Isolation and culture of ceils

Tumour cell lines

The following human cell lines were used for these studies. MDA-MB-231 breast adenocarcinoma cell line, derived from a pleural effusion (a kind gift of E. Hamilton, Zeneca Pharmaceuticals), and RPMI-7951 malignant melanoma cell line, derived from a lymph node metastasis (ATCC HTB 26: American Tissue Type Culture Collection, Rockville, MD). They were grown in Eagle's minimal essential medium (EMEM; Sigma, Poole, Dorset. UK) supplemented with 10% foetal calf serum (PCS; Gibco/BRL, Burlington Canada), 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 1 mM sodium pyruvate and MEM non-essential amino acids (Sigma). Cells were maintained at 37°C in an atmosphere of 5% COg and routinely subcultured using 0.25% trypsin (Sigma) in 1 mM ethylenediamine tetraacetic acid (EDTA) solution.

Human Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were isolated according to the method of Jaffe et al (Jaffe at al., 1973a). Umbilical veins were collected and stored at 4°C for up to three days. Their outer surfaces were washed with 2% activated glutaraldehyde solution (ASEP; Galen Ltd, Graigavon, UK) and their ends were trimmed off to maintain sterility. Each cord was cut into lengths of over 12 cm long which had no obvious signs of damage. Undamaged lengths of cord were used to prevent contamination of the cultures by smooth muscle cells. The vein in each cord was cannulated with a three way stop-cock (Nipro Medical Industries, Tokyo, Japan), which was tied in place using ligature, and then placed into a sterile plastic tray (Nunc, Naperville, IL, USA). The cord was then washed through with 15 ml of calcium-magnesium free Hanks' balanced salt solution (CMF- HBSS; Sigma), followed by 5 ml of 200 U/ml collagenase I (Worthington Biochemical Corporation, Freehold, NJ, USA) made up in CMF-HBSS containing 0.2% N-[2-hydroxyethyl] piperazine-N-[2-hydroxypropane sulphonicacid] (HEPES; Sigma). The end of the cord was clamped to allow the vein to fill up with ~ 5 ml of collagenase I solution. The tray of treated cords was incubated at 37°C for 25 min.

The clamps were then removed and 10 ml of CMF-HBSS containing 10% PCS was washed through the veins while the cords were gently massaged. The cells thus collected were pelleted by centrifugation at 1200 rpm for 5 min. The pellets were resuspended in growth medium and plated onto 25 cm^ flasks (T25; Costar corporation, Cambridge, MA, USA) which had been pre-incubated with 0.2% v/v gelatin solution (Sigma) for 30 min at 37°C. After 12 h at 37°C in an atmosphere of 5% COg the adherent cells were washed with Dulbecco's calcium-magnesium- free phosphate buffered saline (PBS/A; Oxoid, Hampshire, U.K.) and grown to confluence. The growth medium was Medium 199 (M l99; Gibco BRL, Paisley, UK) with 0.014 M HEPES , 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 90 pg/ml heparin (Sigma), 15 pg/ml endothelial cell growth supplement (Boehringer-Mannheim, Mannheim, Germany) and 20% PCS. Primary cultures of HUVEC which had grown to confluence were subcultured with trypsin/EDTA in the same manner as the tumour cell lines. They were split 1:3 into gelatinized 75 cm^ flasks (T75; Costar) and used in experiments at passage three. Their endothelial phenotype was ascertained by their morphology as well as their expression of Platelet-Endothelial cell adhesion molecule (PECAM-1) and von Willebrand Factor (vWP). The monoclonal antibodies used to phenotype the endothelial cells were mouse anti-human PECAM-1 (CD31, clone LI 33.1; Becton Dickinson, Cowley, Oxford, UK) and rabbit polyclonal anti-vWP (Dako, High Wycombe, Bucks, UK).

Préparation of Ulex europaeus I derivatized Dynabeads

Micro vessel endothelial cells were isolated using tosyl-activated superparamagnetic polystyrene beads (Dynabeads M450; Dynal, Merseyside, UK) which had been covalently conjugated to Ulex europaeus-1 lectin (UEA-1; Sigma). The derivatization was carried out according to the method of Jackson et al. (Jackson

etal., 1990). UEA-1 was made up to 0.2 mg/ml in 0.17 M sodium tetraborate buffer

(pH 9.5) and sterile filtered. This was added to an equal volume of Dynabeads and mixed for 24 h on a rotary stirrer at room temperature. The beads were washed four times, for 10 min each time, and then incubated overnight at 4°C in 0.1 % (w/v) bovine serum albumin (BSA; Sigma) in PBS/A. The UEA-1 coated beads were stored at 4°C for up to four months.

Microvessel endothelial cells

Microvessel endothelial cells were isolated from human mammary adipose tissue (HuMMEC) or abdominal adipose tissue (HuAMEC) by the method of Hewett et al. (Hewett at a!., 1993). The tissue was obtained from reductive surgery at the Plastic Surgery Centre, Mount Vernon Hospital, Middlesex. It was washed in 2% antibiotic/antimycotic solution (Sigma) and could be stored in this solution at 4®C for up to two days. The tissue was dissected free of connective tissue and visible blood vessels, and finely chopped. It was then incubated with approximately 10 ml of collagenase type-11 (Sigma) per 25 g fat. The tissue was incubated on a rotary stirrer at 37°C for one hour (until the solution was homogenous) before centrifugation at 400 g for 10 min. Floating adipocytes and oil were then pipetted off the lower aqueous layer which contained the microvessels. This aqueous layer was washed with 10% bovine serum albumin (BSA; Sigma) in PBS/A and centrifuged (200 g, 10 min). The resulting pellet was washed twice with 10% BSA in PBS/A, and then incubated in 5 ml of trypsin/EDTA solution for 15 min at 37°C. After the addition of 30 ml of Hanks balanced salt solution (HBSS, Sigma) containing 5% PCS the digest was filtered through 100 pm nylon mesh (Lockertex, Warrington, Chesire, UK). The filtrate was centrifuged and the pellet resuspended in 1 ml of HBSS/FCS containing - 1 x 1 0 ^ UEA-1 coated Dynabeads. After 10 min incubation at 4°C an extra 10 ml of HBSS/FCS was added, and the microvessel cells selected using a magnetic particle concentrator (MCP-1, Dynal) for 3 min. The microvessel cells were washed three times in HBSS/FCS and then plated onto gelatinized tissue culture flasks. They were grown in the same medium (with 30% PCS) and maintained in the same manner as HUVEC. The identity of the endothelial cells was confirmed as for HUVEC.

Isolation of leucocytes

Freshly collected, heparinized human blood was used as a source of leucocytes which were isolated by density gradient centrifugation after the method of English and Anderson. (English and Anderson, 1974). A double gradient was formed by 3 ml of Histopaque 1077 (Sigma) layered onto 3 ml of Histopaque 1119 (Sigma). Onto these was layered 6 ml of blood. The tubes were then centrifuged at 700 g for 30 min at room temperature. This forms layers of plasma, mononuclear cells/platelets, histopaque, granulocytes, histopaque and erythrocytes from the top

downwards. The required cell layer was then aspirated out and washed in PBS/A. Any erythrocytes included in the cell solution were lysed by incubation in 1:4 PBSiwater.