Conceived and designed the experiments: JOL DK OS MB CM . Performed the experiments: KE DC PS CS LS ED VMcE. Analyzed the data: KE DC PS CS MG AC HK CM. Contributed reagents/materials/analysis tools: LMcE BF BS NC. Wrote the paper: JOL OS DK SOT MB DC KE CM CS HK MG SF AL JD LN.
The authors have declared that no competing interests exist.
Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.
Ovarian cancer is the fifth leading cause of cancer related deaths in women
During hematogenous dissemination, the ability of circulating tumour cells to interact with platelets is believed to promote their survival within the circulation and therefore facilitate metastasis. Pre-clinical animal experiments have demonstrated that pharmacologically
Thrombosis and thrombocytosis are frequent complications of ovarian cancer and are associated with poor prognosis
Firstly, we studied platelet adhesion to ovarian cancer cells under static conditions to determine if an adhesive interaction between platelets and ovarian cancer cells exists. Secondly, we assessed the ability of ovarian cancer cells to induce platelet activation and degranulation [P-selectin expression]. After establishing that platelets adhere to ovarian cancer cells, and ovarian cancer cells are capable of inducing platelet activation and degranulation, we next assessed gene expression changes at the transcriptome level in ovarian cancer cells treated with platelets or platelet releasate. Our results show differential interactions between platelets and ovarian cancer cell lines, not only in terms of platelet adhesion and activation, but also in gene expression changes in cancer cells treated with washed platelets or platelet releasate. Multiple interactions occur between platelets and ovarian cancer cells involving factors released by platelets and cancer cells, as well as direct platelet–ovarian cell interactions. This interaction results in a pro-survival, pro-angiogenic signal for the ovarian cancer cell.
Blood collection for this study was approved by the Royal College of Surgeons in Ireland ethics committee and written informed consent was obtained from all donors prior to phlebotomy.
All reagents were purchased from Sigma-Aldrich [St Louis, MO, USA] unless otherwise indicated. Collagen [soluble calf skin], Adenosine-5′-Diphosphate, Epinephrine, and Arachidonic Acid were obtained from BioData [Horsham, PA, USA]. Alexa Fluor-488-labelled Phalloidin, Calcein AM, and fibrinogen were obtained from Invitrogen [Carlsbad, CA, USA]. Phycoerythrin [PE]-labelled anti human P-selectin [mouse IgG], PE-labelled mouse IgG isotype control, and PE-labelled anti human CD42a [mouse IgG] antibodies were purchased from BD Pharmingen [San Diego, CA, USA].
A selection of ovarian cell lines of epithelial origin were chosen for inclusion in this study as epithelial ovarian cancers are the most common histological type. HIO-80 [a gift from the Fox Chase Cancer Center, Philadelphia, PA] represents a non-tumorigenic normal human ovarian epithelial cell line, which has been immortalised by transfection with a plasmid encoding for the SV40 large T gene. The HIO-80 cells were maintained in a 1∶1 mixture of medium 199 and MCDB-105 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin and 0.2 IU/ml of recombinant human insulin as recommended by Fox Chase. 59M [European collection of cell cultures (ECACC), Salisbury, UK] represents a tumorigenic human ovarian epithelial carcinoma of endometrioid type with clear cell components, originally isolated from ascites of a 65 year old patient with metastatic ovarian cancer. 59M cells were maintained in DMEM, 1 g glucose/L, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin as recommended by ECACC. SK-OV-3 [American Type Culture Collection (ATCC), Manassas, VA, USA] represents a tumorigenic human ovarian epithelial adenocarcinoma, originally isolated from ascites of a 64 year old patient with metastatic ovarian cancer. SK-OV-3 was cultured with McCoy's 5A media, supplemented with 10% fetal bovine serum [Invitrogen, The Netherlands], 100 units/ml penicillin and 100 ug/ml streptomycin as recommended by ATCC. A2780 [ECACC, Salisbury, UK] is derived from an ovarian serous epithelial tumour tissue in an untreated patient. The cisplatin-resistant cell line A2780cis was developed by chronic exposure of the parent cisplatin-sensitive A2780 cell line to increasing concentrations of cisplatin. Both cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin as recommended by ECACC. All cell lines were grown in standard conditions in a humidified atmosphere containing 5% CO2 at 37°C.
