Fig. 1
Generation of tumor-conditioned media and in vitro macrophages. (a) Schematic of the generation of tumor-conditioned media (TCM) using 2 × 105 cells/mL (MDA- MB 231, SK-BR-3 or LK 46) in 0.2% FBS media for 24 h. Media was collected and centrifuged to remove any tumor cells. 10% FBS was added. TCM was stored in 5 mL aliquots in − 80° freezer until use. (b) Schematic of the in vitro generation of macrophages. Monocytes harvested from healthy donor blood were plated at 1 × 106 cells/mL in a total of 10 mL 10% HAB (M1/M2) or 5 mL 10% HAB plus 5 mL TCM (TAM). In M1-like monocyte differentiation, GM-CSF (1 μg/mL) was added to culture for 6 days. On day 6 the media was refreshed with GM-CSF (1 μg/mL) + IFN-γ (20 ng/mL) + LPS (50 ng/mL) for 24 h. In M2-like differentiation, M-CSF (1 μg/mL) was added to culture for 6 days. On day 6 the media was refreshed with M-CSF (1 μg/mL) and IL-4 (1 μg/mL). In TAM differentiation, M-CSF, IL-10, and IL-4 (all at 1 μg/mL) were added to culture in combination with TCM and 10% HAB (1:1). In all differentiation protocols media was refreshed every other day and cells were harvested on day 7