Fig. 2

Flow cytometry analysis of M2 macrophages, GFP-labeled MCF-7 breast cancer cells and MCF-7/macrophage hybrids. a) M2 macrophages exhibit CD45 expression when stained with anti-CD45, showing an increase in fluorescence intensity compared to the isotype and negative controls. b) M2 macrophages exhibit CD163 expression when stained with anti-CD163, showing an increase in fluorescence intensity compared to the isotype and negative controls. c) The transfected MCF-7 cells exhibit GFP expression. The macrophages were not expressing GFP and no GFP were detected in macrophages after co-culture in ThinCert transwell culture system. Regardless if macrophages were cultivated over (upper chamber) or under (lower chamber) GFP-labeled MCF-7 cells in ThinCert transwell cuture system. In this transwell culture system the cells share culture medium, allowing paracrine signaling, but the cells are physically separated by a filter, preventing cellular contact and cell fusion. d) The MCF-7 cells did not express CD163, nor were CD163 expression induced after co-culture with macrophages in the ThinCert transwell culture system, indicating that macrophage traits in cancer cells were not generated by cellular interaction between macrophages and cancer cells. e) Co-cultured MCF-7 cells and macrophages, created hybrids by spontaneous cell fusion. The hybrids expressed phenotypic characteristics from maternal cells, GFP-labeled MCF-7 breast cancer cells and M2 macrophages. Note that the hybrids were identified by exhibiting a double positive phenotype, positive for green fluorescence protein GFP and macrophage-specific antigen CD163, or f) panleukocyte marker CD45