Figure 3. M2 macrophages cause tumor cell budding from the colonosphere bulk.

(A) Representative images of bright field microscopy of the mono-cultures and co-cultures are shown in the upper panel. Green fluorescent images to identify tumor cells are shown in the middle panel, binarized images of the fluorescence are shown in the lower panel, 10x magnification on EVOS microscope; scale bars indicate 400 μm. (B) Adherence assay showing the number of small cell clusters (≤5 cells) per condition; one-way ANOVA, p=<0.0001. (C) Proliferation of colonospheres measured on FACS Caliber after a BrdU pulse chase experiment. (D) Representative bright field microscopy images of the invasive front and the tumor core of T1-tumors using the immunohistochemical stainings Pan-cytokeratin (tumor cells), CD68 and CD163 (macrophages). Quantification of immunohistochemical stainings, depicted as percentage of area on the left CD68 and on the right CD163; n=8 different tumors, one image field per tumor for the invasive field and one field for the tumor core; bar graphs show mean and SD, paired t-test, p=0.0006 and p=0,0052 respectively.