Doxorubicin hydrochloride

The rat cardiomyoblast cell line H9c2 (RRID:CVCL_0286) was purchased from the American Type Culture Collection (Manassas, VA, USA). The DO24 mAb was produced as previously described (Prat, Crepaldi, Pennacchietti, Bussolino, & Comoglio, 1998). Doxorubicin hydrochloride (Pfizer, New York, NY, USA) was obtained from the Pharmacy of Candiolo Cancer Institute, FPO‐IRCCS. HGF was acquired from Tebu‐Bio (Le‐Perray‐en‐Yvelines, France). The MET tyrosine kinase inhibitor JNJ‐38877605 (JNJ) was kindly provided by Janssen Pharmaceutica (Beerse, Belgium). The STAT3 inhibitor Stattic was purchased from Sigma‐Aldrich.

Seleno-L-methionine (≥98% (TLC)) and D-pantethine were purchased from Sigma (St. Louis, MO), and doxorubicin hydrochloride was obtained from Pfizer (New York, NY). Antioxidants were dissolved in sterile 0.9% sodium chloride solution prior to per os treatment of animals or addition to cell culture.

The ratio of foam ingredients were experimentally determined. To create the FBIC solution of taurolidine (Taurolin® Ringer 0.5%, Berlin-Chemie AG, Berlin, Germany), hydrogen peroxide (30% hydrogen peroxide solution, Chempur, Piekary Śląskie, Poland), human serum (from human male AB plasma, Sigma-Aldrich; Merck KgaA, Darmstadt, Germany) and potassium iodide (Sigma-Aldrich; Merck KgaA) was used. The initial liquid solution consisted of 0.045% taurolidine, 22.8% hydrogen peroxide, 12.5% human serum and 12 mM potassium iodide. Additionally, doxorubicin (doxorubicin hydrochloride purchased from PFS®, 2 mg/ml, Pfizer, Sandwich, United Kingdom) and oxaliplatin (Medoxa, Medac GmbH, Wedel, Germany) were added in both ex vivo and in vitro models, respectively. The applied dosage of doxorubicin was chosen based on dosages used in PIPAC, e.g. 3 mg of doxorubicin was used to create foam covering a 4-liter cavity^{11 (link),20 (link),21 (link)}. Oxaliplatin was applied at a total concentration of 26.8 µg/ml.

The cytotoxicity of Q3R against MDCK cells was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [21 (link), 22 ]. The cells were seeded in 96-well microtitre plates (Nunc, Denmark) (3 × 10⁴ cell/well) and incubated at 37 °C in a humidified 5% CO₂ incubator overnight. Then, 2-fold serial dilutions of Q3R in DMEM (100 μl) were added to the cells in triplicate and incubated for more 48 h. Doxorubicin hydrochloride (Pfizer) was used as a positive control. The cells without treatment and cells exposed to dimethylsulfoxide (DMSO) with maximum 0.5% concentration were used as negative and vehicle controls, respectively. After incubation, the colorimetric MTT viability assay was carried out as described before. The cell survival rate was calculated using the following formula: (mean Optical Density (OD) of treated cells/mean OD of control cells) × 100. The 50% cytotoxic concentration (CC₅₀) was defined as the concentration which causes visible morphological changes in 50% of the cells based on the observation under inverted microscope with respect to the control cells. A non-cytotoxic concentration (NCTC) was used for antiviral assays.

MCF7 and T47D ER⁺, and BT-20 and MDA-MB-231 ER^- human breast cancer, and K562 leukemia cells were obtained from commercial sources; the American Type Culture Collection (ATCC), USA. The chemicals Doxorubicin hydrochloride (DOX; Pfizer), Tamoxifen (TAM; Cayman Chemical), phenformin (Sigma), Trichostatin A (TSA; Sigma), estradiol (Cayman Chemical), Apicidin (Sigma), and Troglitazone (TRG; Calbiochem) were acquired from the indicated providers. All treatment compounds were reconstituted in dimethylsulfoxide (DMSO) except metformin (Sigma), which was reconstituted in molecular-grade water (Hyclone). The HDACi assay and in vitro hypoxia experiments were conducted as previously described [14 (link)]. MCF7 and K562 parental cells were selected for drug resistance according to our published methods [14 (link), 15 (link)].

Human packed red blood cells were donated by the Faculty of Medicine Blood bank, at Prince of Songkla University, Thailand. Carboxy-terminated 50:50 poly(d, l-lactide-co-glycolide) (PLGA) polymer 0.66 dL/g was purchased from LACTEL Absorbable Polymers (Birmingham, AL, USA). Doxorubicin hydrochloride was purchased from Pfizer Laboratories (New York, NY, USA). Phosphate buffer solution (PBS) was acquired from Thermo-Fisher (Waltham, MA, USA). Both the bicinchoninic acid (BCA) assay kit and paraformaldehyde were procured from Millipore Sigma (St. Louis, MO, USA). Streptavidin, Streptavidin-Fluorescein Isothiocyanate (Streptavidin-FITC), biotinylated anti-EpCAM monoclonal antibody was bought from Biolegend (San Diego, CA, USA). The MTS Assay Kit (Cell Proliferation) (colorimetric) was purchased from Abcam (Cambridge, MA, USA). MCF7 breast cancer cells and PCS-201-010 dermal fibroblast were purchased from ATCC (Manassas, VA, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotin(polyethylene glycol)-2000] (ammonium salt) (DSPE–PEG-biotin) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). LIVE/DEAD Cell Imaging Kit (488/570) was bought from Thermo-Fisher Scientific (Waltham, MA, USA). CellTiter-Glo^® was purchased from Promega (Madison, WI, USA). Other chemicals and reagents were from Millipore Sigma (St. Louis, MO, USA), or Thermo-Fisher (Waltham, MA, USA).