br Overall IPA incorporated platelet activation across inter
Overall IPA incorporated platelet activation across intervals I1-I5 (Fig. 2A, B); however, given the morphological results we further
Fig. 2. Box and whisker plots indicating the Index of Platelet Activation (IPA) in lysed whole blood (WB) samples incubated with breast cancer 229971-81-7 and hormone-therapy treatment, as presented in intervals I1-I5. Overall IPA presented in pre-treated MCF7 (A) and T47D (B) breast cancer cell lines. Overall IPA was significantly increased following exposure to diluent and media-treated MCF7 (A) and T47D (B) breast cancer cells, compared to the untreated negative control. Neither Anastrozole nor Tamoxifen significantly altered IPA; however, compared to the diluent and media controls Anastrozole did reduce the median IPA induced by both cell lines. Tamoxifen treatment however, reduced the median IPA induced by T47D cells, while increasing the median IPA induced by MCF7 cells. Further analysis of the IPA within I5 (C and D). This interval represents a small percentage of highly active (CD41+CD62P+++) platelets. Hormone-therapy increased IPA; however, this was dependent on cell type, with MCF7 cells (C) showing significantly heightened platelet activation capacity under Anastrozole treatment and T47D cells (D), under Tamoxifen treatment.
investigated IPA within each interval to ascertain how the treatments aﬀected the spread of P-selectin expression. The variance in median IPA observed in the P-selectin high (CD62P+++) interval was particularly large (Fig. 2C, D). MCF7 cells induced significantly heightened IPA under Anastrozole treatment (Fig. 2C), and T47D cells, under Tamox-ifen treatment (Fig. 2D) compared to the untreated control.
Cancer is associated with a hypercoagulable state, which is im-plicated in facilitating the metastatic process [6,17]. Patients presenting with breast cancer have greater risk of developing VTE than healthy women within the same age group . Our in vitro experiment at-tempted to mimic platelet-tumour cell interactions within the tumour microenvironment via leaky blood vessels , and hormone-therapy accumulation within breast tissue itself .
The results obtained indicate that hormone-dependent breast cancer cells induce significant platelet activation comparable to clinical studies [13, 19, 22, 23]. However, we further show that this eﬀect may vary between cell lines, potentially linked to phenotypic diﬀerences. T47D
breast cancer cells present low oestrogen receptor expression, with exceptionally high progesterone receptor expression and are regarded as more aggressive than MCF7 breast cancer cells . This indicates that variation in hormone-receptor profiles and tumour subphenotypes may elicit a diﬀerential thrombotic response, not normally considered in clinical management. Ultrastructural analysis supports these results by showing heightened levels of platelet activation compared to the negative control, platelets expressed greater membrane folds with ex-tension of pseudopodia, which was more visible in samples incubated with the T47D cell line.
Platelet activation is associated with the release of pro-tumorigenic growth factors and chemokines in addition to providing a physical shield for circulating tumour cells [5,15,16]. Activation of platelets is dependent on the synthesis of agonists such as tissue factor, platelet activation factor (PAF) and thrombin facilitating a cascade of events including growth factor and coagulation factor secretion from platelet α-granules [45,46]. The release of granular contents from the OCS fa-cilitates further adhesion and aggregation [45,46]. The heightened level of platelet activation induced by T47D breast cancer cells could be related to the expression of Platelet Activating Factor Receptor (PAF-R),
Fig. 3. Scanning electron microscopy images of platelets in lysed whole blood (WB) samples. A: Untreated, negative control, showing an inactive platelet with a
smooth membrane and membrane folds (*), as well as the presence of microvesicles (black arrows). B: Platelet exposed to 0.1 U/ml thrombin displaying multiple membrane folds (*), with many filipodia (white arrows) extending outward from the platelet body.
Fig. 4. Scanning electron microscopy images of platelets in lysed whole blood (WB) samples co-incubated with MCF7 cells and corresponding treatments. A: Platelets
exposed to diluent-treated MCF7 cells displaying a smooth membrane with membrane folds and extending lamellipodia. B: Platelet exposed to media-treated MCF7 cells displaying membrane folds and multiple filipodia extending outward from the platelet body (white arrows), with the presence of microvesicles (black arrows). C: Platelet incubated with Anastrozole-treated MCF7 cells displaying membrane folds with extending filipodia (white arrow), with very few microvesicles. D: Platelets incubated with Tamoxifen-treated MCF7 cells displaying a spread, rough membrane and extending lamellipodia. The open canalicular system (OCS) with pores (white asterisk *) is evident, with suspected microvesicles and debris adherent to the coverslip (black arrows).