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.
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.
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