Appendicular osteosarcoma (OSA) is a highly malignant primary bone cancer and is both locally aggressive and highly metastatic (22). The treatment of choice, which consists of an amputation followed by adjuvant chemotherapy (carboplatin, cisplatin and doxorubicin), results in a median survival time range of almost 300 to almost 500 days (21). Unfortunately around 90% of patients will ultimately succumb to metastatic disease. Metastases disseminate to regional lymph nodes, the lungs or parenchymal organs such as the liver or spleen (20). Doxorubicin is one of the drugs in the anti-cancer protocol for canine OSA; however, its use is limited by life-threatening side effects such as cardiotoxicity (heart failure or dysrhythmia) (23).
In human medicine, nanoparticles and liposomes have been extensively investigated as effective drug delivery systems for anticancer compounds. Liposomal structures are characterised by their high ability to target neoplastic cells either actively or passively; also the side effects specific to free cytostatic drugs are milder in therapy regimes with this delivery system (32). Liposomes are designed to be multifunctional, with different components providing control over such properties as biodistribution, targeting specificity, permeability and circulation half-lives (2). Doxil (polyethylene glycol (PEG)) was the first nanoparticle anticancer drug that received marketing approval, becoming available in 1995 (1). Currently, 16 clinically approved liposomal drugs are in use, examples of which are DepoCyt (cytarabine), Doxil/Caelyx/Myocet (doxorubicin), DaunoXome (daunorubicin), DepoDur (morphine) and Visudyne (verteporfin) (17). Doxil or Caelyx is a polyethylene glycol–coated (PEGylated) liposomal doxorubicin approved in the USA and Europe, while Myocet, which is a non-PEGylated liposomal doxorubicin, is approved only in Europe. Different loading methods, lipid compositions or sizes of Doxil/Caelyx and Myocet result in different circulation times and toxicities (32). PEG-liposomal doxorubicin is used as a treatment for ovarian cancer in women for whom the first choice treatment has failed and as a treatment for AIDS-related Kaposi’s sarcoma (8). Furthermore, the first-phase clinical trial of PEG-liposomal doxorubicin in people with metastatic OSA focusing mainly on maximal tolerated dose assessment has yielded promising initial results for efficacy against metastatic disease (33). Nevertheless, there is still a lack of studies on the efficacy and safety of PEG-liposomal doxorubicin in OSA (33).
There are few clinical and preclinical studies investigating nanoparticles in veterinary medicine (32). PEG-liposomal doxorubicin (Doxil/Caelyx) has been tested in a canine model to evaluate its bio distribution, pharmacokinetics and safety (5). Vail
Metastasis appears when cancer cells spread from the primary tumour to surrounding tissues and to distant organs. It is the main cause of mortality in cancer patients. Most cancer cells are recognised as foreign and rapidly destroyed in circulation by the host immune system; however, some of them form metastatic foci (10). The ability of cells to extravasate into the surrounding tissue has long been considered a major rate-limiting step in the metastatic cascade (15). Preventing metastatic extravasation seems an appropriate way to reduce metastatic dissemination and potentially may prolong a patient’s life. Although metastatic disease kills almost 90% of cancer sufferers whose cause of death is cancer, most oncological research does not concern
Chick embryo chorioallantoic membrane (CAM), which is highly vascularised, offers a simple, cheap and highly accessible preclinical oncological model and in these aspects compares favourably to other animal models (12). The CAM
The canine OSA cell line D-17 from American Type Culture Collection (ATCC, Manassas, VA, USA) was cultivated under standard conditions (5% CO2, 95% humidity, and 37°C) in Eagle’s minimum essential medium (ATCC) with additions of heat-inactivated foetal bovine serum, penicillin–streptomycin (50 IU/mL), and amphotericin B (2.5 mg/mL). Cells with 90% confluence in a logarithmic growth phase were harvested with trypsin (0.025%) (Sigma Aldrich, St. Louis, MO, USA). The numbers of living and dead cells were counted with a Countess II automatic cell counter (Thermo Fisher, Waltham, MA, USA).
Two drugs were used for the study: PEG-liposomal doxorubicin as the commercial Caelyx PEG-liposomal product containing 2 mg hydrochloride doxorubicin (Janssen-Cilag International, Beerse, Belgium), and conventional doxorubicin as Adriblastina PFS 2 mg/mL, with its active substance doxorubicin hydrochloride (Pfizer, New York, NY, USA).
