Peripheral blood samples from both healthy donors (with no history of malignant disease) and prostate cancer patients were obtained from Virgen de las Nieves University Hospital (Granada) after approval by the ethical Committee of this Hospital, in accordance with the Declaration of Helsinki. Written informed consent was signed from every cancer patient and healthy volunteer prior sample collection.
Samples were processed in the Liquid Biopsies & Cancer Interception laboratory (LiqBiopCI) at GENYO Centre (Granada). All prostate cancer patients were diagnosed and followed-up in the Urology Department and in the Oncology Department of the University Hospital Virgen de las Nieves (Granada).
Circulating Tumor Cells (CTCs) isolation
Peripheral blood samples (10 ml) from prostate cancer patients diagnosed of localized disease or advanced disease (metastatic castration resistant prostate cancer) were collected in EDTA tubes (Vacutainer), stored at room temperature and processed into 4 hours after collection. CTCs were isolated according to the previously established protocol by our group[20,21]. Briefly, blood samples were subjected to density gradient centrifugation and immunomagnetic selection of epithelial cells using the Carcinoma Cell Enrichment and Detection Kit (Miltenyi Biotec) based on multicytokeratin (CK3-11D5) microbeads. Each sample was spun down onto two slides in a cytocentrifuge (Hettich) and stained for confocal microscopy visualization.
Detection and characterization of CTCs from Prostate Cancer Patients
Slides from prostate cancer patients CTCs isolation and fixed cells were stained with mouse anti-human Cytokeratin-FITC (Ref. 130-119-141, Miltenyi), mouse anti-human CD61-Alexa Fluor 647 (Ref. 336408, Biolegend), and Hoechst 33342 (Thermo Fisher). Both sample types were mounted with SlowFade™ Antifade Kit (Invitrogen), for confocal microscopy analysis as previously described. Negative and single stained controls were performed to ensure no fluorescence bleed-through between channels. CTCs were described as CK+ nucleus+ cells and CD61 expression in CTCs was classified into presence or absence.
Platelets were isolated from whole blood collected in EDTA tubes (Vacutainer) by a series of centrifugations at room temperature in a swing-bucket rotor centrifuge. First, leukocyte-rich platelet-rich plasma (L-PRP) was obtained by centrifugation at 120 x g for 10 min without break. Then, remaining white blood cells and erythrocytes were removed by centrifugation at 105 x g for 15 min to obtain pure platelet-rich plasma (P-PRP). Platelets were isolated from P-PRP by centrifugation at 1000 x g for 12 min. Isolated platelets were resuspended in RPMI 1640 at physiological concentration. Experiments were performed immediately after platelet isolation.
Platelet activation induction
Platelet activation was induced by incubation with 15µg/mL of Adenosine 5'-diphosphate sodium salt (ADP) (Sigma-Aldrich) and 1U/mL of thrombin from bovine plasma (Sigma-Aldrich) for 30 min. Activated platelets were washed with PBS-EDTA 2mM and transferred to the cell culture.
Interactions between platelets and cells were evaluated by labeling either cells or isolated platelets using membrane cell tracker (CT) Vybrant™ DiO Cell-Labeling Solution (emission max. 501nm) (Ref. V22886, Invitrogen) and Vybrant™ DiD Cell-Labeling Solution (emission max. 665nm, red) (Ref. V22887, Invitrogen) at 5μM final concentration for 20 min at 37ºC. RNA transference was labeled by incubation with Syto RNASelect™ Green Fluorescent Cell Stain Solution (emission max. 530 nm) (Ref. S32703, Life Technologies) at 500nM (cells) or 10μM (platelets) for 20 min at 37ºC. Alternative staining of cells or platelets before co-culture allowed the measurement of cell tracker transference from platelet to cells and from cell to platelets.
Human cancer cell lines were used according to different tumor types: LNCAP, PC3 and 22RV1 (from prostate cancer); H1975, H1299 and, A549 (from lung cancer); SW480 and SW620 (from colon cancer), and MCF7, MDA-MB-231, and HCC70 (from breast cancer). LNCAP, PC3, 22Rv1, H1975, H1299, SW480, and SW620 were cultivated in RPMI 1640 (BioWest) while A549, MCF7, MDA-MB-231, and HCC70 were maintained in Dulbecco's minimal essential medium (DMEM) (BioWest). Both media were supplemented with 10% of Fetal Bovine Serum (FBS) (BioWest), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were maintained at 37°C in a humidified incubator infused with 5% CO2.
