Figure 6 from Tumor-Secreted ADAMTSL4 Activates Latent TGFβ1 to Drive Cancer Cachexia

<p>ADAMTSL4 induces skeletal muscle fibrosis via local TGFβ signaling and activates fibrosis-related genes in human myotubes and muscles of patients with cancer cachexia. <b>A</b> and <b>B</b>, GSEA (<b>A</b>) of GC muscle from MC38-GFP vs. MC38-ADAMTSL4 tumor-bearing mice showing enrichment of the “TGFβ receptor signaling” pathway and heatmap (<b>B</b>) of the top 10 DEGs within this pathway. <b>C</b> and <b>D</b>, GSEA (<b>C</b>) of GC muscle from C26-shCtrl vs. C26-shAdamtsl4 tumor-bearing mice showing enrichment of “TGFβ receptor signaling activates SMADs” and heatmap (<b>D</b>) of the top 10 DEGs. <b>E,</b> Representative Sirius Red (top) and collagen-I (bottom) staining in GC muscle sections from PBS-injected, C26-shCtrl, and C26-shAdamtsl4 tumor-bearing mice (scale bar, 100 μm). <b>F–K,</b> Human primary myotubes were treated with rhADAMTSL4-CT (250 ng/mL) in the presence or absence of TGFβ pathway blockade: (<b>F</b>) mRNA levels of fibrosis-related genes (<i>TGFB1</i>, <i>CTGF</i>, and <i>TGFBI</i>); (<b>G</b>) atrophy-related genes (<i>FBXO32</i> and <i>TRIM63</i>) measured by qPCR; (<b>H</b>) quantification of myotube diameter; (<b>I</b>) representative images of myotubes treated with vehicle or rhADAMTSL4-CT ± TGFβ1–neutralizing antibody 1D11 (1.5 μg/mL; scale bar, 100 μm); (<b>J</b>) Western blot analysis of phosphorylated SMAD2 (p-SMAD2); (<b>K</b>) qPCR analysis of CTGF and TGM2 mRNA levels in myotubes treated with vehicle or rhADAMTSL4-CT ± galunisertib (10 μmol/L), a TGFBR1 inhibitor. <b>L</b> and <b>M,</b> Relative body weight change and circulating ADAMTSL4 levels measured by ELISA in an exploratory human cancer subcohort including patients with colorectal cancer and PDAC classified as cachectic (cancer cachexia, <i>n</i> = 15) or noncachectic (non–cancer cachexia, <i>n</i> = 7). <b>N–P,</b> qPCR analysis of <i>TGFB1</i>, <i>TGM2</i>, and <i>FBXO21</i> mRNA expression in rectus abdominis muscle biopsies from non–cancer cachexia patients and those with cancer cachexia with colorectal cancer/PDAC. <b>Q,</b> Schematic model depicting how tumor-derived ADAMTSL4 promotes muscle wasting via local activation of TGFβ signaling through LAP engagement and TGFBR1-dependent transcriptional responses.

Data are shown as the mean ± SEM unless indicated otherwise. Each dot represents one biological replicate. Statistical analysis: weighted Kolmogorov–Smirnov test for GSEA enrichment scores (<b>A</b> and <b>C</b>); two-way mixed-effects model with Tukey or Holm–Šídák multiple-comparisons test (<b>F–H</b>, <b>J</b>, and <b>K</b>); for circulating ADAMTSL4 measurements in human plasma (<b>M</b>), ELISA values were log₁₀-transformed and analyzed using a two-tailed Mann–Whitney test; human gene expression comparisons (<b>N–P</b>) were analyzed using two-tailed Mann–Whitney tests.