Differential Platelet Levels Affect Response to Taxane-Based …clincancerres.aacrjournals.org/content/clincanres/21/3/602.full.pdf · Personalized Medicine and Imaging Differential

Personalized Medicine and Imaging

Differential Platelet Levels Affect Response toTaxane-Based Therapy in Ovarian CancerJustin Bottsford-Miller1, Hyun-Jin Choi1, Heather J. Dalton1, Rebecca L. Stone1,Min Soon Cho2, Monika Haemmerle1, Alpa M. Nick1, Sunila Pradeep1, Behrouz Zand1,Rebecca A. Previs1, Chad V. Pecot3, Erin King Crane1,Wei Hu1, Susan K. Lutgendorf4,Vahid Afshar-Kharghan2, and Anil K. Sood1,5,6

Abstract

Purpose: We hypothesized that platelet levels during therapycould serve as a biomarker for response to therapy and thatmanipulation of platelet levels could impact responsiveness tochemotherapy.

Experimental Design: The medical records of patients withrecurrent or progressive ovarian cancer were retrospectively que-ried for changes in platelet and CA-125 levels during primarytherapy. In vitro coculture experiments and in vivo orthotopicmodels of human ovarian cancer in mice were used to test theeffect of modulating platelet levels on tumor growth and respon-siveness to docetaxel.

Results: Thrombocytosis at the diagnosis of ovarian cancerwas correlated with decreased interval to progression (P¼ 0.05)and median overall survival (P ¼ 0.007). Mean platelet levelscorrected during primary therapy and rose at recurrence. Con-trary to treatment-responsive patients, in a cohort of patients

refractory to primary therapy, platelet levels did not normalizeduring therapy. In A2780, HeyA8, and SKOV3-ip1 ovariancancer cell lines, platelet coculture protected against apoptosis(P < 0.05). In orthotopic models of human ovarian cancer,platelet depletion resulted in 70% reduced mean tumor weight(P < 0.05). Compared with mice treated with docetaxel, micetreated with both docetaxel and platelet-depleting antibodyhad a 62% decrease in mean tumor weight (P ¼ 0.04). Platelettransfusion increased mean aggregate tumor weight 2.4-fold (P< 0.05), blocked the effect of docetaxel on tumor growth (P ¼0.55) and decreased tumor cell apoptosis. Pretransfusion aspir-inization of the platelets blocked the growth-promoting effectsof transfusion.

Conclusions: Platelet-driven effects of chemotherapy responsemay explain clinical observations. Clin Cancer Res; 21(3); 602–10.�2014 AACR.

IntroductionThrombocytosis, defined as >450,000 cells/mL, is found in

more than 30% of patients with epithelial ovarian cancer and isassociated with decreased progression-free and overall survival(1). Paraneoplastic thrombocytosis, in addition to hypercalce-mia, leukocytosis, and cachexia, has been shown to occur throughthe generation of IL6 (1, 2). IL6 expression correlates with ovariancancer taxane sensitivity (3). Platelet transfusion leads to

increased tumor cell proliferation (2). Recent clinical work sup-ports the relationship between thrombocytosis and poor prog-nosis in ovarian cancer (4, 5).

The connection between platelets and metastasis is estab-lished (6–14). Platelets have been shown to mediate protectionof micrometastases from NK cell–mediated clearance (15).Direct signaling between platelets and tumor cells contributesto the epithelial-to-mesenchymal transition (16). The role forplatelets in metastasis has proven multifactorial, includingplatelet–tumor interactions involving multiple protein classesand functions (17–21).

Exposure of human adenocarcinoma cells to platelets increasessurvival, proliferation, and in vitro chemoresistance through theupregulation of antiapoptotic pathways, downregulation of proa-poptotic pathways, promotion of DNA synthesis, increased cyclinexpression, increased DNA repair protein expression, and increasedMAPKexpression (22). Inductionof thrombocytopenia in amurinemodel of breast carcinoma results in greater taxane efficacy thatcorrelates with increased vascular leakage at the tumor site (23).Platelets sequester and differentially release angiogenic and mito-genic mediators (24–28). Release of alpha-granule contents andplatelet-driven neutrophil chemotaxis are variable based on pH,suggesting a complex regulatory function (29). Dense granulesrelease agents known to modulate cell growth and migration (30).

Considering the growing evidence for correlation betweenplatelet levels and clinical outcomes, we considered whetherplatelet levels could serve as a biomarker of treatment response.We investigated, using in vitro and preclinical in vivo models,

1Department of Gynecologic Oncology, The University of Texas MDAnderson Cancer Center, Houston, Texas. 2Section of Benign Hema-tology,The University of Texas MDAnderson Cancer Center, Houston,Texas. 3Department of Molecular Therapeutics, University of NorthCarolina Lineberger Comprehensive Cancer Center. 4Department ofPsychology, Obstetrics and Gynecology, and Urology, University ofIowa, Iowa City, Iowa. 5Department of Cancer Biology, The Universityof TexasMDAndersonCancerCenter, Houston,Texas. 6Center forRNAInterference and Non-Coding RNAs, The University of Texas MDAnderson Cancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

V. Afshar-Kharghan and A.K. Sood contributed equally to this article.

Corresponding Author: Anil K. Sood, Departments of Gynecologic Oncologyand Reproductive Medicine and Cancer Biology, The University of Texas MDAnderson Cancer Center, 1155 Herman Pressler Blvd., Unit 1352, Houston, TX77030. Phone: 713-745-5266; Fax: 713-792-7586; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0870

�2014 American Association for Cancer Research.

