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. 2018 Dec 21;8:18024. doi: 10.1038/s41598-018-36626-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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TLR4 deletion attenuates scAT remodeling during cancer cachexia syndrome. (A) Wild-type C57BL/6 and TLR4^−/− mice (8-week old male) were inoculated with LLC tumor cells. Body weight change (excluding tumor weight) was evaluated after 28 days before inoculated tumor cells. N = 10 per group. (B) Histologic sections of scAT in different experimental groups. Histological staining for H/E and picrosirius red were performed, and also, immunohistochemistry for inflammatory profile (TNFα) and immune cell markers (CD68 and CD3). N = 5 per group. (C) The size of adipocytes (cell diameter) from WT and TLR4^−/− mice was quantitatively analyzed (500 adipocytes were measured for each group) after the experimental protocol. N = 5 per group. (D) Stromal vascular fractions (SVF) were isolated from scAT by collagenase digestion for each different group. Flow cytometric analysis of SVF was conducted using fluorescent-conjugated antibodies against CD68, F4/80, CD11c, CD206. Adipose tissue macrophages (ATMϕs) were defined as CD68⁺F4/80⁺ subpopulations and displayed the values as percentage of your respective groups. M1 and M2 ATMϕs were defined as CD68⁺F4/80⁺CD11c⁺CD206⁻ and CD68⁺F4/80⁺CD11c⁻CD206⁺, respectively. Representative flow cytometric dot plots showing the percentage of ATMϕs. N = 4 per group. (E) Quantification of double-positive cells for CD68⁺F4/80⁺ related to dot plots showed in (D). (F) M1/M2 ATMϕs ratio in the scAT. Scale bars, 100 μm and 200 μm. Graphs show the mean ± SEM. Statistical significance was determined by two-way ANOVA. **P < 0.01; ***P < 0.001.