Cell lines and culture
Breast cancer cell lines MCF7 and MDA-MD-231, large vein endothelial cells from human umbilical cords (HUVEC), human microvascular endothelial cells hMEC-1, human telomerase reverse transcriptase immortalised lymphatic endothelial cells (hTERT-LEC) and neonatal dermal lymphatic microvascular endothelial cells (HMVEC-dLy Neo) were used in this study.
Breast cancer cell lines were obtained from the ATCC and used within a 15 passage window. MCF-7 were maintained in RPMI-1640 (Sigma), 10% iron-supplemented donor calf serum (PAA laboratories) with 1% penicillin/streptomycin (Sigma). MDA-MB-231 were maintained in minimal essential medium EAGLE (Sigma), 0.1 mM non-essential amino acids solution (Sigma), 2 mM
l-glutamine (Sigma), 1% penicillin/streptomycin and 1% iron-supplemented donor calf serum. HUVEC were isolated as previously described [
18,
19] and used between passage 2 and 6. HUVEC were maintained in 37% nutrient mixture F-12 HAM media (Sigma) in sterile water containing 3.7% 199 media (Sigma), 20% iron-supplemented donor calf serum, 1% sodium bicarbonate (Sigma), 14 mM HEPES (Sigma), 2 mM
l-glutamine, 1% penicillin/streptomycin, 7.5U/ml heparin (CP Pharmaceuticals), 25 ng/ml epidermal growth factor (EGF) (Peprotech) and 12.5 ng/ml basic fibroblast growth factor (bFGF) (Peprotech). Human microvascular endothelial cells hMEC-1 [
20], obtained from ATCC, were grown in endothelial basal medium (Lonza, USA) with 10% iron-supplemented donor calf serum, 1 μg/ml hydrocortisone (Sigma), 10 ng/ml EGF and 1% penicillin/streptomycin and used between passage 4 and 18. hTERT-LEC were a kind gift from Nissato and Pepper [
19,
21,
22] and were maintained in endothelial basal media (EBM) supplemented with the EGM-2 bullet kit (Lonza) and used between passage 27 and 34. HMVEC-dLy Neo (Lonza), a primary lymphatic cell line, was cultured in the same medium as hTERT-LEC and used between passage 4 and 6. Cell lines were routinely tested for mycoplasma and tumour cells have been subsequently verified using multiplex short tandem repeat (STR) system (Powerplex 16, Promega).
Tumour-conditioned media were generated from confluent tumour cell monolayers which were then cultured in HUVEC or hTERT-LEC basal medium without serum or growth factors for 24 h. Prior to experimental use, tumour-conditioned media were supplemented with iron-supplemented donor calf serum and 50 U/ml polymyxinB-sulphate (Sigma). Tumour-derived lysate (TDL), used for macrophage stimulation, was generated from 1 × 107 cells subjected to five cycles of freeze–thaw using liquid nitrogen and a 37 °C water bath. Debris was removed by centrifugation and cleared lysates stored at −80 °C.
Healthy donor peripheral blood, obtained with the approval of the relevant ethical review board (BT20052010, University of Nottingham Medical School Ethics Committee), was fractionated using Histopaque 1077 (Sigma) to obtain peripheral blood mononuclear cells (PBMC) [
19,
23]. Monocytes were isolated from PBMC using paramagnetic particles conjugated with anti-CD14 antibodies (Miltenyi Biotec) and were >95% pure as determined by flow cytometry. Macrophages were generated by culturing CD14 + monocytes for 6 days in RPMI-1640 medium with 10% foetal calf serum in the presence of 50 ng/ml of macrophage colony stimulating factor (M-CSF) (Peprotech) in Teflon flasks (Thermo Scientific). Macrophage phenotype was confirmed by flow cytometry (CD68+, CD14+, MHC II+) and conditioned media harvested on day 7. IL-1β production by macrophages was induced by stimulating cells with tumour-derived lysate, lipopolysaccharide (LPS) (Invivogen) and a combination of tumour-derived lysate and LPS with and without caspase-1 inhibitor (R&D Systems).
Static adhesion and migration assays
The assays have been described previously [
19]. Briefly, static adhesion assays used a confluent endothelial monolayer that remained unstimulated, or was stimulated with IL-1β for 24 h. Tumour cell adhesion was assessed after 35 min, following cell labelling with 1 μM of Cell Tracker Green CMFDA (Invitrogen). Adherent tumour cells were counted using a fluorescence microscope (Nikon). Two fields of view were counted in each well at 20× magnification. Results were expressed as the percentage of cells adhered relative to control. PBMC adhesion controls were run immediately prior to each experiment to demonstrate that the endothelial cells and cytokine were responding appropriately. In migration assays, a confluent tumour cell monolayer remained unstimulated or was stimulated with IL-1β (5 ng/ml) for 24 h. Mitomycin C (Sigma) was included (10 µg/ml) to inhibit cellular proliferation. Migration was monitored at different time points following a scratch to create an area devoid of adherent cells. Percentage reduction of the scratch area at different time points was measured using ImageJ 1.43u software (National Institute of Health).
Transmigration assay
A confluent endothelial cell monolayer was grown on Boyden Chamber Transwell inserts and remained unstimulated or was stimulated with IL-1β (10 ng/ml) for 24 h. The confluency and integrity of the endothelial barrier was demonstrated by preventing lucifer yellow leakage (Sigma). Tumour cell transmigration was assessed following cell labelling with 5 nM of Cell Tracker Green CMFDA (Invitrogen). Transmigration was monitored, by counting cells on the underside of the chamber, after 16 h using a fluorescence microscope (Nikon). Experiments were conducted twice, both in duplicate.
Elisa
Expression of IL-1β in tumour conditioned media and macrophage conditioned media was conducted using a human IL-1β/IL-1F2 duoset ELISA development kit (R&D Systems) according to the manufacturers’ protocols. Briefly, a 96-well plate was coated with capture antibody overnight and the plate was washed and blocked with bovine serum albumin. Samples and IL-1β recombinant protein standard were added to the plate for 2 h. This was followed by detection antibody for 2 h and streptavidin-HRP for 20 min, with each step preceded by a wash. Colour change was achieved by the addition of substrate followed by a stop solution, and the plate was read at 450 nm on a BMG Fluostar Optima (BMG Labtech).