mentioned in Supplementary Table 2. Following staining, the cells were fixed in standard fixing solution containing 4% paraformaldehyde and taken for flow cytometry. The analysis was done using CYTEK Aurora flow cytometer.
Griess Test
20 μL of Griess Reagent (Thermo Fischer Scientific G-7921) and 150 μL of the nitrite-containing sample were mixed in a microplate (sample capacity at least 300 μL per well). The mixture was incubated for 30 minutes at room temperature in dark. To prepare a photometric reference sample, 20 μL of Griess Reagent was mixed with 280 μL of deionized water. The absorbance of the nitrite-containing samples relative to the reference sample were measured in a spectrophotometric microplate reader at 548 nm. To convert absorbance readings to nitrite concentrations, the method recommended by the manufacturer was used.
Tetrahydrobiopterin (BH4) ELISA
The kit (Catalog No. ABIN6957559, Antibodies online) is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of tetrahydrobiopterin in serum, plasma, tissue homogenates, cell lysates, cell culture supernatants. The amount of lysates used was 100ug of protein.
Human eNOS/NOS3 ELISA
Peripheral blood mononuclear cells (PBMCs) collected from patient blood samples were used for the estimation of NOS3 levels (Catalog # EH169RB, Thermo Fischer Scientific, USA). The steps followed were according to the kit instructions. 100ug of cell lysate was used for quantification.
M-CSF (CSF-1) Human ELISA
Peripheral blood mononuclear cells (PBMCs) collected from patient blood samples were used for the estimation of CSF-1 levels (Catalog # EHCSF1, Thermo Fischer Scientific, USA). 100ug of cell lysate was used for the assay. The steps followed are the same as mentioned in the instruction manual.
Cytokine antibody array
Proteins were isolated from tumors of the CRPC mice that had received treatment with PBS and GSNO. After quantification using Bradford assay, 2.5mg/ml protein lysates were screened for secreted protein using RayBio Human Cytokine Array C5, Code: AAH-CYT-5-2 (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions. The blots were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). A total of 80 molecules were selected for detection namely: ENA-78, G-CSF, GM-CSF, GRO, GRO-alpha, I-309, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL12-p40, IL-13, IL-15, IFN-gamma, MCP-1, MCP-2, MCP-3, M-CSF, MDC, MIG, MIP-1 beta, MIP-1-delta, RANTES, SCF, SDF-1, TARC, TGF-beta 1, TNF-alpha, TNF-beta, EGF, IGF-1, Angiogenin, Oncostatin M, TPO, VEGF, PDGF-BB, Leptin, BDNF, BLC, CK beta 8-1, Eotaxin, Eotaxin-2, Eotaxin-3, FGF-4, FGF-6, FGF-7, FGF-9, Flt-3 Ligand, Fractalkine, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IL-16, IP-10, LIF, LIGHT, MCP-4, MIF, MIP-3-alpha, NAP-2, NT-3, NT-4, Osteopontin, Osteoprotegerin, PARC, PIGF, TGF- b 2, TGF- b 3, TIMP-1, and TIMP-2 respectively.
Biotin switch assay and SNO protein purification
The S-nitrosylated proteins were visualized by biotin-switch assay following the manufacturer’s guidelines (S-Nitrosylated Protein Detection Kit (Biotin Switch), Item No. 10006518, Cayman Chemical, Ann Arbor, MI, USA). A small piece of tumor tissue or cell pellet was taken and washed twice with Wash Buffer. The pellets were resuspended in “Buffer A containing Blocking Reagent” and incubated for 30 min at 4 °C with shaking. The incubated samples were centrifuged at 130,000×rpm for 10 min at 4 °C, and the supernatant was transferred to 15 ml centrifuge tubes. Two milliliters of ice-cold acetone was added to each sample, and the mixture was incubated at − 20 °C for at least 1 h. The protein of each sample was pelleted by centrifugation for 10 min at 4 °C. “Buffer B containing Reducing and Labeling Reagents” was added to resuspend the proteins, with incubation for 1 h at room temperature. The biotinylated protein was precipitated by acetone and rehydrated with the appropriate amount of Wash Buffer.
A total of 40ug of protein was used for labeling and running the standard western blot for detecting the nitrosylated protein.
RNA Sequencing and Enrichment analysis
Fastq files were downloaded from Illumina’s BaseSpace cloud application. FastQC was performed on the fastq files to ensure acceptable base quality and GC content and to check for adapters. Adapters were trimmed using the Trim_Galore software to remove the Illumina Universal Adapter. After adapter trimming, Fastq_screen was performed to check for bacterial contamination. Once no contamination was confirmed, general alignment was performed using the STAR RNAseq aligner to the hg19 genome. During this alignment, raw counts were produced against the GENCODE gene features (v19) and reformatted into a matrix for further statistical processing. After alignment, PicardTools was used to calculate sample by sample alignment quality metrics statistics. Differential expression was performed on the raw count matrix using the edgeR software. Statistical significance was defined as any gene feature that had an FDR value of below 0.05. Gene set enrichment analysis was performed by means of the Broad’s GSEA executable jar file using the MSigDB as the reference database. Normalized log2CPM values were used as the expression values. These CPM values were filtered by log2CPM of 1 to limit the noise of the data that was used for enrichment. Three sets of runs were completed against hallmark genes (h).