The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test was used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. The test also was used to establish the half maximal inhibitory concentration (IC50) and the 20% cell death inhibitory concentration (IC20) (24). Cells of the D-17 line were reseeded into 96-well cell culture plates (Sigma Aldrich) and incubated for 24 h. When the confluency was approximately 90%, the PEG-liposomal doxorubicin and conventional doxorubicin were added in decreasing concentrations of 250, 100, 50, 25, 10, 5, 2.5, 1 and 0.5 μg/mL. After 24 h, 10 μL of the MTT labelling reagent was added to each well. Four hours later, each well was filled with 100 μL dimethyl sulfoxide (Sigma Aldrich) prior to recording absorbance readings. Supernatant optical densities were read at 555 nm using an Infinite M 1000 spectrophotometer (Tecan, Männedorf, Switzerland). Untreated control cells were used to normalise absorbance. The test was performed three times for result reliability.
Flow cytometry with Annexin V and Draq 7 staining was performed to confirm the results obtained from the MTT test. The Annexin V and Draq 7 test allows the quantitative analysis of dead, early and late apoptotic, and live cells. In the study Draq 7 (BioLegend, San Diego, CA, USA) replaced propidium iodide (PI) or 7-AAD, as Draq 7 has no emission overlap with doxorubicin (31). Cells were cultivated in serum-free medium (Sigma Aldrich). After 24 h, the cells were treated with IC50 doses of PEG-liposomal and conventional doxorubicin. On the next day, media with the studied drugs were transferred to cytometry probes (Abcam, Cambridge, UK) with binding buffer. Study samples were divided into four groups: unstained, with addition of Annexin V 450 (Abcam), with addition of Draq 7 (Abcam), and with addition of Annexin V and Draq 7. The percentages of apoptotic, necrotic and viable cells were analysed with an emission wavelength in the 450–750 nm range with a FACS Aria II flow cytometer (BD Biosciences, San Jose, CA, USA). The FACS Diva software application version 6.1.3 (BD Biosciences) was used to analyse the results. The experiment was repeated three times.
The wound-healing assay as an
The
On the 12th day of the experiment, D-17 cells in a concentration of 1.0 × 105/mL were labelled with green 5-chloromethylfluorescein diacetate cell tracking dye (Life Technologies, Paisley, UK). On the same day, the labelled cancer cells were injected into each embryo. Intravenous injections were performed under a microscope (Keyence, Neu-Isenburg, Germany) with 50-μm-diameter glass microneedles. Chick embryos were divided into three groups of 7: a PEG-liposomal doxorubicin group (PEG-liposomal doxorubicin at IC50), a non-liposomal conventional doxorubicin group (doxorubicin at IC50) and a control group. Embryos were injected with 100 μL of phosphate-buffered saline, 30 μg/mL of PEG-liposomal doxorubicin or 6 μg/mL of conventional doxorubicin, according to their group. After injection, labelled D-17 cells were evaluated under a Zeiss Axio Examiner Z1 microscope (Zeiss) to confirm that they were located within the CAM blood vessels, which were labelled with tomato lectin DyLight 649 (Thermo Fisher). The effectiveness of the intravascular administration of the D-17 cells was confirmed under the microscope with a 40× objective (Fig. 2).
Sterile silicon rings (Zegir, Warsaw, Poland) were used to define the evaluation area. The proportion of cancer cells that were initially arrested in the CAM vessels and the proportion of cancer cells that crossed the vessel lumen were determined. To analyse the efficacy of extravasation, all cells within each silicon ring were counted at t1 and t2 using the Zeiss Axio Examiner Z1 with a 4× objective. Extravasation efficacy in each group was calculated using the formula
After the experiment all embryos were euthanised. The experiment was repeated in three biological replicates.
Potent cytotoxic effects of both assessed drugs were observed on the D-17 OSA cell line after 24 h of incubation (Fig. 3); however, the IC50 dose of conventional doxorubicin was more than fourfold lower than that of PEG-liposomal doxorubicin (Table 1).
