Tumor-Released Products Promote Bone Marrow-Derived Macrophage Survival and Proliferation

Abstract

Macrophages play a central role within the tumor microenvironment, with relevant implications for tumor progression. The modulation of their phenotype is one of the mechanisms used by tumors to escape from effective immune responses. This study was designed to analyze the influence of soluble products released by tumors, here represented by the tumor-conditioned media of two tumor cell lines (3LL from Lewis lung carcinoma and MN/MCA from fibrosarcoma), on murine macrophage differentiation and polarization in vitro. Data revealed that tumor-conditioned media stimulated macrophage differentiation but influenced the expression levels of macrophage polarization markers, cytokine production, and microRNAs of relevance for macrophage biology. Interestingly, tumor-derived soluble products supported the survival and proliferation rate of bone marrow precursor cells, an effect observed even with mature macrophages in the presence of M2 but not M1 inducers. Despite presenting low concentrations of macrophage colony-stimulating factor (M-CSF), tumor-conditioned media alone also supported the proliferation of cells to a similar extent as exogenous M-CSF. This effect was only evident in cells positive for the expression of the M-CSF receptor (CD115) and occurred preferentially within the CD16⁺ subset. Blocking CD115 partially reversed the effect on proliferation. These results suggest that tumors release soluble products that not only promote macrophage development from bone marrow precursors but also stimulate the proliferation of cells with specific phenotypes that could support protumoral functions.

Keywords: CD115; CD16; arginase-1; macrophage proliferation; tumor-associated macrophages.

Figure 1

Tumor-conditioned media affect macrophage polarization. (A) Protocol of differentiation and polarization of macrophages with tumor products stimulation. (B–F) Bone marrow hematopoietic progenitor cells were collected and stimulated with M-CSF for 5 days in the presence of 3LL or MN/MCA-conditioned media (CM). After the initial differentiation with M-CSF, IFN-γ plus LPS or IL-4 were added to cultures for a further 24 h to induce polarization. Graphs show the abundance of transcripts encoding inducible nitric oxide synthase (Nos2; (B)), arginase 1 (Arg1; (C)), interferon-β (IFN-β; (D)), transforming growth factor-β (TGF-β; (E)), and found in inflammatory zone 1 (Fizz1; (F)) in M0 (white bars), M1-like (grey bars), M2-like (black bars), and CM alone (striped bars)-stimulated cells. Results are shown as 2^−Δct. n ≥ 4 independent experiments. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Figure 3

Tumor-conditioned media promote macrophage survival and proliferation. Bone marrow hematopoietic progenitor cells were collected and stimulated with M-CSF in the presence of 3LL or MN/MCA-conditioned media (CM) for 3 days. To some cultures PLX-3397 was also added. (A) Flow cytometry strategy to select the populations according to FSC x SSC firstly (left panel), then to exclude cell doublets (middle panel), and finally to separate non-proliferating (R3) and proliferating (R4) groups of cells (right panel). (B) On day 4 of culture, cells were stained with an EdU probe and analyzed by flow cytometry. (C,D) On day 4 of culture, cells were indeed stained with anti-CD115 or anti-CD16 antibodies to analyze proliferating and non-proliferating groups using flow cytometry. Graphs show EdU⁺ (blue and green) and EdU⁻ (purple and orange) cells investigated for CD115 (green and orange; panel C) or CD16 (green and orange; panel D) expression. n ≥ 3 independent experiments. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.

Figure 2

miR-155 and miR-511 expression is modulated by tumor-conditioned media. Bone marrow hematopoietic progenitor cells were collected and stimulated with M-CSF for 5 days in the presence of 3LL or MN/MCA-conditioned media (CM). After this period, IFN-γ and LPS or IL-4 were added to cultures for a further 24 h to induce polarization. Graphs show the abundance of transcripts encoding miR-155 (A) and miR-511 (B) by M0 (white bars), M1 (grey bars), M2 (black bars), and CM alone (striped bars)-stimulated cells. Results are shown as 2^−Δct. n ≥ 3 independent experiments. *: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001.