The TICONC (Ticagrelor-Oncology) Study

Figures

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  Figure 1. Cellular Aggregation in the Platelet-Tumor Cell Microenvironment (A) Aggregation trace depicts typical agonist-induced platelet aggregation (10 μM adenosine diphosphate (ADP), and 1 mM arachidonic acid [AA]) and inhibition by antiplatelet agents (A and B) ticagrelor (Ticag) (10 μM) and aspirin (ASA) (50 μM), respectively. Platelets from normal healthy donors were pre-incubated with Ticag (10 μM), ASA (50 μM), or dual inhibitors, then stirred in the presence of (C) MCF-7 breast cancer cells or (D) HT-29 colorectal cancer cells. Values are mean percent aggregation ± SEM (n = 8). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. DAPT = dual antiplatelet therapy (ticagrelor and aspirin); Plts = platelets; TC = tumor cells.

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  Figure 2. Effect of Antiplatelet Agents on Breast and Colorectal Cancer Cell-Induced Aggregation Platelets, fluorescently labeled in red, were pre-incubated for 10 min at 37 oC with Ticag (10 μM), ASA (50 μM), or dual inhibitors, then stirred for 10 min in the presence of MCF-7 breast cancer cells or HT-29 CRC cells. (A) Samples were stained with platelet-specific CD42a-PE conjugated antibody and images acquired using an EVOS FL cell imaging system (Thermo Fisher Scientific, Glasgow, United Kingdom). (B and C) The perimeter of platelet aggregates and (D and E) diameter of platelet aggregates were analyzed using Image-J (National Institutes of Health, Bethesda, Maryland). Data show mean ± SEM (n = 5). ∗p < 0.05; ∗∗p < 0.01 for MCF-7 (n = 3); and ∗p < 0.05; ∗∗p < 0.01 for HT29. Con = control; CRC = colorectal cancer; other abbreviations as in Figure 1.

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  Figure 3. Tumor Cell Interaction Induces Platelet Activation The amplitude of normal physiological platelet activation leading to expression of (A) P-selectin and (B) Annexin-V binding in response to ADP (10 μM) and AA (1 mM), and subsequent inhibition in the presence of ticagrelor (10 μM) and aspirin (50 μM), were first tested in the absence of TCs. Platelet interaction with TCs induced platelet degranulation (C, D, F) and externalization of phosphatidylserine (C, E, G). Washed platelets were pre-treated with Ticag (10 μM), ASA (50 μM), or DAPT, stirred at 37 oC in the presence of MCF-7 or HT-29 cells and analyzed by flow cytometry for P-selectin and Annexin-V binding. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (n = 6 for both cell lines). FITC = fluorescein isothiocyanate; other abbreviations as in Figure 1.

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  Figure 4. Platelet-Enhanced Tumor Cell−Endothelial Cell Adhesion Platelet-rich plasma was prepared from citrated blood collected from normal healthy donors, and was pre-incubated with Ticag (10 μM), ASA (50 μM), a combination of ASA (50 μM) and Ticag (10 μM), or dimethyl sulphoxide vehicle control, then incubated for 15 min at 37oC with MCF-7 breast cancer cells and HT-29 colorectal cancer cells. Graphs show the (A and B) percentage of TC adhesion for MCF-7 and (C and D) HT-29 following 1-h incubation of each platelet−tumor cell sample with a human umbilical vein endothelial cell monolayer. Values are expressed as mean percentage of adhesion ± SEM. ∗p < 0.05 (n = 3) and (n = 5) for MCF-7 and HT-29, respectively. Abbreviations as in Figure 1.

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  Figure 5. The TICONC Study Design A total of 22 healthy donors, 10 patients with breast cancer, and 6 patients with colorectal cancer were recruited to the TICONC (Ticagrelor-Oncology) study, a randomized, open-label crossover study design. All donors were aged between 18 and 85 years. Following initial screening and baseline sample collection, participants were randomized and received 2 weeks of ASA (75 mg daily) or Ticag (90 mg twice daily) followed by a 2-week washout period. This was followed by 2 weeks of remaining monotherapy, and finally, 2 weeks of DAPT. Blood samples were collected at each visit and assayed for platelet activation.

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  Figure 6. Platelet Activity in Circulating Resting Platelets of Healthy Donors and Patients With Metastatic Breast or CRC Platelet activation markers were measured in the absence of additional exogenous platelet agonists for (A) spontaneous aggregation, (B) P-selectin expression, (C) fibrinogen-binding, and (D) Annexin-V binding. Data show mean ± SEM for healthy donors (n = 22), patients with breast cancer (BC) (n = 10), and patients with CRC (n = 6). ∗p < 0.05; ∗∗p < 0.01. Abbreviations as in Figure 2.

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  Figure 7. The TICONC Trial Results: Effect of Antiplatelet Therapy on Activation Status of Circulating Resting Platelets in Healthy Donors and Patients With Metastatic BC and CRC (A) Mean percentage of spontaneous aggregation, (B) mean percentage of Annexin-V binding, (C) mean percentage of P-selectin positive, and (D) mean percentage of fibrinogen-binding at baseline before drug treatment and during single or DAPT. Blood collected for the washout period (w/o) was drawn 2 weeks after the end of initial antiplatelet therapy. Values are mean ± SEM (healthy donors: n = 22; BC: n = 10, and CRC: n = 6). ∗p < 0.05; ∗∗p < 0.01. Abbreviations as in Figures 1, 2, and 6.

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  Central Illustration. Ticagrelor Reduces Platelet Activation in a Cancer Microenvironment (Left) Summary of the effect of antiplatelets in an in vitro model of platelet−tumor cell interaction within the circulation. Although ticagrelor inhibited aggregation and platelet membrane flipping in both cancer models, there was a lack of effect of ticagrelor or aspirin on breast tumor cell adhesion. In contrast, both drugs were effective in inhibition of colorectal cancer cell adhesion. Neither drug inhibited platelet degranulation. Ticagrelor effects shown in red, aspirin effects in green, − represents drug inhibition, + indicates enhanced activity, x represents no drug effect. (Right) Flowchart of the TICONC (Ticagrelor-Oncology) clinical trial, and levels of activation in circulating platelets isolated from patients with metastatic cancer before commencing antiplatelet therapy. BC = breast cancer; CRC = colorectal cancer; CTC = circulating tumor cells.