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 |
Analysis of the cytometry test confirmed the results of the colorimetric test (MTT assay). The D-17 cells were incubated for 24 h with the studied drugs at IC50 doses. Both drugs exerted comparable apoptotic and necrotic effects (Fig. 4). It was indicated that doxorubicin acted mainly through cell apoptosis on the D-17 OSA cell line, as the number of apoptotic cells was notably higher (P ≤ 0.05) than that of necrotic cells (Fig. 5).
An
Directly after intravenous injection, cancer cells in each study group were spread evenly in the CAM vasculature (Fig. 8 A, C and E). After 24 h there was a notably high percentage of extravasated cancer cells in the control group and a substantially lower amount of extravasated cancer cells in embryos treated with the studied drugs (P < 0.0001). No statistical difference in the percentages of cells with extravasation between those treated with conventional doxorubicin and those treated with liposomal doxorubicin was observed (Figs 8 and 9). The rate of extravasation of D-17 cancer cells in the control group after 24 h was 48%.
The extravasation rates in chick embryos treated with conventional and liposomal doxorubicin were 4.3% and 1.5%, respectively. Nevertheless, it is important to indicate that the obtained result could be partially influenced by the cytotoxic effect of the tested drugs, because IC50 doses of both doxorubicin preparations were used, and the result therefore cannot be simply interpreted only as the inhibition of cancer cell extravasation efficacy. The aim of this study design was to more closely resemble the impact of the drugs injected intravenously in the
Osteosarcoma is a common primary bone tumour in dogs, unfortunately with very high metastatic potential (22). This study intended to compare non-liposomal conventional and liposomal doxorubicin’s effects on the canine D-17 metastatic OSA cell line. So far, therapies of OSA have included amputation or limb sparing surgery with adjuvant therapy like chemotherapy and/or radiotherapy. However conventional cytostatic drugs lack specific biodistribution, non-liposomal doxorubicin among them, which has a low therapeutic index and causes multi drug resistance in cancer cells (6). Liposomes as drug delivery systems may reduce the negative side effects and increase the therapeutic efficacy. Compared with conventional drugs, liposomes can directly deliver drugs to specific cells or tissues, thereby greatly reducing the adverse effects (29). In a community-based observational study, Salzberg
The presented study first evaluated the cytotoxicity after 24 h of conventional and PEG-liposomal doxorubicin on canine metastatic OSA using the MTT assay. The resulting IC50 after 24 h for PEG-liposomal doxorubicin was more than fourfold higher than it was for the conventional form, which may be explained by the slow release rate of free PEG-liposomal doxorubicin after 24 h. Haghiralsadat
In the next step, the accuracy of the IC50 doses was confirmed by flow cytometry with Draq 7 and Annexin V. The results obtained indicated that doxorubicin induced D-17 cell death mainly through apoptosis. There is no current consensus between scientists on the exact doxorubicin mechanism responsible for cancer cell death, and two possible models are indicated: apoptosis and necrosis (18). The specific form of cell death is governed by the drug concentration, the treatment duration and the (7). One of the advantages of the key pharmacological properties of PEG-liposomal doxorubicin was the drug’s ability to suppress anti-apoptotic pathways and further amplify apoptotic activity (18). The apoptotic mechanism of doxorubicin-induced cell death for D-17 canine OSA cells presented in this study matches one posited in our previous research (16).
Cell migration is one of the key steps in the complex metastatic cascade (30). To assess the inhibitory effect of non-liposomal conventional and PEG-liposomal doxorubicin on canine OSA migration, a wound-healing assay was performed, and it indicated significantly (P ≤ 0.01 and P ≤ 0.001) stronger inhibiting effects of the PEG-liposomal doxorubicin than of the conventional preparation after 12 and 24 h incubation (Figs 6 and 7). The wound-healing assay serves as a valuable tool to quantify cell migratory capability following exposures to various chemotherapeutic compounds (28).
The
Kim
The main limitation of the experimental design that should be considered while interpreting the presented results is the use of only one cancer cell line. However, there is only one canine OSA cell line available in the ATCC and European Cell Culture Collection.
The present study provides crucial information about PEG-liposomal doxorubicin. It shows an inhibitory effect of the liposomal form of doxorubicin on the proliferation, migration and extravasation of canine D-17 OSA cells in