Cell lines used in this study were obtained from the American Type Culture Collection (ATCC) and Centre for Scientific Instrumentation (CIC) of the University of Granada; PC3 was kindly donated by Dr. Ignacio Gil Bazo (CIMA, Pamplona). Cell lines were routinely tested for mycoplasma contamination using the Venor®GeM qEP (Minerva Biolabs) and authenticated using AmpFLSTR® Identifiler® Plus (Applied Biosystem).
Most relevant information about Material and Methods was graphically represented in Fig. S1.
For all experiments including tumor cell and platelets co-culture, tumor cells were seeded the day before platelet isolation in order to reach 60-70% of confluence at the time of co-culture. In experiments avoiding direct contact between cells and platelets, 0.4μm membranes Transwell® inserts (Millipore) were placed on well plates and platelets were added onto them.
Flow cytometry experiments were performed using 24-wells plates and 500μl of platelets suspension. Experiments were run in triplicates and collected at different time points. After co-culture, platelets were harvested from cell media after centrifugation at 105 x g for 15 min to eliminate cell fragments. Cells were washed twice with PBS 1X to remove any remaining platelets and cell colonies were dissociated with Tryple Express 1X (Life Technologies). Cells and platelets suspensions were fixed with 3.7% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 20 min at room temperature.
For the analysis of cell activation, platelets were incubated with mouse anti-human PAC-1 (Ref. 340535, BD Pharmingen), and subsequently with goat anti-mouse Alexa Fluor 647 (Ref. A32728, Invitrogen) secondary antibody. After that, platelets were stained with mouse anti-human CD41a-PE (Ref. 555467, BD Pharmingen) for 30 min at room temperature.
In cell tracker transference related experiments, fixed cells and platelets were incubated for 30 min with mouse anti-human CD42b-APC (Ref. 551061, BD Pharmingen), mouse anti-human CD61-Alexa Fluor 647 (Ref. 336408, Biolegend), and rabbit anti-human EpCAM (Ref. ab225894, Abcam). Subsequently, cells and platelets were incubated with goat anti-rabbit Dylight 405 (Ref. 35551, Invitrogen) secondary antibody for 30 min at room temperature.
Between primary and secondary antibody incubations, cells and platelets were washed with FACS Buffer (PBS 1X, 5% FBS, EDTA 2Mm) and PBS-EDTA 2mM, respectively.
Both cells and platelets were analyzed in the BD FACSVerse™ flow cytometer equipped with three lasers: violet (405nm), blue (488nm) and red (633nm) (BD Bioscience) using BD FACSuite™ software (BD Bioscience) for acquisition or by FACS ARIA III™ flow cytometer equipped with four lasers: violet (405nm), blue (488nm), yellow/green (531nm) and red (633nm) (BD Bioscience) using BD FACSDiva™ software (BD Bioscience) for acquisition and FlowJo™ for analysis (FlowJo, LLC-BD Bioscience). Flow cytometry gating strategy is described in Fig. S2.
To study transference mechanisms, cells were seeded onto Poly-L-Lysine (Sigma Aldrich) pre-treated cover slides. Vybrant™ DiO Cell-Labeling Solution (emission max. 501nm, green) (Invitrogen) and Vybrant™ DiD Cell-Labeling Solution (emission max. 665nm, red) (Invitrogen) were used to label platelets and cells respectively.
Transference of RNA was visualized labeling platelets with 10μM Syto RNASelect™ Green Fluorescent Cell Stain Solution and 5μM Vybrant™ DiD Cell-Labeling Solution (emission max. 665nm, red) (Invitrogen) to stain platelet membrane.
After of co-culture, platelets were aspirated, and cell washed once with DPBS Ca+ Mg+ (Gibco), fixed with 3.7% of PFA and stained with Hoechst 33342 (Thermo Fisher). Cover slides were mounted with SlowFade™ Antifade Kit (Invitrogen).
Time-Lapse Assay were performed in Glass Bottom 35mm µ-dish (Ibidi). Tumor cells were labeled with DiD cell tracker (red) and platelets with DiO cell tracker (green), as previously described. Platelets were added a 0 time point and set in the incubation chamber of the confocal microscope at 37°C and 5% CO2 for time lapse monitoring. Five positions were analyzed and a total of 24 images were acquired with 10-min/image-time interval for a total duration of 240 min.
