Canine Cancer Cells Activate Platelets via the Platelet P2Y12 Receptor.

In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets in vitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets in vivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.

Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells.

Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 µg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 µg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 µg/mL of CD80+VEGF and 0.1 µg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 µg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 µg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having spontaneous OSA, such as the dog, and on a human OSA cell line model.

Identification of canine platelet proteins separated by differential detergent fractionation for nonelectrophoretic proteomics analyzed by Gene Ontology and pathways analysis.

During platelet development, proteins necessary for the many functional roles of the platelet are stored within cytoplasmic granules. Platelets have also been shown to take up and store many plasma proteins into granules. This makes the platelet a potential novel source of biomarkers for many disease states. Approaches to sample preparation for proteomic studies for biomarkers search vary. Compared with traditional two-dimensional polyacrylamide gel electrophoresis systems, nonelectrophoretic proteomics methods that employ offline protein fractionation methods such as the differential detergent fractionation method have clear advantages. Here we report a proteomic survey of the canine platelet proteome using differential detergent fractionation coupled with mass spectrometry and functional modeling of the canine platelet proteins identified. A total of 5,974 unique proteins were identified from platelets, of which only 298 (5%) had previous experimental evidence of in vivo expression. The use of offline prefractionation of canine proteins by differential detergent fractionation resulted in greater proteome coverage as compared with previous reports. This initial study contributes to a broader understanding of canine platelet biology and aids functional research, identification of potential treatment targets and biomarkers, and sets a new standard for the resting platelet proteome.

Ultra-pure platelet isolation from canine whole blood.

BACKGROUND: Several research applications involving platelets, such as proteomic and transcriptomic analysis, require samples with very low numbers of contaminating leukocytes, which have considerably higher RNA and protein content than platelets. We sought to develop a platelet purification protocol that would minimize contamination, involve minimal centrifugation steps, and yield highly pure platelet samples derived from low volume whole blood samples from healthy dogs. RESULTS: Using an optimized OptiPrep density gradient technique, platelet recovery was 51.56% with 99.99% platelet purity and leukocyte contamination of 100 leukocytes per 108 platelets, on average. Platelet samples were subjected to additional purification with CD45-labeled Dynabeads after density barrier centrifugation resulting in a 95-fold depletion of residual leukocytes. Platelets purified using these methods remained inactivated as assessed by Annexin V and P-selectin labeling with flow cytometry. CONCLUSIONS: The use of OptiPrep density gradient is a quick method for obtaining highly purified platelet samples from low volumes of canine whole blood with minimal contamination. Additional depletion of residual leukocytes can be achieved using CD45-labeled beads. These platelet samples can then be used for many downstream applications that require ultra-pure platelet samples such as RNA and protein analysis.