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whether modulation of platelet counts could influence responseto chemotherapy, and whether such effects could be blocked toimprove sensitivity to taxane-based chemotherapy.

Materials and MethodsApprovals

Approval for relevant studies was obtained from the Universityof Texas at M.D. Anderson Cancer Center Institutional ReviewBoard (IRB). All animal experiments were approved and super-vised by the MDACC Institutional Animal Care and UseCommittee.

Clinical analysisPatients were retrospectively identified at the University of

Texas at M.D. Anderson Cancer Center (MDACC), the Universityof Iowa, and the University of Virginia who were diagnosed withovarian, primary peritoneal, or fallopian tube carcinoma. Thisdatabase was partially overlapping with that reported by Stoneand colleagues (1). Patients were excluded if they did not receiveprimary therapy or follow-up at the institution of record. Toexplicitly focus onpatterns of recurrence andprogression, patientswere excluded who did not develop tumor recurrence or progres-sion. Exclusions were made for a history of other malignancy,myeloproliferative disease, inflammatory disease, splenectomy,or other confounding cause of thrombocytosis. All patients weretreated by surgical cytoreduction performed by a gynecologiconcologist in addition to adjuvant or neoadjuvant taxane- and/or platinum-based chemotherapy. Clinical data collected includ-ed patient demographics, tumor characteristics, details of treat-ment, and outcomes data. Platelet levels and CA-125 measure-ments were recorded at the time of primary evaluation, throughtherapy, after the completion of surgery and 6 cycles of cytotoxicchemotherapy, during the posttherapy monitoring period, and atthe time of diagnosis of ovarian cancer recurrence. Thrombocy-tosis was defined as a platelet count greater than 450,000/mL (31).Interval toprogressionwasdefined starting at the conclusionof sixcycles of primary therapy and ending at the clinical diagnosis ofrecurrence by physical exam, laboratory evaluation, and/or imag-

ing. The survival interval was also defined as starting at theconclusion of six cycles of primary chemotherapy. Patients whowere known to be alive at the time of last contact were censoredaccordingly.

Preclinical analysisDocetaxel.Docetaxel (Sanofi-Aventis) is a commonly used taxanechemotherapy shown in phase III clinic trials to be equivalent topaclitaxel in the primary therapy of ovarian cancer (32).Docetaxelwas obtained from surplus clinical samples from the clinicalpharmacy associated with the University of Texas M.D. AndersonCancer Center.

Cell lines and culture conditions. The derivation of the humanovarian cancer cell lines A2780, HeyA8, and SKOV3-IP1 arepreviously reported (33). Cell lines were obtained from theinstitutional Cell LineCore laboratory andper institutional policy(MD Anderson policy ACA#1044) cell line authentication wasperformed at least once per year. In this case, authentication wasperformedwithin6months of theworkdescribed. Authenticationwas performed by the short tandem repeat method using thePromega Power Plex 16HS kit (Promega). Somatic mutationswere detected using a Sequenom MALDI TOF MassArray system(Sequenom). Mycoplasma detection was performed using theMycoAlert Kit (Lonza). The cell lines were maintained in RPMI-1640 with 15% FBS. Cell lines were routinely genotyped toconfirm identity and tested to confirm absence of mycoplasma.Cells were maintained at 37�C in a humidified incubator infusedwith 20% O2 and 5% CO2.

Platelet isolation for in vitro assays. Platelets were prepared for invitro assays in a manner that would remove plasma contents andnucleated cells. Whole blood was drawn from the inferior venacava of anesthetized nude mice into a syringe preloaded with 1:9v/v 3.8% sodium citrate and mixed 1:1 v/v with tyrodes bufferlackingMg2þ and Ca2þ. Blood was centrifuged at 1,100 rpm for 3minutes, twice, at room temperature. The platelet-rich plasmafraction was passed through a filtration column of Sepharose 2Bbeads (SigmaAldrich) loaded into a siliconized glass columnwitha 10-mm nylon net filter (Millipore) and sepharose 2B beadspreviously washed in acetone 1:1 v/v, followed by 0.9%NaCl 1:1v/v, and "Buffer 1" 1:1 v/v. Platelet-containing eluent was diluted1:200 andplateletswere countedwith ahemocytometer byphase-contrast microscopy at 400� magnification.

In vitro assays. To examine potential effects of platelets on apo-ptosis and response to chemotherapy, we incubated cancer cellswith platelets using a tissue coculture system and observedconsistent protection against apoptosis. To assess the effect ofplatelets on apoptosis, cells were plated in 6-well plates at 50,000cells per plate. At 50% confluence, media was changed to serum-free for 24 hours before starting treatment. After serum starvation,platelets were isolated and added to achieve a final number of 1�108 platelets/mL. Docetaxel was dosed at 5 mmol/L based onpreviously published IC50 levels. Controls utilized an equivalentvolume of the appropriate buffer. All treatments were performedin triplicate. After 72 hours of platelet and docetaxel exposure,apoptosis and cell viability were assessed using Annexin V and 7-amino-actinomycin-D staining (BD Pharmingen) by flow cyto-metry. Indirect contactwithplateletswasprovidedby theuseof anintervening cell culture insert with 0.4-mm pores (BD Falcon).

