EMT6 and 4THM tumor models and analysis of micro‐/macrometastasis

Both EMT6 and 4THM tumors were grown following injection of 2 × 10⁵ cells into the mammary fat pad of BALB/c female mice (WT or CD200RKO). Except where specified, tumors were resected at 14 days and 10 days later macrometastases to lung/liver were enumerated visually on tissues harvested and fixed in Bouin's solution. Micrometastases to draining lymph nodes (DLNs) were also measured at this time on cells harvested from individual mice and cloned at limiting dilution in microtitre plates for 21 days as described previously ¹³. In this case, a mAb (clone 35C1) for a breast‐cancer‐amplified kinase (BTAK‐1) obtained from Hycult Biotechnology (5405 PB Uden, the Netherlands) was used with permeabilized cloned cells to confirm tumor cells in all colonies as discussed in earlier reports ¹³, ¹⁴.

Where the frequency of circulating tumor cells (CTCs) was assessed in peripheral blood (PBL), PBL were harvested by cardiac puncture and spun over mouse hypaque (Cedarlane Labs, Hornby, Ontario) to deplete red cells. CD45⁻ cells (obtained as the eluate after passage twice over a CD45⁺ enrichment column; Miltenyi, San Diego, CA) were used as a surrogate population containing CTCs, and were cloned at limiting dilution on irradiated (3000 Rads) feeder layers of BALB/c bone marrow (5 × 10⁵/well), with colonies enumerated at 21 days. Again tumor cells in PBL‐derived colonies were confirmed by staining with anti‐BTAK‐1 ¹³.