Drugs and compounds. DL-PPN hydrochloride lyophilized powder (Sigma-Aldrich, Missouri, US) was first solubilized using citrate buffer (pH 3) and further diluted with phosphate buffer saline (PBS) to reach working concentrations. Cisplatin (CDDP) and methotrexate (MTX) were purchased from Fada (Buenos Aires, Argentina) and Abbot laboratories (Illinois, US), respectively. Catecholamines epinephrine and norepinephrine were purchased from BIOL laboratories (Buenos Aires, Argentina).
Tumor cell lines. Human MG-63 (ATCC® CRL-1427™) and U-2OS (ATCC® HTB-96™) OSA cell lines and glioma U-87MG cells (ATCC® HTB-14™) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Massachusetts, US) plus 10% fetal bovine serum (FBS) (Natocor, Córdoba, Argentina), 2 mM glutamine and 80 µg/ml gentamycin in monolayer culture, at 37˚C in a humidified atmosphere of 5% CO2. All cells were harvested using a trypsin/EDTA solution (Gibco, Massachusetts, US) and routinely tested por mycoplasma.
OSA cell growth and citotoxicity. Direct in vitro effects of PPN, alone or in addition to chemotherapy, or catecholamines, against rapidly growing MG-63 or U-2 OS OSA cells were assessed by crystal violet staining after a 72-h treatment (for detailed methodology see Additional file 4. Supplementary methods). Before evaluating the effects of PPN on cell cycle phase distribution or migration, lack of direct short-term cytotoxicity of PPN was confirmed using the trypan blue dye exclusion assay (for detailed methodology see Additional file 1. Supplementary methods).
Cell cycle distribution. Cell cycle phase distribution and hypodiploid population quantification in starved OSA cells following a 24 h PPN (50 µM) treatment was evaluated by flow cytometry as previously reported  (for detailed methodology see Additional file 1. Supplementary methods).
Apoptosis. After a 48-h treatment with PPN (50 µM), apoptotic cells in OSA cultures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and fluorescence microscopy (for detailed methodology see Additional file 1. Supplementary methods).
In vitro mitosis. PFA-fixed and DAPI-stained OSA cell cultures were analyzed for mitotic index calculation after a 48-h exposure to PPN (50 µM). Mitotic figures in all four basic stages of mitosis were counted using a high-power field (HPF) of 400-fold magnification (X400) and the percentage of mitotic cells was obtained after quantification of total cell number. Ten pictures per group were used for quantification.
Clonogenic growth. Effect of PPN (1, 10 or 50 µM) on OSA colony forming ability was assessed using a 7-d clonogenic assay, as previously reported  (for detailed methodology see Additional file 1. Supplementary methods).
OSA spheroid growth. Impact of PPN (50 µM) treatment on 3D growth was evaluated on MG-63 multicellular spheroids generated by the hanging drop method . After confirming appropriate sphericity and size (> 200 µm), fully-formed spheroids were incubated with PPN during one week, and volume was periodically assessed as a direct parameter of 3D OSA growth (for detailed methodology see Additional file 1. Supplementary methods).
Cell chemotaxis. Migration was assessed in serum-starved MG-63 cell after 24-h treatment with PPN (50 µM) using the Transwell® migration assay, as previously reported  (for detailed methodology see Additional file 1. Supplementary methods).
Animals. Outbred athymic female N:NIH(S)-nu mice aged 8 weeks with a weight of approximately 23 g, were purchased from the School of Veterinary Sciences Animal Facility at National University of La Plata (Buenos Aires, Argentina), and, after randomization, housed at 5 mice per cage in our animal facility at the National University of Quilmes. Food and water were provided ad libitum and general health status of the animals was monitored daily.
OSA xenograft progression. Human OSA tumors were heterotopically generated after subcutaneous injection of MG-63 cells in athymic mice . In vivo combination studies were performed by administering PPN 10 mg/kg i.p. in a 5-day-on, 2-day-off schedule, alone or in combination with 2 mg/kg i.p. doses of CDDP three times per week during 4 weeks, until the end of the protocol. PPN [29, 30] and CDDP [31, 32] dosage were defined according to previously reported preclinical studies following a metronomic rationale. Tumor growth rates and volume, as well as total animal weight were recorded or calculated throughout the protocol. Histopathological assessment of OSA tumors involved mitotic index quantification in viable sections of H&E-stained tumor slides and determination of adjusted tumor necrotic rate after treatment  (for detailed methodology see Additional file 1. Supplementary methods).
Statistics. Statistical analysis was performed using the PRISM 6 (GraphPad Software Inc., San Diego, US) or Compusyn software (Combosyn Inc., New Jersey, US). To compare differences between two experimental groups Mann Whitney or t tests were used for non-parametric or normal distribution of data, respectively. In case of more than two experimental groups, ANOVA analysis with Tukey's multiple comparisons post-test was used when normal distribution of data was determined. Kruskal-Wallis analysis with Dunn's multiple comparisons post-test was used in case of non-parametric distribution of data. Differences were considered statistically significant at a level of p < 0.05. Data correspond of at least 2 or 3 independent experiments unless stated otherwise. Data were presented as mean ± standard deviation (SD) or standard error of mean (SEM).