Translational Relevance

Thrombocytosis is known to correlate with poor clinicaloutcomes in cancer and to be caused by tumor cells, andplatelets are known to participate in metastasis. In this work,we show that in patients with recurrent ovarian cancer, ele-vated platelet counts at diagnosis correlated with decreasedinterval to progression anddecreasedoverall survival. Changesin platelet counts during and after therapymay be a biomarkerfor response to that therapy and for recurrence. Plateletsprotect ovarian cancer cells from apoptosis in a manner notrequiring a direct contact. Platelet transfusion results inincreased tumor growth that can be at least partially blockedwith aspirin. Furthermore, platelet transfusion decreases theefficacy of taxane-based chemotherapy, and platelet depletionincreases the efficacy of the same therapy. These findings arguefor reconsideration of the risk of platelet transfusion andthrombopoietin receptor agonists, as well as for considerationof antiplatelet reagents as chemosensitizers.

Platelet Levels Affect Response to Taxanes in Ovarian Cancer

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Proliferation was measured by flow cytometry (Click-iT EdUKit; Invitrogen). For platelet fixation experiments, plasma-freeplatelets were incubated in 1% paraformaldehyde (2). To testthe effect of aspirin in this system, a 325-mg tablet of aspirinwas dissolved in deionized, distilled water and filter sterilized.Cancer cells were plated, plasma-free platelets were isolatedand coincubated with aspirin 30 mmol/L, and in vitro experi-ments were performed as described. Internal controls (n ¼ 3)were performed for each experiment given the variability inbaseline apoptosis and proliferation rates seen between experi-ments to avoid batch error.

Orthotopic model of ovarian cancer in nude mice. Female athymicnude (NCr-nu) were purchased from Taconic Farms, Inc. Thedevelopment and characterization of the orthotopic mouse mod-el of ovarian cancer has been previously described (34). SKOV3-IP1 (1 � 106 cells/mouse), A2780 (1 � 106 cells/mouse), orHeyA8 (0.25 � 106 cells/mouse) were lifted with trypsin/EDTA,washed with PBS, and resuspended in 200 mL of Hank's balancedsalt solution (HBSS, Mediatech, Inc.) and were injected into theperitoneal cavity of female nude mice.

Platelet-depleting antibody. To deplete platelets in mice forin vivo experiments, we used a commercially available ratanti-mouse monoclonal antibody directed against mouseGP1b-alpha (CD42b, Emfret Analytics) that causes irreversibleplatelet depletion within 60 minutes of administration with-out inducing platelet activation. Dose kinetics are previouslyvalidated (1).

Thrombocytosis, thrombocytopenia, and effect on chemotherapyin vivo. The cell lines A2780 and SKOV3-IP1 were used in theorthotopic model of nude mice. The animals were injected withtumor on day 0 as described above. Starting on day 7, animalswere randomized into the following groups: twiceweekly tail veininjections of control IgG (0.5 mcg/gram); twice weekly controlIgG via tail vein injection and weekly docetaxel 35 mcg i.p.;platelet-depleting antibody (0.5 mcg/gram) via tail vein injectiontwice weekly; platelet-depleting antibody plus docetaxel; tail veintransfusion of platelet rich plasma isolated from nude mice;platelet transfusion and docetaxel. Mice were treated until theybecame moribund and then sacrificed.

Aspirinization of platelets. Pharmacy grade aspirin was acquired,and a single 325-mg tablet was dissolved in 500 mmol/L sodiumacetate (pH 5.6). This was added 1:10 v/v to platelet rich plasmaand the combination was incubated at 37�C for 15 minutes.Incubation with an equivalent sodium acetate solution withoutaspirin was used for control.

Effect of aspirin on thrombocytosis and malignancy in vivo. Usingthe A2780orthotopicmodel of ovarian cancer,micewere injectedwith tumor onday 0.Onday 7, the animalswere randomized intothe following groups: untreated control; intraperitoneal aspirin20 mg/kg twice per week; 500 mL of platelet rich plasma isolatedfrom nude mice and incubated with sodium acetate for 15minutes (as described above) via tail vein injection weekly; tailvein transfusion of platelet rich plasma that had been incubatedfor 15 minutes with a 50 mmol/L solution of aspirin in sodiumacetate (as described above). Mice were treated until they becamemoribund and then sacrificed.

Cleaved caspase-3 immunohistochemistry. Immunohistochemistryfor cleaved caspase-3wasused tomeasure apoptotic rates ex vivo intumor using a rabbit polyclonal anti-human antibody to cleavedcaspase-3 (BioCare Medical; #CP229B). Paraffin-embeddedtumor sections were heated, deparaffinized, and antigen retrievalwas performed by steaming, and endogenous peroxides wereblocked with 3% hydrogen peroxide in methanol. Nonspecificproteins were blocked with 4% fish gelatin in PBS. Slides wereincubated in primary antibody (1:100), and the secondary anti-body (ready-to-use) was followed by streptavidin horseradishperoxidase (ready-to-use). Slides were quantified by counting thenumber of positively staining cells per 200� field.

