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PEG-liposomal doxorubicin as a potential agent for canine metastatic osteosarcoma – in vitro and ex ovo studies

Magdalena Walewska
Magdalena Walewska
, 
Anna Małek
Anna Małek
, 
Bartosz Taciak
Bartosz Taciak
, 
Anna Wojtalewicz
Anna Wojtalewicz
, 
Sylwia Wilk
Sylwia Wilk
, 
Agata Wojtkowska
Agata Wojtkowska
, 
Katarzyna Zabielska-Koczywąs
Katarzyna Zabielska-Koczywąs
 et 
Roman Lechowski
Roman Lechowski
   | 16 juin 2023
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Publié en ligne: 16 juin 2023
Pages: 297 - 305
Reçu: 15 févr. 2023
Accepté: 19 avr. 2023
DOI: https://doi.org/10.2478/jvetres-2023-0026
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© 2023 Magdalena Walewska et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1.

Embryos prepared for the ex ovo experiment: A – the chorioallantoic membrane and cancer cell injection site; B – embryos in weighing boats covered with plastic Petri dishes
Embryos prepared for the ex ovo experiment: A – the chorioallantoic membrane and cancer cell injection site; B – embryos in weighing boats covered with plastic Petri dishes

Fig. 2.

Stained D-17 cell located intravascularly in the lumen of the CAM blood vessel. Scale bar 20 μm
Stained D-17 cell located intravascularly in the lumen of the CAM blood vessel. Scale bar 20 μm

Fig. 3.

Viability of canine D-17 cells (osteosarcoma cell line) after exposure to 100 μL of polyethylene glycol–liposomal doxorubicin (Caelyx) or conventional doxorubicin for 24 h. Curved lines represent the log (inhibitor) vs normalised response curve. Standard deviation is represented by the error bars
Viability of canine D-17 cells (osteosarcoma cell line) after exposure to 100 μL of polyethylene glycol–liposomal doxorubicin (Caelyx) or conventional doxorubicin for 24 h. Curved lines represent the log (inhibitor) vs normalised response curve. Standard deviation is represented by the error bars

Fig. 4.

Scatterplots showing D-17 subpopulations distinguishable as necrotic (Q1), late apoptotic (Q2), live (Q3) and early apoptotic (Q4) cells after incubation for 24 h with the tested drugs at IC50 doses. Scatterplots are for: A – control group cells (no treatment); B – polyethylene glycol–liposomal doxorubicin–treated cells; C – conventional doxorubicin–treated cells. Horizon V450-A refers to Annexin and APC-A to Draq 7 detection filters
Scatterplots showing D-17 subpopulations distinguishable as necrotic (Q1), late apoptotic (Q2), live (Q3) and early apoptotic (Q4) cells after incubation for 24 h with the tested drugs at IC50 doses. Scatterplots are for: A – control group cells (no treatment); B – polyethylene glycol–liposomal doxorubicin–treated cells; C – conventional doxorubicin–treated cells. Horizon V450-A refers to Annexin and APC-A to Draq 7 detection filters

Fig. 5.

The effects of polyethylene glycol–liposomal and conventional doxorubicin on apoptosis and necrosis of canine D-17 osteosarcoma cells. From left to right, the bars represent the percentage of apoptotic and necrotic untreated control cells, cells treated with conventional doxorubicin, and cells treated with PEG-liposomal doxorubicin (Caelyx) at IC50 doses after 24 h incubation. Error bars represent standard deviation. P-value ≤ 0.05 (*) was assigned significance
The effects of polyethylene glycol–liposomal and conventional doxorubicin on apoptosis and necrosis of canine D-17 osteosarcoma cells. From left to right, the bars represent the percentage of apoptotic and necrotic untreated control cells, cells treated with conventional doxorubicin, and cells treated with PEG-liposomal doxorubicin (Caelyx) at IC50 doses after 24 h incubation. Error bars represent standard deviation. P-value ≤ 0.05 (*) was assigned significance

Fig. 6.