Blood was obtained from healthy donors who had not taken medications known to affect platelet function for at least 10 days. Blood was collected by venipuncture through a 19-gauge butterfly needle without a tourniquet, to avoid platelet activation. Platelets were prepared as previously described
The platelet adhesion assay was performed as previously described
The ability of ovarian cancer cells to induce platelet activation (P-selectin expression) was assessed by a flow cytometry based assay modified from Nylander et al
Washed platelets [2.5×108/ml] were stimulated with both TRAP [Thrombin receptor activating peptide, 20 μM] and Collagen [190 μg/ml] and stirred in a BioData PAP-4 light transmission aggregometer [Horsham, PA, USA] at 37°C for 15 minutes. The platelet aggregate was centrifuged at 720 g for 10 minutes. The supernatant was then aspirated and filtered through a syringe filter with a 0.22 μm PVDF membrane to remove platelet microparticles.
To optimise the concentration of platelet releasate for tumour cell exposure, a series of MTT cell proliferation assays [Roche Diagnostics Ltd, United Kingdom] were performed according to the manufacturer's instructions to determine the maximum concentration that could be applied to cells without negatively impacting on cell growth or survival. Cells were seeded into 96-well cell culture plates at 2×104 cells/well and cultured for 24 hours to allow cell attachment. Once attached to the plates, growth media was aspirated and cells were briefly washed with 200 μl of pre warmed PBS. Cells were then exposed to serial dilutions of platelet releasate from 1∶10 to 1∶10,000 in full media, serum free media or JNL buffer to allow optimisation of concentration to be used in subsequent gene expression based studies.
The optimal concentration of platelet releasate [determined by MTT] was applied to the cell lines and the levels of apoptosis were compared against cells grown in full media only using a Roche Apodirect TUNEL/Propidium iodide kit. As the 1∶1000 dilution of platelet releasate in full growth media was observed to be the highest concentration of platelet releasate in full media that did not impact on cell growth/viability for all cell lines and was subsequently shown to result in no significant difference in apoptosis compared to cells grown in full media alone it was determined that it was suitable to proceed with this concentration. Given that the platelet releasate was prepared from identical washed platelet preparations, the 1∶1000 dilution of platelet releasate directly informs the use of a 1∶1000 dilution of washed platelets for comparative study. Optimal concentrations of platelet releasate and washed platelets [1∶1000] were suspended in full culture media and applied to cultured cells in 75 cm2 flasks in triplicate. Cells were exposed to releasate or washed platelets for 6 hours after which total RNA was extracted from cells. Transcriptome analysis was performed using Affymetrix Human Exon Arrays.
Cells were washed briefly in PBS, trypsinised and centrifuged to remove supernatant. RNA was extracted using RNeasy mini kit [Qiagen Ltd., West Sussex, UK] according to the manufacturer's protocol. RNA quantity was assessed using a Nano-Drop ND-1000 Spectrophotometer [Wilmington, USA] and quality by an Agilent Bioanalyser 2100 and RNA 6000 Nano microfluidic chip assay [Santa Clara, USA]. RNA was stored at −80°C.
100 ng of total RNA extracted from control cells and cells exposed to washed platelets/platelet releasate was labelled using the Ambion WT Expression kit [Ambion/Life Technologies, Austin, TX, USA] including the labelling controls from the Affymetrix Gene Chip Poly-A RNA Control Kit. As suggested by Affymetrix/Ambion, at each step of the sample preparation protocol, progress was monitored using both the Agilent 2100 Bioanalyzer and the Nanodrop spectrophotometer. Quality control [QC] required assessment after the first cycle RNA cleanup, after the second cycle single-strand cDNA cleanup and following ssDNA fragmentation. Prepared fragmented ssDNA was hybridised to the Affymetrix Human Exon 1.0 ST Array at 45°C for 16 hours following Affymetrix protocols for their GeneChip WT Terminal Labeling, GeneChip Hybridization Control and GeneChip Hybridization, Wash, and Stain kits [Affymetrix, Santa Clara, USA]. Following hybridization, the chips were washed and stained using the Affymetrix GeneChip Fluidics Station with appropriate 64 format assay protocol. Following staining and washing steps the Affymetrix GeneChip Scanner 3000 and Affymetrix GeneChip Operating Software was used for the management and initial processing of the expression data. The data from 45 exon arrays was subject to array quality control performed using the Affymetrix Expression Console. All controls were within the parameters suggested by Affymetrix. Following successful quality control standards assessment, the chip data was then analysed in depth using Biotique Systems XRAY analysis software. Each cell line was examined under three different conditions; resting cells, cells exposed to platelet releasate, and cells exposed to washed platelets. Each cell line and condition was assayed in triplicate. The software was used to compare the two exposure cohorts against the resting control and genes exhibiting a positive or negative fold change of greater than 1.5 and a significance of p≤0.05 examined.