Statistical analysisA two-sided long-rank statistic was used to compare Kaplan–

Meier survival curves. Variables estimated to have a normaldistribution were compared using the Student t test using Excel(Microsoft). The F test was used to compare variances whereindicated. A P of <0.05 was considered statistically significant.Formouse experiments, sample sizewas estimatedutilizing a two-way ANOVAmodel. For an effect size of 0.65, a sample size of 10mice per group was considered sufficient to provide 80% powerfor a ¼ 0.05 anticipating less than 10 groups.

ResultsThrombocytosis is associated with resistance to chemotherapy

We first identified patients known to have recurrent or pro-gressive epithelial ovarian cancer (n ¼ 355) for whom adequatelaboratory data before treatment, treatment data, and posttreat-ment follow-up data were available. Demographics (Supplemen-tary Table S1) indicated a median age 61 years (range 31–88years). Ninety percent had advanced stage (III or IV) and 89%hadhigh-grade disease. For primary therapy, all patients underwent acombination of surgical cytoreduction (60%had "optimal" cytor-eduction to <1 cm gross residual disease) and taxane-basedchemotherapy, most commonly paclitaxel and carboplatin. Inthis population, in which all patients developed disease recur-rence, the mean platelet level was 409,000/mL (range 134,000–1,122,000 cells/mL) at diagnosis. Thirty-two percent had amean platelet level of >450,000 cells/mL at the time of diagnosis.Even after patients without diagnosed recurrence were excluded,thrombocytosis at diagnosis was associated with worsemedian progression-free survival (12.9 vs. 14.7 months, P ¼0.05; Fig. 1A) and median overall survival (16 vs. 20.8 months,P ¼ 0.007; Fig. 1A).

A subgroup of 96 patients was identified whose availablelaboratory data were adequate to consider platelet and CA-125trends through primary diagnosis, primary treatment, surveil-lance, and until the clinical diagnosis of recurrence (Fig. 1B,Supplementary Table S2). CA-125 is a standard tumor markerfollowed in ovarian cancer to track the efficacy of primarytherapy and in surveillance for recurrence. In this group ofpatients, 86% of patients had a normal CA-125 level (<35 U/mL) at the conclusion of primary therapy. In contrast, allpatients had a normal platelet count <450,000 cells/mL (mean206,000 cells/mL) after primary therapy. At the clinical diag-nosis of disease recurrence or progression, CA-125 was elevatedin 75% of patients. In parallel, at the diagnosis of recurrence,mean platelet counts were found to be increased 57.8% to262,000 cells/mL compared with nadir levels found after

Bottsford-Miller et al.

Clin Cancer Res; 21(3) February 1, 2015 Clinical Cancer Research604




primary therapy was completed (P < 0.001; Fig. 1B; Supple-mentary Table S2). Among patients with a CA-125 <35 U/mL atthe time of recurrence, platelet levels were increased by 49%(mean increase 108,400 cells/mL, P < 0.01) at the diagnosis ofrecurrence compared with the conclusion of primary therapy.

Among patients with ovarian cancer, approximately 10%willnot respond to primary therapy and are considered to have"refractory" disease. From the 96 patients with complete lon-gitudinal data, 10 patients were identified who had diseaserefractory to primary treatment. Ten additional patients(matched for stage, grade, histology, and primary therapy)were identified for comparison who experienced a completeresponse to primary therapy that was durable for at least 6months. In the patients who experienced a compete response totherapy that was durable for >6 months, 50% had thrombo-cytosis at diagnosis, and all of these patients consistentlynormalized platelet counts by the end of primary therapy (Fig.1C). In the treatment-refractory cohort, all patients had throm-bocytosis at the time of diagnosis, and platelet counts were farmore heterogeneous during primary therapy, with only 50%having normalized platelet counts by the completion of pri-

mary therapy (Fig. 1C). These data suggest a correlationbetween the normalization of platelet counts during primarytherapy and disease response to that therapy.

Platelets mediate resistance against chemotherapy-inducedapoptosis in vitro

Tissue coculture with platelets demonstrated consistent protec-tion against apoptosis, both directly and indirectly, and with orwithout exposure to docetaxel. Platelet activation was evident bythe aggregation of platelets within the initial hours of 37�Cincubation. Direct incubation of the A2780, HeyA8, andSKOV3-ip1 cells with platelets in serum-free conditions reducedapoptosis by 46.7% (P¼ 0.002), 64.4% (P < 0.001), and47.3% (P¼ 0.004), respectively (Fig. 2A). After incorporating docetaxel,direct incubation of the same cell lines with platelets reducedapoptosis by 20.4% (P¼ 0.004), 74.0% (P < 0.001), and15.1% (P¼ 0.007), respectively (Fig. 2A). To consider whether direct contactbetween platelets and tumor cells was required to observe thesechanges in apoptotic rates, ovarian cancer cells were indirectlyincubated with platelets across a barrier with 0.4-mm pores for72 hours in a serum-free environment with or without docetaxel

