Mda mb 231 human breast carcinoma

EMT-6 and 4T1 murine breast carcinoma and MDA-MB-231 human breast carcinoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 1% L-glutamine, 1% sodium-pyruvate and 1% streptomycin. Human umbilical vein endothelial cells (HUVECs) (Lonza, Switzerland) were cultured in plates covered with 10% fibronectin (1 mg/ml Biological Industries, Beit HaEmek, Isreal) following 37°C incubation for 30 min. HUVECs were cultured in M199 medium (Sigma-Aldrich, Rehovot, Israel) supplemented with 20% heat inactivated fetal calf serum (FCS), 50 mg/ml endothelial cell growth supplement (ECGS), 50 mg/ml heparin, 10 mM Hepes, 1% L-glutamine, 1% sodium-pyruvate and 1% streptomycin.

The primary objective is to characterize TP cells in tumor tissue, ascites, and blood from EOC patients. This study was approved by the institutional review board overseeing human research at Stony Brook University. Female patients at least 18 years of age were included. Patients consented to participate in this study including collection of blood and otherwise discarded ascites and tissue specimens and their clinical status and treatment plan without their personal information.
Blood collection and transport were previously described.14 Briefly, 2‐20 mL of blood was collected from patients using Vacutainer® tubes (Becton Dickinson; green top, sodium heparin as anticoagulant) and processed within 48 hours from collection. Blood was kept at 2‐8°C when storage longer than 4 hours was needed.
LOX human malignant melanoma line was obtained as described 20 and MDA‐MB‐231 human breast carcinoma and other tumor cell lines used in this study were obtained from American Type Culture Collection (Manassas, VA). Choice of LOX cells in this study is that the tumor cell line is the most reliable line to generate up to 25% of the cellular population that are positive in the CAM invasion assay and the spontaneous metastasis mouse model used in this study.

To characterize TP and bulk tumor cells, we used CAM‐coated Vita‐Assay™ plates (Vitatex Inc, Stony Brook, NY) for the prospective isolation of CAM‐avid tumor cells from LOX human malignant melanoma20 and MDA‐MB‐231 human breast carcinoma and other cell lines (American Type Culture Collection, Manassas, VA), as well as from primary/metastatic tumors, ascites and blood from 49 EOC patients. Aliquots of CAM‐avid cells were left in the plate for 1‐3 days to enhance signal of cellular functions, that is, CAM uptake. Numbers of TP and bulk tumor cells were counted manually by microscopy or by automated flow cytometry according to methods previously described.11