Gene expression was validated using Fluidigm's high throughput qPCR 48.48 dynamic array system. Genes displaying the most significant fold changes in addition to genes involved in biologically relevant pathways were selected for validation. TaqMan® real-time PCR expression assays were used in conjunction with Fluidigm's microfluidic Biomark system. Samples were analysed in triplicate and results were compared with Affymetrix expression data. RNA was reverse transcribed to single stranded cDNA using a High Capacity cDNA RT Kit [Applied Biosystems, CA, USA] in 100μl reactions. Reactions contained 10 μl of buffer [10×], 4 μl of deoxynucleotide triphosphate [25×], 10 μl of random primers [10×], 5 µl of multiscribe RT enzyme [50 U/μl], 21 μl of nuclease-free water and 50 μl of extracted total RNA [20 ng/µl]. Prior to Fluidigm, high throughput qPCR, a pre-amplification step was performed following Fluidigm protocols; 1 ml of TaqMan assay for each of the genes of interest identified through XRAY analysis and endogenous controls were pooled and made to a final volume of 100 ml in 1× TE Buffer, pH 8.0. 1.25 ml of each sample was added to 2.5 ml Preamp Master Mix [AB] and 1.25 ml pooled assay mix to give a 5 ml reaction volume. This mixture was then subject to 14 amplification cycles of 95°C, 15 seconds and 60°C, 4 minutes before being diluted 1∶5 with DNAse and RNase free H20 to give a final volume of 25 ml of pre-amplified cDNA. Fluidigm data was analysed using Fluidigm Real-Time PCR Analysis software [ver3.02] to yield relative quantitation values calibrated to normal [HIO-80] cells. Fold changes returned from Affymetrix analysis were plotted against those calculated from Fluidigm data and the correlation between the values was calculated using Graph Pad Prism [ver 5.02].
The process of EMT is critical in the metastatic cascade, by facilitating entry of cells into the vascular system. RNA was reverse transcribed to single stranded cDNA using a High Capacity cDNA RT Kit [Applied Biosystems, CA, USA] in 100 µl reactions as above. A combination of the primary players in the EMT process
GAPDH - Hs99999905_m1
AKT1 - Hs00178289_m1
ALDH1A1 - Hs00167445_m1
PIK3CA - Hs00180679_m1
SNAIL1 - Hs00195591_m1
SNAIL2 - Hs00950344_m1
ZEB1 - Hs01566407_m1
ZEB2 - Hs00207691_m1
TWIST - Hs00361186_m1
VIMENTIN - Hs00958116_m1
CDH1 - Hs01023895_m1
MYD88 - Hs00182082_m1
TLR4 - Hs00152939_m1
Data were analysed using GraphPad Prism 5.0 software [GraphPad Software Inc., San Diego, CA, USA]. Results are expressed as mean ± standard error of the mean.
Platelet adhesion to ovarian cancer cell lines under static conditions was quantified based on the fluorescent detection of labelled platelets [
[
The ability of ovarian cancer cells to induce platelet activation was quantified by flow cytometry. Increasing concentrations of ovarian cancer cells [0 – 1.5×106 cells/ml] were added to PRP and platelet activation was assessed based on P-selectin expression. Ovarian cancer cells induced a dose-dependent increase in platelet activation [
Platelet activation [P-selectin expression] induced by a range of ovarian cell lines over a large concentration range [0.1–1.5×106/ml] was measured by flow cytometry, based on platelet P-selectin surface expression. The most significant platelet activation was seen in response to the metastatic ovarian cancer cell lines SK-OV-3 and 59M.
Since 59M cells caused the most significant platelet activation, they were used to test the effect of a range of platelet inhibitors on ovarian cancer cell induced platelet activation. 59M ovarian cancer cells [1.5×106 cells/ml, response normalised to 100% activation] were added to PRP pre-treated with inhibitors and platelet activation was measured by flow cytometry [P-selectin expression]. The concentrations of inhibitors were chosen based on preliminary flow cytometry and platelet aggregometry results that showed them to be antagonistic (data not shown). Antagonists against Thrombin [hirudin], integrin αIIbβ3 [Reopro and RGDS peptide], Cox-1 [aspirin], and calcium [EDTA], had no effect on 59M cell induced platelet activation [
This suggests a mechanism of platelet activation dependant on the platelet receptors P2Y12 and P2Y1, and ADP released by 59M cells into their supernatant. Other platelet antagonists such as hirudin, EDTA, abciximab, RGDS, and aspirin had no effect on 59M cell induced platelet activation.