Figure 1.A, patients with recurrent ovarian cancer (n ¼ 341) were identified and stratified according to their platelet counts at diagnosis into two groups: those withthrombocytosis (>450,000 cells/mL) and those with normal platelet counts (<450,000 cells/mL). The patients with thrombocytosis at the time of diagnosishad a significantly shortermedian interval to progression (12.9 vs. 14.7months,P¼0.05). The patientswith thrombocytosis at the timeof diagnosis had a significantlyshortermedianoverall survival (16 vs. 20.8months,P¼0.007). B, patientswith recurrent ovarian cancer forwhom longitudinal datawere available through treatmentand surveillance (n ¼ 96). The mean platelet level at diagnosis was 403,000 cells/mL. During primary therapy, the mean platelet nadir was 198,000 cells/mL.At the conclusionof therapy, the averageplatelet levelwas 221,000 cells/mL, and this remained stable in the surveillanceperiod,with themeannadir count of 166,000cells/mL during this time. At recurrence, mean platelet counts increased 27% to 262,000 cells/mL (P < 0.001). Of the patients with available longitudinal data,the mean CA-125 level at diagnosis was 332 U/mL (normal <35 U/mL). Only 86% had a normal CA-125 level at the conclusion of primary therapy with a mean level of63 U/mL, and the mean posttreatment nadir was 23 U/mL. At the clinical diagnosis of disease recurrence, CA-125 was elevated in 75% of patients, with a medianof 229 U/mL. C, a subgroup of the patients with complete longitudinal datawas identified who experienced progression of disease through firstline therapy (n¼ 10).These patients were matched to a cohort who experienced a durable response to therapy lasting more than 6 months. In the subgroup of patients with adurable response, only 50% had thrombocytosis (>450,000 cells/mL) at the time of diagnosis, and all patients in this subgroup achieved normal platelet countsduring therapy. In the treatment-refractory cohort, all patients had thrombocytosis at the time of diagnosis, and platelet levels were more heterogeneousduring primary therapy, with only 50% having normalized platelet counts by the completion of primary therapy.






5 nmol/L. Indirect incubation of A2780, HeyA8, SKOV3-ip1, and2774 cells with platelets in serum-free conditions reduced apo-ptosis by 60.8% (P < 0.001), 80.7% (P ¼ 0.001), 82.3% (P <

0.001), and 25.3% (P ¼ 0.002), respectively (Fig. 2B). Afterincorporating docetaxel, direct incubation of the same cell lineswith platelets reduced apoptosis by 17.4% (P < 0.001), 31.9% (P <

Figure 2.A, in vitro, A2780 directly coculturedwith platelets (plt) 10� 107/mL� docetaxel 5 nmol/L, platelets decreased apoptosis from 10.7% to 5.7% (P¼0.004) comparedwith serum-free media (SFM). With the addition of docetaxel, platelets decreased apoptosis from 46.6% to 37.1% (P ¼ 0.007). HeyA8 directly cocultured withplatelets � docetaxel, platelets decreased apoptosis from 21.8% to 7.8% (P < 0.001) compared with serum-free media. With the addition of docetaxel, plateletsdecreased apoptosis from 28.6% to 7.5% (P <0.001). SKOV3-ip1 directly coculturedwith platelets�docetaxel, platelets decreased apoptosis from 17.1% to 9.0% (P¼0.008) compared with serum-freemedia.With the addition of docetaxel, platelets decreased apoptosis from 70.1% to 59.5% (P¼0.013). B, in vitro, A2780 indirectlycocultured with platelets � docetaxel, platelets decreased apoptosis from 48.5% to 19.0% (P < 0.001) compared with serum-free media (SFM). With theaddition of docetaxel, platelets decreased apoptosis from 70.4% to 58.2% (P < 0.001). HeyA8 indirectly cocultured with platelets � docetaxel, platelets decreasedapoptosis from 46.1% to 8.9% (P < 0.001) compared with serum-free media. With the addition of docetaxel, platelets decreased apoptosis from 75.8% to 51.6% (P¼0.001). SKOV3-ip1 indirectly cocultured with platelets� docetaxel, platelets decreased apoptosis from 34.3% to 6.1% (P < 0.001) compared with serum-free media.With the addition of docetaxel, platelets decreased apoptosis from 60.8% to 40.2% (P ¼ 0.001). 2774 indirectly cocultured with platelets � docetaxel, plateletsdecreased apoptosis from 9.9% to 7.4% (P ¼ 0.003) compared with serum-free media. With the addition of docetaxel, platelets decreased apoptosis from25.9% to 18.8% (P¼ 0.033). C, in vitro, HeyA8 cells were incubated in serum-free media, platelets, and buffer or paraformaldehyde-fixed platelets. Normal plateletsdecreased apoptosis from 9.6% to 3.3% (P < 0.001). In contrast, platelet fixation had no significant effect on tumor cell apoptosis (10.4%, P ¼ 0.28). SKOV3-ip1cells were incubated with serum-free media, platelets, and/or aspirin (ASA) 30 mmol/L. EdU incorporation was used to measure proliferation by flowcytometry. Platelet coculture increased proliferation from 21.8% to 34.1% (P¼ 0.004) , whereas aspirin had no effect. When aspirin was added to the platelets, thedegree of proliferation was decreased to 27.3% (P ¼ 0.22, compared with serum-free medium control).






