Background: Apoptotic cell clearance by phagocytes (efferocytosis) is essential in tissue homeostasis. However, the effects of efferocytosis in the multistep process of cancer cell dissemination, leading to cancer metastasis, have not been studied thus far.
Materials and Methods: To determine whether interaction of macrophages with dying cancer cells inhibits epithelial-mesenchymal transition (EMT) in various epithelial cancer cells, and decreases cancer cell migration and invasiveness, conditioned medium (CM) from murine macrophages (RAW, BMDMs and M2-like cells) or human blood MDMs exposed to apoptotic cancer cells was added to target cancer epithelial cells in the absence or presence of TGF-β1. Western blot and Real-time quantitative PCR were employed to determine changes in epithelial and mesenchymal markers. Cell migration and invasion were tested using Transwell chambers. Standard 3D culture was performed.
Results: Condition medium from RAW cells exposed to apoptotic 344SQcells (ApoSQ-exposed CM) inhibited TGF-β1-induced EMT, based on morphological cellular alterations, and EMT marker mRNA and protein expression profiles. We confirmed the anti-EMT effects of various types of apoptotic cancer cell-exposed CM in the human non-small cell lung cancer (NSCLC) cell line A549 and other human cancer cells lines, including breast (MDA-MB-231), colon (COLO320HSR), and prostate (PC3) cancer cells. In addition to RAW cells, CM from ApoSQ-exposed primary isolated mouse bone marrow-derived macrophages (BMDMs), or their IL-4-derived M2 phenotype, substantially inhibited TGF-β1-induced changes in EMT markers in 344SQ cells. CM from blood monocyte-derived macrophages (MDMs) from healthy humans and NSCLC (adenocarcinoma) patients exposed to apoptotic A549 cells (ApoA) showed anti-EMT effects. ApoSQ-exposed CM partially blocked Smad-independent TGF-β1 signaling, including the p38 MAP kinase and Akt pathways, in 344SQ cells. ApoSQ- -exposed CM prevented TGF-β1-induced cancer cell migration and invasion. In addition, TGF-β1-induced gene-level enhancement of cancer stem-like cell markers was reduced by treatment with ApoSQ-exposed CM. Moreover, 3D Matrigel culture confirmed the anti-invasive effect of ApoSQ-exposed CM, which caused 344SQ cells to recover their lost polarity and acinus-like colonies, and caused invasive projection by TGF-β1.
Conclusions: programming macrophages by apoptotic cancer cells might create a tumor microenvironment antagonizing cancer progression.