Since thrombosis is a complex process that involves multiple agonists
Activation occurs in a dose dependent manner [n = 3, + SEM]. At low concentrations, 59M [0.5–1×105/ml], cells significantly potentiated TRAP, PAR4 agonist, and Arachidonic acid induced platelet activation [P-selectin expression]. This suggests a synergistic relationship between PAR-1, PAR-4, and TxA2 receptor mediated platelet activation and 59M induced platelet activation. Similar results are seen for A2780 cells [1–5×105 cells/ml, data not shown]
All arrays passed QC using Affymetrix QC software. Biotiques X-Ray software plug-in for Microsoft Excel was used to interrogate gene expression changes between treatments and cell types [Fold change >1.5 and p<0.05]. Analytical stringency was relaxed to fold changes of >1.5 to accommodate subtle but meaningful biological variation [p<0.05] that was contingent upon treatment.
Affymetrix | Fluidigm | |||||
Cell Line | Exposure | Gene | Fold Change | Significance ( |
Fold Change | Significance ( |
HIO-80 | Platelet Releasate |
|
1.50 | 0.0275 | 1.612 | 0.0000 |
Washed Platelet |
|
1.76 | 0.0016 | 1.6909 | 0.0000 |
|
IDI1 | 1.70 | 0.0218 | ||||
PMM2 | 1.60 | 0.0188 | ||||
|
1.59 | 0.0372 | 1.137 | 0.0077 | ||
CCDC68 | 1.56 | 0.0245 | ||||
|
1.55 | 0.0318 | 1.1571 | 0.0000 |
Data presented according to whether cells directly exposed to platelets or releasate. Genes highlighted in bold were selected for validation. Fluidigm validation fold change and t-test
Affymetrix | Fluidigm | |||||
Cell Line | Exposure | Gene | Fold Change | Significance ( |
Fold Change | Significance ( |
59M | Platelet Releasate |
|
1.94 | 0.0137 | 2.83 | 0.0000 |
|
1.92 | 0.0013 | 3.50 | 0.0000 |
||
|
1.67 | 0.0034 | 2.01 | 0.0000 |
||
|
1.64 | 0.0487 | 2.09 | 0.0000 |
||
ANKRD1 | 1.60 | 0.0341 | ||||
|
1.59 | 0.0018 | 2.51 | 0.0000 |
||
|
1.58 | 0.0303 | 1.88 | 0.0000 |
||
ICAM1 | 1.57 | 0.0012 | ||||
IRAK2 | 1.56 | 0.0050 | ||||
|
1.53 | 0.0060 | 2.20 | 0.0000 |
||
CSF2 | 1.52 | 0.0188 | ||||
GCH1 | 1.52 | 0.0211 | ||||
HIVEP1 | 1.51 | 0.0212 | ||||
Washed Platelet |
|
1.90 | 0.0056 | 2.87 | 0.0000 |
|
|
1.78 | 0.0281 | 2.67 | 0.0000 |
||
|
1.63 | 0.0048 | 1.86 | 0.0000 |
||
|
1.50 | 0.0397 | 2.45 | 0.0000 |
||
|
-1.60 | 0.0350 | -1.09 | 0.0036 |
Data presented according to whether cells directly exposed to platelets or releasate. Genes highlighted in bold were selected for validation. Fluidigm validation fold change and t-test
Affymetrix | Fluidigm | |||||
Cell Line | Exposure | Gene | Fold Change | Significance ( |
Fold Change | Significance ( |
SK-OV-3 | Platelet Releasate |
|
1.91 | 0.0346 | -1.09 | 0.0338 |
UBLCP1 | 1.82 | 0.0256 | ||||
RPL7A | 1.72 | 0.0290 | ||||
AK3 | 1.66 | 0.0432 | ||||
MGC71993 | 1.66 | 0.0299 | ||||
|
1.64 | 0.0225 | 1.04 | 0.3191 | ||
|
1.58 | 0.0417 | -1.05 | 0.1362 | ||
PCNP | 1.52 | 0.0455 | ||||
SLC9A8 | 1.52 | 0.0480 | ||||
TPM4 | 1.51 | 0.0356 | ||||