0.001), 33.9%(P<0.001), and27.5%(P¼0.03), respectively (Fig.2B). These data suggest that platelets have an antiapoptotic effecton cancer cells, and they suggest that this effect does not requiredirect contact between platelets and tumor cells.

To determine whether platelet activation was necessary for theapoptosis protection, the above experiments were repeated usingplatelets fixed with paraformaldehyde. Fixation of platelets abro-gated the antiapoptotic effect (P ¼ 0.28, Fig. 2C), suggesting thatplatelet activation is necessary for the antiapoptotic effects. Plate-let coculture has previously been shown to increase tumor cellproliferation, which was abrogated by platelet fixation (2).Acknowledging that aspirin is a moderate inhibitor of plateletactivation, aspirin pretreatment of platelets was utilized to blockthe proproliferative effects of platelets in vitro. The cell lineSKOV3-ip1 was cocultured with platelets with or without aspirin30 mmol/L for 24 hours and evaluated by flow cytometry for EdUincorporation as a proxy for proliferation. As anticipated, plateletcoculture increased proliferation by 56.5% (P¼ 0.004). Inclusionof aspirin abrogated the effect of platelet coculture (Fig. 2C).

Effects of platelets on tumor growth and response tochemotherapy in vivo

All ovarian cancer cell lines utilized here are known to causeincreased platelet counts (1). To simulate the effects of excessplatelet counts, allogeneic platelet transfusions were performed.Noting that platelet activation was apparently necessary for theantiapoptotic effects in vitro, we considered whether in vivo effectsfrom platelet transfusion might be blocked by utilizing aspirin.Nude mice were given intraperitoneal injections of A2780 cells,and 7 days later, theywere randomized to the following treatmentgroups (n ¼ 10 mice/group): untreated control, intraperitonealaspirin, platelet transfusion, and aspirinized platelet transfusion.Platelet transfusion resulted in a 1.9-fold increase in the aggregatemean tumor weight compared with control (P ¼ 0.01; Fig. 3A).Intraperitoneal aspirin therapy did not have any significant effecton aggregate tumor weight. In contrast, preaspirinization of theplatelets blocked the progrowth effect of platelet transfusion (P¼

0.01 compared with platelet transfusion; P ¼ NS compared withcontrol; Fig. 3A).

In resected tumor specimens, ex vivo immunohistochemistrydemonstrated that platelet transfusion resulted in a 37% lowerrate of apoptosis compared with control (P ¼ 0.009; Fig. 3B).Aspirin delivered intraperitoneally did not significantly changethe apoptotic rate in tumor (P ¼ 0.86; Fig. 3B). In contrast,aspirinizing platelets before transfusion blocked the antiapopto-tic effect of platelets on tumor (P ¼ 0.11; Fig. 3B).

We next studied the effect of platelets on response to taxane-based chemotherapy in vivo by reducing platelet counts using ananti-platelet antibody (APA) that sequesters circulating plateletsand has been previously validated in our laboratory (1).Seven days after intraperitoneal injection of A2780 cancer cells,mice were randomized to the following treatment groups: controlIgG, APA, control IgGwith docetaxel, or APAwith docetaxel. After5 weeks, mice treated with APA had 65% decrement in meanaggregate tumor weight compared with control (P ¼ 0.008, Fig.4A) that was similar to the 70% decrease that resulted fromtreatment with docetaxel (P ¼ 0.004, Fig. 4A). There was nostatistical difference between the APA treatment and docetaxeltreatment (P ¼ 0.35, Fig. 4A). In comparison, mice treated withboth the APA and docetaxel had an additional 62% reduction inaggregate tumorweight comparedwith that achievedbydocetaxelalone (P ¼ 0.04, Fig. 4A).

To confirm this finding and consider the effect of platelettransfusion, nude mice were given intraperitoneal injectionsof SKOV3-ip1 cells, and after 7 days were randomized tothe following groups: control IgG, APA, twice weekly platelettransfusion, control IgG with docetaxel, APA with docetaxel,and platelet transfusion with docetaxel. Platelet depletionand docetaxel resulted in similar reductions in tumor size atnecropsy (Fig. 4B). Mice given platelet transfusions had a 2.4-fold increase in mean aggregate tumor weight compared withcontrols (P ¼ 0.01, Fig. 4B). Compared with mice treated withdocetaxel, treatment with docetaxel and platelet transfusionresulted in a 4-fold increase in mean aggregate tumor weight

Figure 3.A, in vivo, A2780-bearing nude mice were allocated into the following groups (n ¼ 10): untreated control, intraperitoneal aspirin, platelet transfusion, andtransfusion of aspirinized platelets. Aspirin by itself had no significant effect on mean aggregate tumor weight at necropsy. Platelet transfusion increased meanaggregate tumor weight from 2.1 to 4.1 g (P ¼ 0.03). Pretransfusion aspirinization of platelets abrogated the increased tumor growth (2.1 vs. 2.1 g, P ¼ NS). B,immunohistochemistry for cleaved caspase-3 demonstrated reduction in apoptosis in tumors of mice receiving platelet transfusion from 28.1/hpf to 17.6/hpf(P ¼ 0.009). Pretransfusion aspirinization of platelets partially abrogated the reduction of apoptosis by 22.2% (P ¼ 0.11 compared with control). Intraperitonealaspirin had no statistically significant effect on the rate of apoptosis.






