Fig. 2

myCAF-derived exosome PWAR6 enhanced glutamine uptake and promote CRC cell migration. A–C Evaluation of invasion and stemness of DLD1 Cells after GW4869 treatment using Transwell and spheroid formation assays. Scale bars = 100μm(upper), Scale bars = 50μm(lower). (Representative images are shown, data are from five biologically replicates). D PDO 3D invasion assay to evaluated the invasion of PDOs after GW4869 treatment. Scale bars = 50μm. (Representative images are shown, data are from three biologically replicates). E Workflows of the extraction of fibroblasts from LM tissues (myCAFs) and adjacent normal tissues (NFs) in CRLM patients, followed by the extraction of exosomes for lncRNA sequencing and metabolomics analysis. F Expression of the exosome markers TSG101, HSP70 and Alix confirmed by western blot. (n = 3, one of three biological replicates). G Transmission electron microscopy (TEM) images of exosomes secreted from myCAFs and NFs. Scale bars = 100nm. (n = 3, one of three biological replicates). H Top 100 upregulated and downregulated lncRNAs in exosomes secreted by myCAFs from CRLM tissues and paired NFs. I The expression levels of PWAR6 among normal colonic epithelial cell line (NCM460), CRC cell lines, CRC tissues (T), normal intestinal epithelial tissues (N), NFs, NF-derived exosomes, myCAFs, and myCAF-derived exosomes. (n = 3 technical replicates, one of three biological replicates). J ISH experiments in CRC cells and PDO indicate that PWAR6 is localized in both the nucleus and the cytoplasm. Scale bars = 100μm. (n = 3, one of three biological replicates). K Knockdown of PWAR6 in myCAFs. (n = 5, one of three biological replicates). L Knockdown of PWAR6 in myCAFs reduces its level in exosomes secreted by myCAFs. (n = 5, one of three biological replicates). M Incubation with exosomes from PWAR6-knockdown myCAFs significantly reduces PWAR6 levels in DLD1 cells. (n = 5, one of three biological replicates). N, O PDO 3D invasion assay to evaluated the invasion of PDOs after knocking down PWAR6. Scale bars = 50μm. (Representative images are shown, data are from three biologically replicates). P, Q PWAR6 knockdown significantly reduces the size of subcutaneous tumors. (n = 5 mice per group). R, S PWAR6 knockdown significantly reduces the size of liver metastases. (n = 3 mice per group). Data are shown as mean ± s.e.m. For A-D, I and K–O, data were analyzed by one-way ANOVA. For P-S, data were analyzed by two-tailed Student’s t-test. P values. *P < 0.05, **P < 0.01, ***P < 0.001