2.1 Cell cultures
143B (ATCC® CRL-8303) and MG-63 (ATCC® CRL-1427) human OS cell lines were purchased from the American Type Culture Collection [ATCC; (www.lgcstandards-atcc.org)]. OS cells were grown in Eagle’s minimal essential medium (ΕMEM) supplemented with 10% fetal bovine serum (FBS) and 100 u/ml penicillin - 100 mg/ml streptomycin. Cells were kept at 37°C in a humidified atmosphere of 5% CO2.
2.2 Cell viability Assay (Trypan blue exclusion)
OS cells were plated at a cell density of 3.5 × 104 cells/well in 6-well plates in EMEM supplemented with 10% FBS. Twenty-four and 48 h after seeding cells, trypan blue exclusion assay was used to determine the number of living MG-63 and 143B cells exposed to 0, 5, 10 and 20 µg/ml of Dox .
2.3 Cell migration assay (Wound healing assay)
OS cells cultured in EMEM supplemented with 10% FBS were seeded into 24-well tissue culture plate wells at a density of 2×106 cells/well so that after 24 h of growth, they should reach 90-95% confluency as a monolayer. The monolayers were scratched vertically with a sterilized 200 µl pipette tip across the center of the well. After scratching, the wells were washed twice with 1x phosphate-buffered saline (PBS) to remove the detached cells and replenished with fresh medium containing Dox (20 µg/ml) or PBS (control). Cells were photographed at 0, 8, 16, and 24 h after scratching, using an Olympus Bx40 microscope. TScratch software version 7.8 (Computational Science and Engineering Laboratory, Swiss Federal Institute of Technology, Zurich, Switzerland) was then used to perform image analysis and measure the gap areas [21, 22].
2.4 Apoptosis Assay (Fluorescence-activated cell sorting/FACS)
The effect of Dox in OS cell lines apoptosis was carried out using flow cytometry. Briefly, 143B and MG-63 cells were fixed overnight at 40 C in 70% ethanol. The fixed cells were stained with RNase-containing propidium iodide (PI) and Annexin V FITC solution (TACS Annexin V FITC, Apoptosis Detection Kit, Gaithersburg, MD, USA). Cells were then separated as early apoptotic (Annexin V FITC-stained), late apoptotic (Annexin V FITC and PI-stained), necrotic (PI-stained) or non-apoptotic/live cells (no staining). DNA content was analyzed using a FACS caliber flow cytometer (Becton Dickinson, San Jose, CA) and MoD Fit software (Verity Software House, Topsham, ME) .
2.5 In vivo orthotopic implantation of 143B human OS cells in mice xenografts
143B is a highly tumorigenic OS cell line with high metastatic rate [24, 25, 26]. The injection of 143B cells in mice tibias generates either spontaneous (tumor cells interact with their native microenvironment, invade local vessels, and move to distant sites) or experimental (direct seeding of tumor cells in lungs during the injection procedure) pulmonary metastases [27, 28, 29].
Thirty-two 6 to 8-week-old female severe combined immunodeficient (SCID) mice were obtained from the National Center for Scientific Research (Demokritos, Greece). Mice were acclimatized for 10 days without any interventions after transportation to the Laboratory for Experimental Surgery and Surgical Research “N.S. Christeas”, Medical School, University of Athens, Greece, where the in vivo study was conducted. The animals were housed individually in clean metabolic cages placed in a well-ventilated house with optimum conditions (temperature 23 ± 1°C; photoperiod 12 h natural light and 12 h dark; humidity 45-50%) with access to food and water ad libitum during the entire study period. On day 0, the animals were anesthetized using a ketamine (100 mg/kg, Imalgene 1000; Merial, France) - xylazine (10 mg/kg Rompun; Bayer Animal Health GmbH, Germany) mixture injected intraperitoneally (IP). 143B OS cells (1 x 106 in 100 µl of PBS) were injected into the left proximal tibia of SCID mice using a 25-G needle . All procedures were approved by the Regional Veterinary Service (no:366107/July 9, 2019) the Ethics Committee of the NKUA/Medical School (no:163/September 18, 2019) and were in accordance with National Legislation and European Directive 63/2010.
2.6 Study design
Mice were randomly divided into four groups. Mice in group A, Dox−/Amp− (n=7), comprised the untreated control group. Mice in group B, Dox+/Amp− (n=8), received Dox hyclate 50 mg/kg (D9891; Sigma Aldrich Chemical Co., St Louis, MO, USA) diluted in water and injected IP daily for 28 consecutive days . Mice in group C, Dox−/Amp+ (n=7), underwent transfemoral amputation using a standardized method (Day 5) . Mice in group D, Dox+/Amp+ (n=6), underwent transfemoral amputation (Day 5) and received Dox hyclate (50 mg/kg) IP daily for 28 consecutive days.
Four weeks after tumor inoculation (Day 28), all animals were anesthetized, blood was drawn from the orbital sinus and mice were euthanized via cervical dislocation [34, 35]. During the inoculation process 4/32 mice died after the acute onset of tachypnea, possibly due to pulmonary embolism. The left tibias (primary tumor sites) were evaluated with x-rays (Chirana type RK 75-10 EKv 2mm AL, Film Agfa 100NIF 25x30, Cassette Agfa CR MD 4.0 General) three weeks after engraftment of 143B cells to assess tumor formation at primary sites.