(P ¼ 0.004, Fig. 4B). Mice given platelet transfusions and treatedwith docetaxel had a similar mean aggregate tumor weight to thatof untreated controls (P ¼ 0.55, Fig. 4B). Compared with micetreated with docetaxel, mice treated with APA and docetaxel had a51% decrease in mean tumor weight (P ¼ 0.02, Fig. 4B). In aconfirmatory experiment using the SKOV3-ip1 model, the ani-mals were randomized to control IgG, twice weekly platelettransfusion, or platelet transfusion with APA. Platelet transfusionresulted in a 70% increase in mean aggregate tumor weight (P ¼0.001, Fig. 4C), whereas the combination of platelet transfusionwith APA resulted in a nonsignificant decrease in mean aggregatetumor weight compared with control (P ¼ 0.06, Fig. 4C).

DiscussionIt is increasingly recognized that there are multiple biological

components that participate in a cooperative relationshipbetween the host and tumor cells. Crosstalk between various celltypes, including platelets, leukocytes, and endothelial cells, hasbeen shown to participate in the epithelial-to-mesenchymal tran-sition, metastasis, as well as arrest of tumor emboli with theestablishment of themetastatic niche (16, 35). Platelets have beenshown to sequester angiogenesis regulators in addition to othermitogens (24) and release these compounds from alpha-granulesin a manner that modulates angiogenesis (27). There is evidencethat exposure to anticoagulants decreases platelet release of VEGF,suggesting that anticoagulants may alter the potential of plateletsto facilitate angiogenesis (36).

In a cohort of patients enriched for recurrence of disease, wefound that elevated platelet counts correlated with a decreasedinterval to progression and decreased overall survival. Overallsurvival as a trial endpoint is influenced by therapeutic crossover;therefore, it is notable that thrombocytosis correlates with wors-ened overall survival, suggesting that platelet effects may beagnostic to the types of therapy used. Furthermore, we demon-

strated that platelet counts might be useful as a tumor marker, inparallel to CA-125 levels, to follow treatment response and followin surveillance for recurrence. These data were limited by providervariation in the frequency of both CA-125 and CBC checks.Standardization as well as prospective analysis could allow thedevelopment of prospective algorithms to test for the predictivevalue of platelet response as a biomarker for tumor response.

In breast cancer models, chemotherapy was found to be moreeffective in the context of thrombocytopenia, and the effect wasattributed to intratumoral hemorrhage facilitated by leukocytesand deficiency in b-2 or b-3 integrins (26, 37). On the basis of ourobservation in patients with ovarian cancer that elevated plateletcounts are associated with higher rates of relapse and lower ratesof response to chemotherapy, we hypothesized and confirmedthat platelets might confer resistance to apoptosis, including thatinduced by taxane chemotherapy. Coincubation of cancer cellswith platelets resulted in platelet aggregation, and blockade ofplatelet activation abrogated these effects. Aspirin at least partiallyblocked the increased tumor cell proliferation attributed to plate-let coculture.

A series of meta-analyses of randomized and case–controlstudies have indicated a significantly reduced risk of malignancyin individuals treated with low-dose aspirin (38–41). In ourmodel, platelet transfusion resulted in accelerated tumor growththat was partially blocked by pretreatment of the platelets withaspirin; however, intraperitoneal administration of aspirin didnot have a clear effect. Aspirin is a moderate inhibitor of plateletactivation and aggregation, and it is known that other activatingstimuli (e.g., shear force, catecholamines, thrombin, andADP) arecapable of activating platelets despite aspirinization throughnon–thromboxane-dependent mechanisms (42). The intraperi-toneal aspirin dose utilized here may not have been adequate toovercome these mechanisms.

The potential impact of platelet transfusion on cancer progres-sion or survival has not been well studied. Concern has been

