Ex vivo interaction between blood components and hormone-dependent breast cancer cells induces alterations associated with epithelial-mesenchymal transition and thrombosis.

The prevalence of breast cancer is steadily increasing with metastasis and thromboembolic complications identified as the most common causes of death. The acquisition of an aggressive phenotype by hormone-dependent breast cancers is mediated by Transforming Growth Factor Beta 1 (TGF-ß1) expression and is associated with epithelial-mesenchymal transition (EMT) and, potentially, increased propensity for thrombosis. We investigated this phenomenon by assessing the effect of platelet-rich plasma (PRP) and whole blood (WB) on parameters of EMT and hypercoagulation in vitro. MCF-7 breast cancer cells were cultured under standard conditions, followed by co-culture with PRP or WB. Cells were processed for real-time PCR (TGF-ß1 and vimentin), electron microscopy or immunocytochemistry (TGF-ß1). Micrographs were qualitatively assessed, and real-time PCR data analyzed with PAST Statistical Software. The addition of blood components heightened TGF-ß1 immunolocalization and significantly increased corresponding gene expression. Both PRP and WB significantly increased vimentin expression and induced a shape change from a typical epithelial phenotype to a spindle-shape morphology, indicative of EMT. Fibrin fiber, network and plaque formation indicated a hypercoagulatory environment. The results thus show that in preparation for hematogenous metastasis, hormone-dependent breast cancer cells assume an aggressive phenotype associated with EMT, simultaneously increasing the propensity for the formation of thrombo-emboli.

Evaluation of the morphological changes in the lungs of BALB/c mice after inhalation of spherical and rod-shaped titanium nanoparticles.

Titanium nanoparticles are widely used by industry in consumer products such as sunscreens and some cosmetic products due to their specifically engineered properties. Some of these properties may, however, increase the toxicity of the nanoparticles which in turn may affect human and environmental health. Therefore, it is of utmost importance to study the possible effects of these particles through in vivo studies, which might produce different results than in vitro cell studies. The current study aimed to investigate the possible remodelling in the lungs of BALB/c mice by means of light and transmission electron microscopy after inhalation of spherical and rod-shaped titanium nanoparticles at two different concentrations. The focus of this paper was to demonstrate whether whole body exposure to different concentrations of the said nanoparticles could induce an inflammatory response in the lungs and no inter particle comparison was done or retention investigated. Animals were divided into five experimental groups: control, high and low concentration groups exposed to the spherical-shaped particles, as well as high and low concentration groups exposed to the rod-shaped particles. Histological and ultrastructural changes, typical of an inflammatory response, were noted in the lungs of the exposed animals. These changes were not observed in the lungs of the control animals. It can be concluded from this study that titanium nanoparticles may cause inflammatory reactions in the lungs of animals exposed through inhalation, as indicated by the presence of inflammatory cells and congestion of inter-alveolar areas. This has implications for individuals who may be potentially exposed during the production and use of titanium nanoparticles.

Comparing techniques: the use of recalcified plasma in comparison with citrated plasma alone and in combination with thrombin in ultrastructural studies.

Fibrin plays a vital role in the coagulation process and fibrin fiber morphology can be studied using ultrastructural techniques. When studying the ultrastructure of fibrin networks, thrombin may be added to the plasma, ensuing fibrin network formation. The question that arises is whether there are differences in morphology when thrombin is added to plasma, versus morphology observed when plasma from citrated or recalcified citrated whole blood, is studied. The current study therefore aimed to compare ultrastructure of platelets and fibrin networks from these three techniques. Results indicated comparable platelet ultrastructure between smears formed from the plasma of citrated blood and that of the citrated recalcified blood. This method might give us further information regarding the 'natural state' fibrin assembly and association with platelets, when studying haemostasis. However, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibrin fibers.

Investigating the effect of a platelet additive solution on apheresis platelet and fibrin network ultrastructure.

In thrombotic events and diseases such as cancer, HIV/AIDS, dysfibrinogenaemia, as well as acute incidents (e.g. burn wounds), ultrastructure of platelets and fibrin networks change. In the current study, we compare the ultrastructure of platelets and fibrin networks of apheresis platelets stored in citrated human plasma (CP) and in a first-generation platelet additive solution (PAS) (T-Sol), to that of fresh donor plasma (FP). Eighteen apheresis platelet donors donated platelets on Trima®-Accel™ V5.2 and V5.1 cell separators. Six collections were stored for five days in autologous citrated plasma (CP); six collections were stored in 40% citrated human plasma and 60% PAS solution (CP/PAS) controlled, for the duration of storage, at a constant temperature (22±2°C) with continuous flat-bed agitation; and six collections were stored in conditions uncontrolled for temperature and without continuous agitation. On days 1, 3 and 5, equal volumes of human thrombin were mixed with platelets collected in either CP or CP/PAS to form a coagulum (fibrin network containing platelet aggregates), followed by preparation for scanning electron microscopy. Results were compared with platelets and fibrin networks in FP. Typically, in FP, platelet aggregates with smooth membranes and pseudopodia are seen and fibrin networks arrange to form major, thick fibers and scattered, minor, thin fibers. On day 1, in CP and in all CP/PAS units, platelet ultrastructure compared well to that of FP, although the fibrin fibers were denser, with the minor fibers forming a matted layer over the major fibers. On day 3, in platelet units uncontrolled for temperature and without continuous agitation during storage, some platelet aggregates in CP/PAS showed typical apoptotic morphology, with shrinkage and membrane damage, but comparable fibrin networks were present. On day 5 however, in those units where storage conditions were uncontrolled and where the pH had decreased to below 6.4, no platelet aggregates were seen and fibrin was arranged into short, lumpy masses with no separate major or minor fibrin fibers visible. In those units stored at 22°C with continuous flat-bed agitation, where pH was maintained >7.0, ultrastructure of platelets and fibrin network in CP/PAS was typical and similar to FP and CP at the end of five days of storage. Examining platelet and fibrin network ultrastructure may be useful, in addition to conventional laboratory analysis, in assessing the viability and potential clinical efficacy of platelets for transfusion and could play a role in the evaluation of new generation platelet additive solutions.