Stavrou, Evi X.

Abstract Sex differences in cancer outcome, including that of glioblastoma (GBM), are shaped by biological, hormonal, and immunological factors and impact disease progression, treatment responses, survival, and tumor microenvironment (TME) interactions. Platelets regulate the immune responses and tumor progression in many cancers, but it is not clear how they contribute to these sex-based differences by affecting the dynamics of the TME. Here, we show that GBM patients exhibit heightened platelet reactivity driven by PAR4 signaling. In murine GBM models, both pharmacological inhibition of PAR4 using BMS986120 and genetic deletion of PAR4 significantly prolong survival in females but not males. This survival advantage is estrogen dependent: it is preserved in chromosomal male-hormonal female mice within the four-core genotype model and is rescued in ovariectomized mice treated with estrogen. The survival benefit is TME specific and is mediated by platelet-driven enhancement of CD8⁺ T cell infiltration into the tumor. Inhibition of platelet PAR4 signaling increases calcium signaling through an estrogen-dependent interaction between PAR4 and estrogen receptor β (ERβ)—a receptor interaction not previously described. PAR4-activated platelets within the TME suppress CD8⁺ T cell function, and depletion of CD8⁺ T cells abolishes both the tumor-induced platelet reactivity and the survival benefit conferred by PAR4 inhibition. These findings establish platelet-mediated PAR4 signaling as a critical driver of tumor progression and identify sex-specific immune responses as key to therapeutic efficacy.

Host and Tumor Factor XII Drive Ovarian Cancer Maintenance and Progression Introduction . Epithelial ovarian cancer (EOC) is the leading cause of cancer death in women.Effective strategies to treat this disease are lacking due to the complexity of pathways involved and the multitude of cells that contribute to EOC biology. To this end, coagulation factor XII (FXII) and its receptor urokinase plasminogen activator receptor (uPAR) represent very promising therapeutic targets because i) uPAR is overexpressed in more than 90% of ovarian cancer patients, ii) FXII has been shown to be upregulated in the peritoneum of EOC patients and promotes EOC dissemination and iii) FXII-uPAR signal and upregulate key neutrophil functions, recently linked to tumor growth and metastasis. Through analysis of the TCGA ovarian cancer patient cohort, we have found that FXII and uPAR are co-expressed in ovarian tumors and their overexpression is associated with decreased overall survival (Fig 1A-B). Given that FXII and uPAR can be produced by both the EOC tumor and the host, the contribution of each of these compartments which has not been examined to date needs to be explored. Thus, we asked if the FXII-uPAR axis in neutrophils and EOC cells synergistically influences tumor biology. Methods - Results. We utilized a tissue microarray to determine FXII and uPAR expression. Both FXII and uPAR were co-expressed in all major histologic subtypes of EOC tumors but not in normal ovarian epithelium (Fig C-D). We adopted a composite expression score system and found significantly increased expression of FXII and uPAR in high grade tumors compared to low grade (grade 1-2) tumors, independently of tumor subtype and stage (Fig 1C-D). Given prior reports that neutrophils are enriched in the ovarian tumor microenvironment (TME) and our findings that FXII-uPAR are integral to neutrophil activation, tumors were also examined for their neutrophil content. Invariably, as tumor grade advanced, Neutrophil Elastase expression significantly increased (data not shown). We next asked if neutrophils contribute to EOC tumor progression or their recruitment merely correlates with advanced disease. We found that in the presence of neutrophils, EOC cells migrate significantly faster (p<0.0001). Since enhanced tumor cell migration implies a pro-mesenchymal phenotype, we examined if neutrophils facilitate EOC cell epithelial-to-mesenchymal transition (EMT). Murine EOC cancer cells (ID8) were co-cultured with wild type (WT) neutrophils for 24 h; cells were harvested and used for immunoblotting and mRNA studies. We found that WT neutrophils significantly increased the expression of mesenchymal marker(s) vimentin and N-cadherin while they decreased the expression of E-cadherin, suggesting that neutrophils promote EMT of EOC cancer cells (Fig 1E). To investigate whether the FXII-uPAR axis contributed to neutrophil-induced EMT, we next co-cultured ID8 cells with neutrophils from tumor-bearing FXII deleted (F12-/-)mice. In ID8 cells co-cultured with F12-/-neutrophils, EOC induced EMT was blocked. Similarly, treatment with 2 peptide inhibitors that block the FXII-uPAR interaction (collectively termed, SMPAs), reversed the pro-invasive effects of both ID8 cells and WT neutrophils (Fig 1F). Next, in order to assess whether host FXII could contribute to EOC dissemination and progression we utilized WT, F12-/-, and uPAR deficient (Plaur-/-) mice for in vivo EOC tumor model. After orthotopic injection of ID8 EOC cells, WT mice exhibited faster rates of tumor development and ascitic fluid accumulation (13.4 ± 0.92 ml), relative to F12-/- (0.37 ± 0.26 ml) and Plaur-/- (0.5 ± 0.5 ml) mice (Fig 1G). Blinded examination of tumors showed significantly higher tumor burden in WT mice compared to F12-/-and Plaur-/- mice, with F12-/-mice exhibiting the highest degree of protection (Fig 1H). Conclusions . Our studies indicate three novel aspects: i) a direct crosstalk between ovarian cancer cells and neutrophils, that enhances tumor cell migration through FXII-uPAR signaling; ii) the presence of neutrophils in the TME induces EMT of cancer cells which is abolished when the FXII-uPAR interaction is inhibited and; iii) abrogation of the FXII-uPAR axis in EOC tumor cells and neutrophils, synergistically restricts the pro-invasive phenotype of ovarian tumor cells. Disclosures No relevant conflicts of interest to declare.

Background Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets. Methods and Results Recombinant Escherichia coli ‐expressed exopolyphosphatase deletion mutant PPX_Δ12 labeled with fluorescent Alexa488 dye was used as a polyphosphate probe in flow cytometry. PPX_Δ12‐Alexa488‐signal dose‐dependently increased with long‐chain polyphosphate binding to platelets. In contrast, short‐chain polyphosphate that is found in the supernatant of activated platelets, did not bind to the platelet surface. Both exopolyphosphatase treatment and polyphosphate pre‐incubation abolished PPX_Δ12‐Alexa488 binding to polyphosphate on platelets. Stimulation of platelets with thrombin receptor agonist Trap6, and P2Y 12 receptor activator ADP increased polyphosphate accumulation on platelet surfaces and PPX_Δ12‐Alexa488 signal in a dose‐dependent manner. Conclusion This study indicates that long‐chain polyphosphate binds to platelet plasma membranes and presents a promising diagnostic assay to measure this interaction on human platelets in platelet‐rich plasma. Future investigations will aim to determine if polyphosphate can be used as a novel biomarker of thrombosis. © 2016 International Clinical Cytometry Society