Wound-healing assay microscopic images showing inhibition of canine D-17 osteosarcoma cell migration. A – untreated control cells at t1 (0 h); B – these cells at t2 (12 h); C – these cells at t3 (24 h); D – cells incubated with conventional doxorubicin at t1; E – these cells at t2; F – these cells at t3; G – cells incubated with polyethylene glycol–liposomal doxorubicin at t1; H – these cells at t2; I – these cells at t3
Wound-healing assay microscopic images showing inhibition of canine D-17 osteosarcoma cell migration. A – untreated control cells at t1 (0 h); B – these cells at t2 (12 h); C – these cells at t3 (24 h); D – cells incubated with conventional doxorubicin at t1; E – these cells at t2; F – these cells at t3; G – cells incubated with polyethylene glycol–liposomal doxorubicin at t1; H – these cells at t2; I – these cells at t3

Fig. 7.

Bar graphs showing the effect of the tested drugs inhibiting the migration of canine D-17 osteosarcoma cells at: A – t1 (0 h); B – t2 (12 h); and C – t3 (24 h). From left to right, the bars represent untreated and incubated control group cells, conventional doxorubicin–treated and incubated cells, and polyethylene glycol–liposomal doxorubicin–treated and incubated cells at each time point *** – very high statistical significance at P ≤ 0.001; ** – high statistical significance at P ≤ 0.01; * – statistical significance at P ≤ 0.05
Bar graphs showing the effect of the tested drugs inhibiting the migration of canine D-17 osteosarcoma cells at: A – t1 (0 h); B – t2 (12 h); and C – t3 (24 h). From left to right, the bars represent untreated and incubated control group cells, conventional doxorubicin–treated and incubated cells, and polyethylene glycol–liposomal doxorubicin–treated and incubated cells at each time point *** – very high statistical significance at P ≤ 0.001; ** – high statistical significance at P ≤ 0.01; * – statistical significance at P ≤ 0.05

Fig. 8.

Evaluation of polyethylene glycol–liposomal and conventional doxorubicin’s inhibitory effects on cancer cell extravasation in the chorioallantoic membrane. Fluorescently labelled canine osteosarcoma D-17 cancer cells: A – in untreated control cells at t1, the time of cell injection and 0 h; B – in these cells at t2, after 24 h; C – in conventional doxorubicin–treated cells at t1; D – in these cells at t2; E – in PEG-liposomal doxorubicin–treated cells at t1; F – in these cells at t2. Dyed cancer cells are visible as light green dots. Scale bars 200 μm
Evaluation of polyethylene glycol–liposomal and conventional doxorubicin’s inhibitory effects on cancer cell extravasation in the chorioallantoic membrane. Fluorescently labelled canine osteosarcoma D-17 cancer cells: A – in untreated control cells at t1, the time of cell injection and 0 h; B – in these cells at t2, after 24 h; C – in conventional doxorubicin–treated cells at t1; D – in these cells at t2; E – in PEG-liposomal doxorubicin–treated cells at t1; F – in these cells at t2. Dyed cancer cells are visible as light green dots. Scale bars 200 μm

Fig. 9.

Bar graphs showing the influence on the extravasation rate of Caelyx polyethylene glycol–liposomal doxorubicin and conventional doxorubicin on the canine D-17 osteosarcoma cell line after 24 h of incubation *** – very high statistical significance at P < 0.001. Results are presented as mean ± standard error of the mean
Bar graphs showing the influence on the extravasation rate of Caelyx polyethylene glycol–liposomal doxorubicin and conventional doxorubicin on the canine D-17 osteosarcoma cell line after 24 h of incubation *** – very high statistical significance at P < 0.001. Results are presented as mean ± standard error of the mean

Half maximal inhibitory (IC50) doses of polyethylene glycol–liposomal and conventional doxorubicin after 24 h incubation for the canine D-17 osteosarcoma cell line

IC50 after 24 h (μg/mL)
PEG-liposomal doxorubicin 28.862
Conventional doxorubicin 6.090
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Langue:
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Sujets de la revue:
Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
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