Mda mb 231 human breast cancer cells

MDA-MB-231 human breast cancer cells (ATCC, Virginia, USA) were used throughout the study due to their well-characterized ability to invade confined spaces^{14 (link)}. Cells were cultured in DMEM media (high glucose cat #11965, GibcoTM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin. All experiments were performed at 37 °C in a 95% air/5% CO₂ environment.

The murine dendritic cell line, JAWS II, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). JAWS II was cultured in the presence of alpha-minimum essential media (Lonza, Basel, Switzerland) complemented with 20% fetal bovine serum (FBS, Life Technologies; Grand Island, NY, USA), 1% penicillin-streptomycin (Sigma-Aldrich, St. Quentin Fallavier, France), 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), and 5 ng/mL of Granulocyte-Macrophage Colony Stimulating Factor (Miltenyi Biotec; San Diego, CA, USA). The cells were maintained in culture in a humified incubator at 37 °C and 5% CO₂.
The MDA-MB-231 human breast cancer cells were purchased from ATCC and cultured at 5% CO₂ at 37 °C in Dulbecco’s modified Eagle media (Lonza), supplemented with 10% FBS, 2 mM glutamine, 10% penicillin-streptomycin.
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown at 37 °C and 5% CO₂ in endothelial basal cell growth medium, supplemented with 2% FBS, 1% streptomycin-penicillin, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% heparin, 0.1% VEGF, 0.1% gentamicin-amphotericin B, 0.04% hydrocortisone, 0.4% human bFGF, and 0.1% R3-IGF-1 (all from Lonza).

MDA-MB-231 human breast cancer cells (ATCC, Manassas, VA, USA) were engineered to stably express VEGF as previously described [21 (link)]. Briefly, cDNA for VEGF165 (Genentech Inc, South San Francisco, CA, USA) was cloned into the eukaryotic expression vector pCR3.1 under the control of a constitutive CMV promoter. MDA-MB-231 cells were cultured using RPMI 1640 medium (Sigma^®, Saint Louis, MO, USA) supplemented with 10% fetal bovine Serum (FBS, Sigma^®, Saint Louis, MO, USA). MDA-MB-231 VEGF cells were cultured with the same medium with 400 μg/mL of G418 Sulfate (Corning™, Glendale, AZ, USA). Expression of VEGF was routinely checked by RT-PCR.
Tumors were obtained by inoculating 10⁶ MDA-MB-231 wild-type (231 WT) or MDA-MB-231 VEGF (231 VEGF) cells, orthotopically, in female severe combined immunodeficient (SCID) mice. Tumors reached volumes of 300–400 mm³ within 4–6 weeks after inoculation, at which point the animals were used for study. All animal studies were done in compliance with a protocol approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.

We purchased Jurkat human T-lymphoblast cells, EL4 mouse T-lymphoblast cells, HepG2 human hepatocellular carcinoma cells, MDA-MB231 human breast cancer cells, and Ln229 human glioblastoma cells from ATCC (Manassas, VA). We also purchased the following: Bodipy®.FL.L-cystine (BFC) from Life technologies (Grand Island, NY); Propidium Iodide (PI) from Sigma-Aldrich (Milwaukee, WI); Alexa Fluor Annexin V from BioLegend (San Diego, CA); Cell Titer Blue (CTB) from Promega Corporation (Madison, WI); MTT from Life Technologies (Eugene, OR); DCFDA from Sigma-Aldrich (Milwaukee, WI); Calcein AM from Life Technologies (Eugene, OR); etoposide, paclitaxel and hydroxyTamoxifen from Sigma-Aldrich (St. Louis, MO); methotrexate from Tocris Bioscience (Bristol, UK); DMSO from Fisher Scientific (Santa Clara, CA); and cell culture medium, FBS, penicillin, streptomycin, sodium bicarbonate, and all cell culture plates from GIBCO BRL (Frederick, MD). Plasmid vector expressing p53 protein under a Tamoxifen regulatable gene expression system was constructed by standard gene cloning procedures in our laboratory^{29 (link)}.