Fig. 3

MCT-1 promotes breast tumor progression and M2 macrophage polarization. (a and b) MDA-MB-231 (control vs. MCT-1) and IV2–3 (scramble vs. shMCT-1#3) cells were injected into the mammary fat pad of nude mice. Tumor growth and burdens were analyzed at the indicated time. (c) Tumor immunohistology revealed the levels of MCT-1, E-cadherin, vimentin, CD31 (indicated by arrowheads) and F4/80 (enclosed in circles). Scale bars, 50 μm. (d) Immunohistochemistry characterized the tumor-associated CD163-positive M2 macrophages (enclosed in circles). The images were captured with 40X objective lens and macrophage numbers were counted (n = 6). (E and F) Levels of the M2-specific markers CD163 and CD206 were compared after THP-1 coculture with RPMI medium, MDA-MB-231 (control vs. MCT-1) or IV2–3 (scramble vs. shMCT-1#3) cells for 48 h. (g) The pan-macrophage (F4/80) and M1-like macrophage (CD86) markers were analyzed after THP-1 coculture with RPMI or with MDA-MB-231 cells (scramble vs. shMCT-1#3) for 48 h in a Boyden chamber. (h) THP-1 polarization into M2-like macrophages was signified by an increase in Arginase-1 and IL-10 after coculture with the indicated cells (control vs. MCT-1; scramble vs. shMCT-1#3). Different blots with normalized internal control were used to present the data of the experiment. (I and J) Quantitative RT-PCR analysis evaluated the CD86 and CD163 expression after THP-1 priming by RPMI or by the MDA-MB-231 cells (control vs. MCT-1) pretreated with an IL-6R mAb (tocilizumab) or stimulated with IL-6 for 48 h. The results are expressed as the mean ± SD (n = 3). One-way ANOVA with a post hoc two-tailed t-test was used to calculate the statistical significance of pairwise comparisons. (*p < 0.05; **p < 0.01; *** p < 0.001)