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. 2019 Aug 19;9(8):575.
doi: 10.3390/ani9080575.

Extracellular Vesicles in the Blood of Dogs with Cancer-A Preliminary Study

Magdalena Żmigrodzka  1 , Olga Witkowska-Piłaszewicz  2 , Alicja Rzepecka  2 , Anna Cywińska  2 , Dariusz Jagielski  3 , Anna Winnicka  2
Affiliations

Extracellular Vesicles in the Blood of Dogs with Cancer-A Preliminary Study

Magdalena Żmigrodzka et al. Animals (Basel). .

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of submicron-sized structures released during the activation, proliferation, or apoptosis of various types of cells. Due to their size, their role in cell-to-cell communication in cancer is currently being discussed. In blood, the most abundant population of EVs is platelet-derived EVs (PEVs). The aim of this study was to estimate the absolute number and the origin of EVs in the blood of healthy dogs and of dogs with various types of cancer. The EV absolute number and cellular origin were examined by flow cytometry technique. EVs were classified on the basis of surface annexin V expression (phosphatidylserine PS+) and co-expression of specific cellular markers (CD61, CD45, CD3, CD21). The number of PEVs was significantly higher in dogs with cancer (median: 409/µL, range: 42-2748/µL vs. median: 170/µL, range: 101-449/µL in controls). The numbers of EVs derived from leukocytes (control median: 86/µL, range: 40-240/µL; cancer median: 443/µL, range: 44-3 352/µL) and T cells (control median: 5/µL, range: 2-66/µL; cancer median: 108/µL, range: 3-1735/µL) were higher in dogs with neoplasia compared to healthy controls. The estimation of PEV and leukocyte-derived EV counts may provide a useful biological marker in dogs with cancer.

Keywords: cancer; canine; flow cytometer; platelet microparticles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Flow cytometry gates and thresholds for characterizing blood microvesicles in dogs. (A) Characteristic dot plot of Megamix SSC beads; P1—rectangular region for extracellular vesicles (EVs) located between beads 0.2–0.5 µm on the y-axis H-SSC; P5 region—rectangular region of TruCountTM beads. (B) Characteristic dot plot of TruCountTM beads in filtered DPBS. (Dulbeccos’ PBS).
Figure 2
Figure 2
Characteristic of dot plot of microvesicle regions in healthy dog. (A) P1—rectangular region of EVs 0.24–0.5µm on the SSCH scale; and P5—rectangular region of TruCountTM beads. (B) Characteristic of dot plot events from P1 EV region labeled with annexin V (PS+)—region P3. (C) Microparticles with CD61 (PE) and/or CD45 (APC) expression in healthy dogs from the P3 region. Events in quadrant 1 (Q1) are positive for CD61 and PS, and quadrant 2 (Q2) events are positive for CD61, CD45, and PS. The microparticles in (Q4) are CD45+ PS+. Events in quadrant 3 (Q3) represent debris, machine noise, and microparticles that did not bind either fluorescent label. (D) Exemplary dot plot of annexin-V-positive microparticles with CD61 (PE) and/or CD45 (APC) expression in dogs with cancer.
Figure 3
Figure 3
(A) CD61 + PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15); (B) CD45 + PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15); (C) CD3 +PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15). * p < 0.05 between control dogs and dogs with cancer.
Figure 3
Figure 3
(A) CD61 + PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15); (B) CD45 + PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15); (C) CD3 +PS + EVs number/µL in healthy control dogs (n = 13) and dogs with cancer (n = 15). * p < 0.05 between control dogs and dogs with cancer.
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