Resected xenograft tibias and tumor dimensions were measured using a digital caliper according to the formula: volume = (L + W) (L) (W) (0.2618). The value assigned as width (W) was the average between the anterior - posterior and medial - lateral planes of the proximal tibia. The value assigned as length (L) was the distance between the most proximal and the most distal tumor margin . The lung volumes were calculated with a digital weight scale, and lung tissues were investigated for macroscopically detectable metastases using a stereoscope.
2.7 Surgical technique
Mice were anesthetized using a ketamine - xylazine mixture, as described above, injected IP . A small animal heating pad was used during surgery to maintain normothermia. While under anesthesia, the left tibia of the mouse was shaved, cleaned with povidone iodine, and then rinsed with alcohol. The knees were flexed in 900 and tibias were removed after midshaft femur cut to achieve tumor-free margins. The skin was closed with size 4-0 monofilament nylon sutures . During this procedure, a mouse from group C died due to excessive bleeding (group C, new n=6). Buprenorphine (0.1 mg/kg, IP every 8 h; Bupaq® 0.3, Neocell Ltd.) was used for pain control over the first 24 h after amputation (Day 5) .
2.8 Quantitative measurement of blood biomarkers MMP9 and VEGFA
On day 28, blood samples were aspirated from mice under anesthesia with retro-orbital technique using fine-walled glass Pasteur pipettes (diameter:150mm). The blood samples were collected in heparin coated microhematocrit tubes and stored on crushed ice for no longer than 30 min before being centrifuged at 8000 rpm at 4°C for 10 min. Mouse serum was examined for MMP9 and VEGFA protein levels by ELISA (#ab100610 and #ab100662 respectively, Abcam, Cambridge, UK), according to manufacturer’s instructions. The cell line we used to generate the in vivo OS model was human and we used human anti-VEGFA and anti-MMP9 ELISA kits to avoid cross-reaction with mouse VEGFA and MMP9 .
2.9 Histological Examination And Immunohistochemistry (Ihc)
Primary tumors and lungs were collected from mice, fixed in 4% paraformaldehyde in Tris-buffered saline (TBS) at 4°C for 18 h. Paraffin embedded tissue specimens were sectioned at a thickness of 3.0 µm, followed by deparaffinization in xylene and dehydration in a graded series of ethanol solutions
Each section was photographed in its entirety and subsequently, digitally processed utilizing the IpWin6 program. Tumorous areas were manually demarcated, and the program proceeded to assess the percentage of tumor areas to overall tissue surface. The same procedure was conducted to assess the percentage of necrotic tumor areas to the overall tumor surface in primary sites.
IHC: Antigen retrieval was performed by heating the samples for 20 min at 95°C in citrate buffer (pH 6.0), and endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min at room temperature. Afterwards, the sections were incubated with anti-MMP9 antibody (rabbit polyclonal, #ab38898, Abcam), anti-MMP2 antibody (rabbit polyclonal, #ab97779, Abcam), anti-vimentin antibody (rabbit monoclonal [EPR3776], #ab92547, Abcam), anti-E-cadherin antibody (mouse monoclonal [4A2], #ab231303, Abcam), anti-Ki67 antibody (rabbit polyclonal, #ab15580, Abcam), anti-Ezrin antibody (mouse monoclonal, #ab4069, Abcam), anti-VEGFA antibody (rabbit polyclonal, cat. #ab46154; Abcam), all at a dilution of 1:300 at 4°C overnight. After washing with PBS, the sections were incubated with biotinylated secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG, cat. no. 20775; Millipore, Burlington, USA) for 10 min at room temperature (RT). They were then incubated with Streptavidin HRP (#20774, Millipore, Burlington, USA) for 10 min at RT, and the reaction was visualized using 3,3'-diaminobenzidine (#D12384, Sigma-Aldrich, Gillingham, UK). Eventually, the specimens were counterstained with Mayer’s hematoxylin at RT for 1 min. Images were photographed using a Nikon Eclipse 80i microscope equipped with a digital camera image system (Cellsens). The immunostaining of antibodies was scored on the scale of semi-quantitative assessment by evaluating the intensity and percentage of positively stained cells. The intensity of antibodies staining was scored as follows: 0, none; 1, weak; 2, moderate; and 3, strong. Samples were blindly inspected by two independent experienced pathologists .
2.10 Statistical analysis
All statistical analysis was performed using SPSS version 26 (IBM SPSS Statistics for Windows; IBM Corp., Armonk, NY). All data were tested for normality using a Kolmogorov–Smirnov test and displayed parametric or nonparametric distributions (p < 0.05). The measurements of blood biomarkers, number of lung metastases, lung weight and quantitative IHC in lungs were analyzed using one-way analysis of variance (ANOVA) (Kolmogorov–Smirnov significance p >0.05) followed by a post-hoc Bonferroni test. The measurements for the tumor volume, tumor weight, tumor necrosis and quantitative IHC in primary tumors were analyzed using an independent samples t test, as the data showed normality. P<0.05 was considered as a statistically significant difference.