Activation of Monocytes for M1/M2 polarization
U937 cells were activated using different cytokines as done by previous studies . The cells were treated in RPMI 1640 medium supplemented with 5% cFBS, in 100mm flat-bottom culture plates. Activation treatments consisted of (1) no stimulation control (mock); (2) PMA 20 ng/mL for 48 h (PMA-only control); (3) pre-treatment with PMA 20 ng/mL for 48 h, followed by LPS 50 ng/mL and IFN-γ10 ng/mL for 48 h (condition favoring M1 polarization, M1 cocktail); (4) pre-treatment with PMA 20 ng/mL for 48 h, followed by IL-4 25 ng/mL and IL-13 25 ng/mL for 48 h (condition favoring M2 polarization, M2 cocktail). In addition to these treatments, three drug combinations, i.e., GSNO (50uM), CSF1Ri (0.5uM) and a combination of both GSNO and CSF1Ri were also used to check their effect on monocyte activation as well as macrophage polarization.
Macrophage polarization study for studying the interaction of AR and GSNO
For this study, U937 cells (purchased from ATCC) were used as a model for monocyte-macrophage differentiation. The study was divided into two steps: a) Collection of conditioned media and cell lysates from 22Rv1 cells and b) Treatment of U937 cells with conditioned media from 22Rv1. For preparing the 22Rv1 cell conditioned media (CM), 3.0 × 105 cells were seeded into 6-well culture plates and allowed to adhere overnight. Next day, the cells were starved using charcoal stripped FBS for about 12 hours and then 2 ml of medium containing 1% FBS. The cells were treated with 4µM enzalutamide (ENZA, Selleckchem, MDV3100) in the presence or absence of 50 µM GSNO. The untreated 22Rv1 cells served as control. The media was collected 48 h later. The supernatant was centrifuged at 1800 rpm for 10 min and was collected as CM. U937 cells were treated with the collected CM from these different treatment conditions. The cells were collected at 48 hrs for analysis of M2 macrophage marker, CD206 (Abcam, ab64693) and AR (Abcam, ab74272).
Organoid cultures using 22Rv1 cells for Immunohistochemical (IHC) staining
The organoid cultures using 22Rv1 cells were generated using the protocol followed by Ma et al (2017). A total of 250,000 cells/ml were suspended in thawed Matrigel and a drop was added in each well of a 24 well plate. Organoids were maintained in adDMEM/F12 media with growth factors and 10 μM Y-27632 dihydrochloride. The media was refreshed every 2-3 days. From 7 days after initial plating, an organoid culture medium without Y-27632 dihydrochloride was used. The culture was ended on day 14. Later the organoids were fixed in 4% paraformaldehyde and processed for paraffin embedding and sectioning.
Site directed mutagenesis
CSF1R ORF clone (OHu24034, NM_001349736.1) was procured from GenScript Biotech (NJ, USA). Site directed mutagenic changes were done to incorporate 3 cysteine deletions at C224, C278 and C830 (as determined by GPS-SNO 1.0 software with high threshold) using Quick Change Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, USA). Briefly, 40 ng plasmid was subjected to PCR amplification as per standard kit guidelines using mutagenic primers 5'-tgcccagatcgtgtcagccagcagcg-3', 5'-cgctgctggctgacacgatctgggca-3'; 5'- cgttgctggccacggagtagttgccg-3', 5'-cggcaactactccgtggccagcaacg-3' and 5'-ctctgaaccgtgtagacgtcaaagatgctctctg-3', 5'-cagagagcatctttgacgtctacacggttcagag-3' for cysteine del1, del2 and del3 respectively. Following PCR, 10ul of the product was subject to DpnI digestion for 5min at 37oC and transformed into chemically competent DH5α cells (NEB, USA) by heat shock at 42oC for 30 sec. Resulting transformants were grown in SOC media for 1hr at 37oC and selected overnight on LB agar plates containing 100μg/ml Ampicillin. Following day, single colonies were selected and further grown in LB broth containing 100μg/ml Ampicillin for 12 hours. Plasmid isolation and purification was done using plasmid miniprep kit (Qiagen, Germany) as per standard instructions. Sanger sequencing for confirmation of deletion was done by Genewiz, USA. For verification of these deletions, the confirmed wild type as well as mutant clones were transfected in 22Rv1 cells using Lipofectamine 3000 reagent. After transfection, cells were treated with/without 50µM GSNO and cells were collected after 48 hrs to do western blots using anti-Androgen Receptor (Abcam, ab74272) and anti-beta Actin antibody (Cell Signaling Technology, 4970) antibodies.
Statistical analysis and calculation of sample size
GraphPad Prism (GraphPad Software) was used for statistical analysis. All data are presented as means ± SEM. The statistical significance between two groups was determined by unpaired two-tailed t test. Multiple group comparisons were performed using a one-way analysis of variance with Tukey least significant difference test. In all cases, p < 0.05 was considered statistically significant.