4-iodo-6-phenylpyrimidine and Carcinoma--Squamous-Cell

Tumor-associated macrophages (TAMs) are important determinants of intratumoral immune evasion, neoangiogenesis, extracellular matrix remodeling and dysregulated tumor cell proliferation. Our prior studies revealed that macrophage-derived, but not tumor cell-derived, macrophage migration inhibitory factor (MIF), is an important determinant of TAM alternative activation and M2 polarization.. Because MIF is historically thought to initiate signaling via a receptor-dependent, outside-in mode of action, we wished to investigate the specific contributions of tumor-derived vs. macrophage-derived MIF to M2 marker expression during macrophage polarization.. Murine oral squamous cell-carcinoma cells (SCCVII) were co-cultured with either the RAW 264.7 mouse macrophage cell line or mouse primary bone marrow-derived macrophages in the context of MIF genetic loss/inhibition individually or in combination each cell type.. Twelve well Transwell plates were used to co-culture SCCVII cells and RAW 264.7, MIF+/+ or MIF-/- macrophages treated with/without the small molecule MIF inhibitor, 4-iodo-6-phenylpyrimidine and incubated in the presence or absence of interleukin (IL-4) for 48 h. Macrophages were analyzed by quantitative real-time polymerase chain reaction and/or immunoblotting for relative macrophage polarization marker expression.. IL-4 treatment synergizes with SCCVII co-culture in inducing the expression of macrophage M2 markers and loss or inhibition of macrophage-derived MIF significantly reduces both IL-4 alone and IL-4/SCCVII co-culture-induced macrophage M2 marker expression.. These studies identify an important and dominant requirement for macrophage MIF in maximal Th2-cytokine and oral squamous carcinoma cell-induced macrophage polarization and M2 marker expression.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Interleukin-4; Intramolecular Oxidoreductases; Macrophage Migration-Inhibitory Factors; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Neovascularization, Pathologic; Pyrimidines; RAW 264.7 Cells

Recent clinical observations and experimental studies of our group indicate that macrophage migration inhibitory factor (MIF) may contribute to tumor progression in head and neck squamous cell carcinomas (HNSCC). The present study was undertaken to examine the effects of the irreversible MIF inhibitor 4-iodo-6-phenylpyrimidine (4-IPP) on proliferation and invasiveness of the squamous carcinoma cell line SCCVII. Cell counting, crystal violet assay and flow cytometry were used to analyze the effects of 4-IPP on SCCVII cell growth. The impact of 4-IPP on cell invasiveness was assessed by Boyden chamber assay. Knockdown of the MIF receptor CD74 was achieved by transduction with lentiviral vectors encoding anti-CD74 shRNAs. As shown by immunofluorescence staining, SCCVII cells express both MIF and CD74. Decreased MIF immunoreactivity as a result of exposure to 4-IPP suggested a covalent modification of the cytokine. 4-IPP inhibited SCCVII cell proliferation and invasiveness. Moreover, the cytostatic effect of 4-IPP was enhanced by CD74 knockdown. The inhibitory effects of 4-IPP on cell proliferation and invasiveness strongly suggest that MIF is involved in proliferative activity and invasive properties of squamous carcinoma cells. In conclusion, MIF inhibition may open possibilities for target-directed treatment of head and neck squamous cell carcinoma.

Topics: Antigens, Differentiation, B-Lymphocyte; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Knockdown Techniques; Head and Neck Neoplasms; Histocompatibility Antigens Class II; Humans; Macrophage Migration-Inhibitory Factors; Neoplasm Invasiveness; Pyrimidines