ACTL6A | 1.51 | 0.0380 | ||||
C14ORF153 | 1.50 | 0.0190 | ||||
|
1.50 | 0.0222 | -1.09 | 0.0809 | ||
|
-1.55 | 0.0105 | -1.23 | 0.0007 | ||
|
-1.69 | 0.0036 | -1.09 | 0.0000 |
||
|
-1.69 | 0.0402 | -1.03 | 0.5467 | ||
Washed Platelet |
|
1.58 | 0.0030 | -1.03 | 0.9001 |
Data presented according to whether cells directly exposed to platelets or releasate. Genes highlighted in bold were selected for validation. Fluidigm validation fold change and t-test
Affymetrix | Fluidigm | |||||
Cell Line | Exposure | Gene | Fold Change | Significance ( |
Fold Change | Significance ( |
A2780cis | Washed Platelet |
|
1.63 | 0.0239 | 1.44 | 0.0309 |
|
1.52 | 0.0497 | 1.23 | 0.0000 |
||
ABCD3 | -1.50 | 0.0431 | ||||
RMND1 | -1.50 | 0.0335 | ||||
ARL15 | -1.50 | 0.0137 | ||||
RAD18 | -1.51 | 0.0327 | ||||
CAPZA1 | -1.51 | 0.0324 | ||||
SPAST | -1.52 | 0.0200 | ||||
C11ORF54 | -1.53 | 0.0316 | ||||
MAD2L1 | -1.53 | 0.0407 | ||||
MRPL33 | -1.53 | 0.0328 | ||||
RNASEH2B | -1.53 | 0.0384 | ||||
DAB1 | -1.54 | 0.0204 | ||||
SMCHD1 | -1.54 | 0.0359 | ||||
DPY19L4 | -1.56 | 0.0383 | ||||
EIF2B3 | -1.56 | 0.0310 | ||||
C1ORF112 | -1.56 | 0.0305 | ||||
ZNF271 | -1.56 | 0.0131 | ||||
VGLL3 | -1.56 | 0.0115 | ||||
NDUFAF1 | -1.58 | 0.0443 | ||||
LARP7 | -1.58 | 0.0400 | ||||
SNRPA1 | -1.59 | 0.0276 | ||||
MED10 | -1.60 | 0.0114 | ||||
PTBP2 | -1.60 | 0.0337 | ||||
|
-1.61 | 0.0304 | 1.13 | 0.0019 | ||
PRIM2A | -1.62 | 0.0464 | ||||
|
-1.67 | 0.0259 | 1.02 | 0.4974 | ||
ZNF706 | -1.68 | 0.0259 | ||||
CCDC76 | -1.70 | 0.0263 | ||||
RP9 | -1.71 | 0.0387 | ||||
|
-1.81 | 0.0082 | -1.19 | 0.0054 |
Data presented according to whether cells directly exposed to platelets or releasate. Genes highlighted in bold were selected for validation. Fluidigm validation fold change and t-test
In HIO-80 cells, following treatment with washed platelets or platelet releasate, seven genes were significantly up-regulated, with the greatest difference seen in response to washed platelets. The upregulated genes encode for proteins associated with ovarian cancer metastasis [SERPINB2/PAI2], metabolic activities [IDI1, PMM2] and gene expression/transcription [PCGF6, ZNF267].
59M cells also exhibited gene expression changes following treatment with platelet releasate and washed platelets. The biological processes affected involved anti-autophagy, anti-apoptotic and pro-angiogenic signalling pathways [TRAF2, CCL2, TNFAIP2, PDGFb]. Pro-proliferative [HBEGF, CSF2/GMCSF, IRAK2] immune suppression [CD274/PDL1] anti-apoptotic [BIRC3/CIAP], cell adhesion and migration [ICAM1] signalling was also altered in 59M cells treated with platelet releasate.
SK-OV-3 cells exhibited a greater difference in gene expression following treatment with platelet releasate compared to treatment with washed platelets. The largest fold change was observed for the gene encoding for Cardiolipin Synthase [CRLS1] responsible for cardiolipin [CL] production.
