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. 2014 Sep 24;124(11):4737–4752. doi: 10.1172/JCI76375

Figure 5. MerTK-dependent efferocytosis of dying breast cancer cells induces production of TGF-β, IL-10, and IL-4.

Figure 5

(A) Schematic of a novel breast cancer efferocytosis coculture assay. PyVmT cells expressing mCherry and HSV-TK were cocultured with GFP-Raw264.7 macrophages. Gancyclovir caused cell death in HSV-TK+ PyVmT cells. BMS-777607 or neutralizing anti-MerTK antibody was used to block MerTK-dependent efferocytosis. (B) Gancyclovir-inducible cell death in PyVmT cells. Original magnification, ×400. (C) Western blot analysis of whole-cell lysates or MerTK immunoprecipitates from cocultures treated with or without BMS-777607 (1 μM) for 16 hours. (D) Representative fluorescent images of cocultures. White arrows indicate condensed mCherry+ tumor cells within cytoplasmic vacuoles of GFP+ macrophages; yellow arrows indicate condensed mCherry+ cells free in culture. Original magnification, ×400. (E) mCherry+ cells remaining after 32 hours in coculture. ****P < 0.0001 by Student’s t test. (F) Remaining MCF7-mCherry cells were quantified. Values represent the average ± SD. **P < 0.01; ***P < 0.001. n = 6. (G) MCF7-mCherry cells were cultured for 4 hours in suspension with BKM120 (1 μM) plus ABT-263 (2 μM) to induce cell death, then seeded over adherent Raw264.7 cells and cocultured for 16 hours with or without BMS-777607 or anti-MerTK antibody. (H) Mouse IL-4 and IL-10 ELISA of cultured media and qPCR to measure mouse Tgfb1 transcripts in whole coculture RNA. Values represent the average ± SD. n = 4, each assessed in duplicate. **P < 0.01. (I) Mouse IL-4 ELISA of cocultured media. n = 4, each sample assessed in duplicate. Values are the average ± SD. **P < 0.01 by Student’s t test. PyV-mCh-TK, PyVmT-mCherry-TK; PyV-mCh, PyVmT-mCherry.