identify vascular parameters measurable with MRI that are most likely to detect therapeutic response in patients. The in vivo imaging results were correlated with ex vivo IHC staining of CD34 in tumor sections, indicative of the microvascular density. We also investigated the effect of our peptide on the proliferation, migration and adhesion of vascular cells, lymphatic endothelial cells, and tumor cells. Based on the changes observed following treatment of LEC in vitro, we investigated the changes in lymphatic vascular density in tumor sections, and found a reduction in lymphatic vascular density; however, the reduction did not reach the level of statistical significance. Our results identify SP2024 peptide as a promising new candidate for breast cancer therapy. Methods and Materials Cell culture Human umbilical vein endothelial cells, and Human Lymphatic Endothelial Cells were purchased from Lonza. HUVEC were maintained using Endothelial Basal BioPQQ web PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19690147 Media supplemented with the Bullet Kit, the LEC were grown in Microvascular Endothelial Cell Growth Medium-2 . Triple negative human breast cancer cells, MDA-MB-231, were provided by Dr. Zaver Bhujwalla. The breast cancer cells were grown in RPMI-1640 medium supplemented with 10 tm% fetal bovine serum and antibiotics. Cells were maintained under standard conditions of 37uC and 5% CO2, the passage numbers of the endothelial cells were between 2 and 7. Peptide synthesis The peptide, SEQ: LRRFSTMPFMFININNVINF-NH2, and the scrambled peptide, SEQ: LRRFSTAPFAFIPEAKVINF-NH2 were synthesized using solid-phase synthesis and were supplied as a Trifluoroacetic salt with an amidated C-terminus by New England Peptide. The purity of the peptides was.95% and the suppliers provided product characterization for molecular weight and purity accuracy. The peptides stock was solubilized in 5% DMSO and water due to their hydrophobic profile. The pH of the solutions was measured before injection, and found to be around pH 7. For all in vitro experiments the DMSO % was maintained at non-toxic threshold as determined by toxicity assays of DMSO on cells. The in vivo control contained the same DMSO %, as the peptide solution. In vitro assays Proliferation assay. Colorimetric-based proliferation assay using WST-1 proliferation reagent was applied to HUVEC, LEC and MDA-MB-231 cells. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19690518 2000 cells/ well were plated in 96-well plates and allowed to adhere overnight. The following day, the media were exchanged with fully supplemented media containing the SP2024 peptide or equivalent DMSO vehicle for the controls. Three days later, the media was replaced with serum-free EBM-2 media containing WST-1 reagent, and incubated for 4 hours at 37uC. The plates were read on a Victor V fluorescence plate reader by measuring the absorbance at 450 nm. Migration assays. The effect of the SP2024 peptide on cells migration was investigated using two different assays; a real time migration assay system based on electrical impedance and a wound healing type assay. The RTCIM assay uses a CIM 16-well plate composed of a top and bottom chamber separated by a microporous polycarbonate membrane. The membrane was coated with fibronectin and 45,000 cells/well in serum free media, with or without peptide was added to the top compartment. Media with chemoattractant was added to the bottom compartment of the chamber, and the plate was incubated at 37uC for 20 hours. The sensors integrated on the bottom side of the membrane monitored and continuously recorde