Confocal images were obtained using a LSM 710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany) equipped with an incubation chamber (Pecon, Germany). Images were acquired with a Zeiss Plan-Apochromat 63x/1.40 NA DIC M27 oil-immersion objective and ZEN 2010 software (Carl Zeiss, Jena, Germany). Cells were excited with a 405 nm diode laser line, a 488 nm argon laser line, a 543 nm HeNe laser line and a 594 HeNe laser line.
Monolayer cell cultures of LNCAP cells and platelets were conducted in 8 wells Permanox Lab-Tek® Chamber Slides (NUNC) and fixed at 4ºC in 1.5% glutaraldehyde, 1% formaldehyde, 0.05M cocodilate buffer. Fixed cells were post-fixed in 1% osmium tetroxide for 1 hour at 4ºC, washed in distilled water, and treated with 0.15% tannic acid and 2% uranyl acetate. Then, dehydration through graded alcohols and propylene oxide, and then embedding in EMbed (Electron Microscopy Sciences) was done. Ultrathin sections (50-70nm) were stained with 1% uranyl acetate and lead citrate. Samples were prepared and examined in a Transmission Electron Microscope using the Libra 120 (Zeiss) ITEM Imaging Platform Software (Olympus) at the Centre for Scientific Instrumentation (CIC) of the University of Granada.
After co-culture, platelets and LNCAP cells were collected as previously described for antibody staining, after which cells and platelets were fixed using the FIX & PERM® Cell Permeabilization kit (Invitrogen) following manufacturer instructions. Fixed cells and platelets were incubated for 30 min with mouse anti-human CD42b-APC (Ref. 551061, BD Pharmingen). Platelets were also incubated with rabbit anti-human EpCAM (Ref. ab225894, Abcam) for 30 min and subsequently incubated with goat anti-rabbit Dylight 405 (Ref. 35551, Invitrogen) secondary antibody for 30 min at room temperature. Cell DNA was labeled by cell resuspension in 1X of Hoechst 33342 (Thermo Fisher) and incubation for 5 min. Finally, platelets and cells were resuspended in FACS Buffer for cells and PBS-EDTA 2mM for platelets. Both cells and platelets data were acquired in an ImageStream® Mark II Imaging Flow Cytometer with four lasers; violet (405nm), blue (488nm), yellow (561nm) and red (642nm) (Amnis) and analyzed with the software IDEAS.
Cell Proliferation Assay
The effect of co-culturing platelets with tumor cells on cell proliferation was evaluated with the real-time cell monitoring assay (RTCA) (xCELLigence; ACEA Biosciences, Inc.). Cells were seeded in the RTCA plates and isolated platelets were added 24h afterwards. Cell growth was monitored from seeding in a 4 hour interval on the RTCA system for 72 hours and impedance was recorded as a measurement of Cell Index (CI). Experiments were performed in quadruplicates and outliers were removed.
Total RNA from cancer cells cultured alone or in co-culture with resting platelets was extracted with TRIzolTM Reagent (Invitrogen), according to manufacturer’s instructions. RNA concentration and purity were determined using NanoDrop 2000c Spectrophotometer (ThermoFisher Scientific) and 1µg of total RNA was converted to complementary DNA (cDNA) using the Transcriptor First Strand cDNA Synthesis Kit (Roche) for subsequent RNA expression.
qRT-PCR primers previously described elsewhere were used (Sigma-Aldrich), details in Table S1. Gene expression was measured using iTaq™ Universal SYBR® Green Supermix (Biorad) on a 7900 HT Real-Time PCR system (Life Technologies). Each test was run three times including non-template controls (NTC). GAPDH was selected as endogenous control. Expression levels are shown as 2−ΔΔCt paired for control and co-cultured cells for normalization at selected time points (1h, 24 and 48h).
Statistical analyses and graphs were performed using IBM SPSS Statistics (version 22.0 for Windows, IBM Corp.) and GraphPad Prism (version 7.04 for Windows, GraphPad software). One-way ANOVA and Two-way ANOVA (Multiple comparisons test) were used. Two-tailed unpaired t-test, was performed. CTCs were assessed as a continuous (number of CTCs) and CD61 expression was defined as a dichotomous variable (positive or negative). Dynamics of tumor cell growth after platelets addition were studied using non-linear regression (second order polynomial, quadratic) best-fit modeling, moreover tumor cell growth with or without platelets were compared at 24h and 48h. P values less than 0.05 were considered statistically significant.