Figure 4 from Tumor-Secreted ADAMTSL4 Activates Latent TGFβ1 to Drive Cancer Cachexia

<p>Stable knockdown of tumor-derived ADAMTSL4 attenuates adipose tissue wasting, prolongs cachexia-related survival, and protects against cancer-induced cardiac atrophy. <b>A–R,</b> Mice were subcutaneously injected with PBS, control C26 cells (C26-shCtrl), or ADAMTSL4-depleted C26 cells (C26-shAdamtsl4). <b>A,</b> Schematic overview of the preclinical model used to assess the impact of tumor-derived ADAMTSL4 on adipose wasting, survival, and cardiac atrophy. <b>B,</b> Relative fat mass changes measured by EchoMRI. <b>C,</b> Mass of individual fat depots: iWAT, eWAT, and BAT. <b>D,</b> Plasma glycerol levels normalized to final fat mass. <b>E,</b> Selected GO terms from GSEA of eWAT comparing C26-shAdamtsl4 vs. C26-shCtrl tumor-bearing mice (see also Supplementary Data S7 and S8 for full GO term analysis results). <b>F,</b> mRNA expression of lipolysis-related genes and (<b>G</b>) fatty acid oxidation genes in eWAT, validating GSEA-enriched “fatty acid oxidation” and “lipid oxidation” terms. <b>H,</b> Western blot analysis of ATGL protein levels. <b>I,</b> Western blot analysis of autophagy-related proteins in eWAT, supporting the “autophagy” GO term from GSEA. <b>J–R,</b> Mice were subcutaneously inoculated with PBS (<i>n</i> = 5), C26-shCtrl cells (<i>n</i> = 6–7), or C26-shAdamtsl4 cells (<i>n</i> = 6–9). <b>J,</b> Tumor mRNA expression of <i>Adamtsl4</i>, <i>Mki67</i>, <i>Bbc3</i>, and <i>Pmaip1</i> assessed by qPCR. <b>K,</b> Plasma ADAMTSL4 protein levels measured by Western blot. <b>L,</b> Kaplan–Meier curve showing percentage of mice developing cachexia over time. <b>M,</b> Percentage of cachexia-free mice at endpoint. <b>N,</b> Representative heart sections stained with WGA Alexa Fluor 488 and DAPI (200× magnification; scale bar, 100 μm).

Representative structural features are highlighted by arrows (white, PBS/control; red, cachectic condition; blue, ADAMTSL4 knockdown). <b>O,</b> CSA quantification of cardiomyocytes (<i>n</i> = 3 per group, >200 cells per mouse). <b>P,</b> Cardiac expression of autophagy-related genes (<i>Bnip3</i>, <i>Map1lc3</i>, and <i>Gabarapl1</i>) by qPCR. <b>Q,</b> Western blot analysis of cardiac autophagy (LC3-I and LC3-II) and (<b>R</b>) apoptosis (BAX) markers.