The A2780 cell line and its cisplatin resistant daughter cell line A2780cis used here as a model for recurrent chemo-resistant disease returned different responses to treatment with washed platelets or platelet releasate. The A2780 cell line did not show significant alteration of gene expression in response to either treatment. Conversely, the A2780cis cell line revealed a panel of genes up and down regulated following treatment with washed platelets but none following treatment with platelet releasate. Increased expression was observed in genes for cancer associated proteases [KLK1], cell adhesion/migration molecules [ITGB2/LFA-1], and reduced expression of genes involved in maintaining genomic instability [GMNN], inhibition of gene transcription/expression [CCDC7B, ZNF271, ZNF706, LARP7, RNASEH2B], pro-apoptotic regulation [STK17B/DRAK2] and immune response/evasion [CD58/LFA3].
Gene expression array results were validated using Fluidigm high throughput qPCR technology. Twenty five genes were selected along with two endogenous controls for analysis using Fluidigm's 48×48 Dynamic Array. The genes selected and the cell lines and conditions in which significant alteration of expression were observed by Affymetrix analyses are highlighted in bold in
This figure displays some examples of the correlation between the affymetrix array data and the fluidigm validation data. Correlation coefficients are displayed in the legend.
Ovarian cancer cells exposed to platelets and platelet releasate exhibit a variety of gene expression changes summarised in this figure.
EMT profiling was performed to see if interaction with platelets would induce EMT. Expression analysis of EMT associated genes demonstrated constitutive expression of the majority of EMT associated genes [
This figure displays fold change difference in relation to p value for EMT genes assessed in the cell lines.
The results of the present investigation demonstrate a differential interaction between platelets and ovarian cancer cell lines, not only in terms of the effect of ovarian cancer cells on platelets, but also in the effect of platelets on ovarian cancer cells. Firstly, we used a panel of ovarian cell lines to assess platelet adhesion under static conditions. Adhesion across the 5 cells lines was extremely heterogeneous with significant platelet adhesion to the 59M, A2780, and A2780cis cell lines, while adhesion to the HI0-80 and SK-OV-3 cell lines was not significant compared to the BSA negative control [
Using 59M cells we then investigated the effect of different platelet inhibitors to determine the mechanism of ovarian cancer cell induced platelet activation. Following treatment with cangrelor [P2Y12 antagonist], MRS2179 [P2Y1 antagonist], or apyrase [ADP/ATPase], platelet activation in the presence of 59M ovarian cancer cells was greatly diminished, suggesting a P2Y12/P2Y1 dependent mechanism of activation, mediated by the release of ADP by 59M cells, as the 59M cell supernatant induced comparable platelet activation [
Ovarian cancer cells alone induce activation of platelets. However, thrombosis is a complex process that involves many different agonists
Having established that ovarian cancer cells interact with platelets, we next assessed the effect of platelet adhesion and platelet granule release on ovarian cancer cells. Overall analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed an upregulation of anti-apoptotic, anti-autophagy, pro-angiogenic, pro-cell cycle and metabolic genes in the treated ovarian cancer cells [
In HIO-80 cells, there was significant upregulation of genes encoding for proteins associated with ovarian cancer metastasis [SERPINB2/PAI2], metabolic activities [IDI1, PMM2] and gene expression/transcription [PCGF6, ZNF267]
59M cells exhibited aberrant upregulation of genes involved in anti-autophagy, anti-apoptotic and pro-angiogenic signalling [TRAF2, CCL2, TNFAIP2]
SK-OV-3 cells exhibited significant overexpression in the Cardiolipin Synthase [CRLS1] gene, which is responsible for cardiolipin [CL] production. Anti-cardiolipin antibodies are associated with both solid and non-solid tumours and are associated with increased thrombocytosis
The A2780 cell line and its cisplatin resistant daughter cell line A2780cis displayed different responses to the exposure treatments. The A2780 cell line did not show significant alteration of gene expression following treatment with either platelet releasate or washed platelets. Conversely, the A2780cis cell line revealed a panel of dysregulated genes following treatment with washed platelet but none following treatment with platelet releasate. Increased expression was observed in genes for cancer associated proteases [KLK1],cell adhesion/migration molecules [ITGB2/LFA-1], and reduced expression of genes involved in maintaining genomic instability [GMNN], inhibition of gene transcription/expression [CCDC7B, ZNF271, ZNF706, LARP7, MASEH2B], in pro-apoptotic regulators [STK17B/DRAK2] and immune response/evasion [CD58/LFA3]
TaqMan expression analysis of EMT associated genes demonstrated constitutive expression of the majority of EMT associated genes [
In summary, our data shows for the first time that there is a potent dynamic interaction between ovarian cancer cells and platelets
(TIF)