Figure 4.A, in vivo, A2780-bearing nudemicewere treated with a control IgG, a platelet-depleting anti-platelet antibody (APA), docetaxel, or a combination of docetaxel andAPA. Animals treated with APA had a 65% decrease in mean aggregate tumor weight compared with control (P ¼ 0.008) that was similar tothe 70% decrease that resulted from treatment with docetaxel (P¼ 0.004 compared with control). There was no statistical difference between the APA treatmentand docetaxel treatment (P ¼ 0.35). Mice treated with both APA and docetaxel had an additional 62% reduction in aggregate tumor weight comparedwith that achieved by docetaxel alone (P ¼ 0.04). B, in vivo, SKOV3-ip1–bearing nude mice were treated with control IgG, APA, and docetaxel, and/or platelettransfusion. Platelet depletion with APA resulted in a 43% decrease in mean aggregate tumor weight of borderline significance (P ¼ 0.07). Docetaxel resulted in asimilar reduction in mean aggregate tumor weight (69%, P ¼ 0.006). Mice given platelet transfusions had a 2.4-fold increase in mean aggregate tumorweight compared with control (P¼ 0.01). Compared with mice treated with docetaxel, mice treated with docetaxel and platelet transfusion had a 4-fold increase inmean aggregate tumor weight (P ¼ 0.004). Mice given platelet transfusions and treated with docetaxel had a mean aggregate tumor weight similar tothat of untreated controls (P ¼ 0.55). Compared with mice treated with docetaxel, mice treated with APA and docetaxel had a 51% decrease in mean tumorweight (P¼ 0.02). C, in vivo, SKOV3-ip1–bearing nudemice were treated with control IgG, platelet transfusion, or platelet transfusion with APA. Platelet transfusionresulted in a 70% increase in mean aggregate tumor weight (P¼ 0.001), whereas the combination of platelet transfusion with APA resulted in a nonsignificant 40%decrease in mean aggregate tumor weight compared with control (P ¼ 0.06).






identified that erythropoiesis-simulating agents are associatedwith tumor progression and decreased survival (43–45). In thiscontext, some centers are exploring the effects of agents such asromiplostim (a thrombopoietin receptor agonist) to maintainplatelets >100 � 109/L in patients being treated with cytotoxicchemotherapy. Limited data report a 15% DVT rate and are notadequate to consider impact on progression and/or survival (46).Ourmodel would suggest that care should be taken when platelettransfusions or thrombopoietin receptor agonists are consideredin cancer patients.

We further demonstrated that reduction of platelet counts invivo reduced tumor growth to the same extent as chemotherapy,and platelet transfusion strongly counteracted the antitumoreffect of chemotherapy. Thrombocytopenia is a common toxicityof frontline chemotherapy, and clinical trials will decline toenroll, delay therapy, or remove patients from protocols basedon persistent platelet levels less than 10 � 105 cell/mL (47). Theeffect of relative thrombocytopenia and platelet transfusion onthe response to chemotherapies needs to be investigated in alarger number of patients in a controlled setting. If our results areconfirmed, the risks of platelet transfusion in a patient populationmay be greater than previously thought. Furthermore, relativethrombocytopenia may be of therapeutic benefit, and withincarefully defined safety parameters, the use of antiplatelet reagentsmay be considered as chemosensitizers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Bottsford-Miller, H.-J. Choi, H.J. Dalton, R.L. Stone,E.K. Crane, V. Afshar-Kharghan, A.K. SoodDevelopment of methodology: J. Bottsford-Miller, H.-J. Choi, H.J. Dalton,R.L. Stone, C.V. Pecot, A.K. Sood

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Bottsford-Miller, H.-J. Choi, H.J. Dalton, M.S. Cho,M. Haemmerle, S. Pradeep, B. Zand, R.A. Previs, C.V. Pecot, E.K. Crane,A.K. SoodAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Bottsford-Miller, H.-J. Choi, H.J. Dalton, M. Haem-merle, C.V. Pecot, E.K. Crane, V. Afshar-Kharghan, A.K. SoodWriting, review, and/or revision of the manuscript: J. Bottsford-Miller,H.-J. Choi, R.L. Stone, A.M. Nick, B. Zand, R.A. Previs, E.K. Crane, W. Hu,S.K. Lutgendorf, V. Afshar-Kharghan, A.K. SoodAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Bottsford-Miller, H.-J. Choi, M.S. Cho, W. Hu,A.K. SoodStudy supervision: J. Bottsford-Miller, V. Afshar-Kharghan, A.K. Sood

Grant SupportThis work was supported in part by NIH grants (CA177909,

P50CA083639, CA109298, P50CA098258, U54CA151668, UH2TR000943,CA016672, U54CA96300, and U54CA96297), CPRIT RP110595 andRP120214, an Ovarian Cancer Research Fund Program Project DevelopmentGrant, Department of Defense grants (OC120547 and OC093416), the BettyAnn Asche Murray Distinguished Professorship, the RGK Foundation, theGilder Foundation, the Judi A. Rees Ovarian Cancer Research Fund, theChapman Foundation, the Meyer and Ida Gordon Foundation, and theBlanton-Davis Ovarian Cancer Research Program. M. Haemmerle is sup-ported by a Research Fellowship of the Deutsche Forschungsgemeinschaft(DFG). STR DNA fingerprinting was done by the Cancer Center SupportGrant–funded Characterized Cell Line core, NCI #CA016672. J. Bottsford-Miller, H.J. Dalton, R.L. Stone, B. Zand, R.A. Pervis, and E.K. Crane aresupported by NIH T32 training grant CA101642.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 8, 2014; revised August 11, 2014; accepted November 12,2014; published OnlineFirst December 3, 2014.

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2015;21:602-610. Published OnlineFirst December 3, 2014.Clin Cancer Res Justin Bottsford-Miller, Hyun-Jin Choi, Heather J. Dalton, et al. Therapy in Ovarian CancerDifferential Platelet Levels Affect Response to Taxane-Based

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