CD133 identifies CSCs in colorectal cancer.

<p>(A)Schematic of CD133⁺ and CD133^-/lo tumor cells sorting from dissociated colorectal xenograft tumor by FACS. (B) A representative example of post-sorting analysis of the sorted CD133⁺ and CD133^-/lo XhCRC cells.(C) Tumor-initiating frequency of CD133⁺ and CD133^-/lo CRC cells in immunodeficient mice (D-G)Serial sphere formation assays for purified CD133⁺ and CD133^-/loCRC cells (i.e., XhCRC and SW620). Spheres were enumerated (D, F) and representative images are shown (E, G).Scale bars, 100μm.***<i>P</i>< 0.001.</p

CSCs display cell-autonomous resistance to chemotherapy.

<p>(A, B) Cell death analysis of CSCs and non-CSCs from XhCRC or SW620 cells was assessed by CCK-8 activity assay upon chemotherapeutic treatment (5-Fu or OXA).**<i>P</i>< 0.01, ***<i>P</i>< 0.001. (C, D) Enrichment of CSCs in bulk cells from XhCRC (C) and SW620 cells (D) was assessed by FACS analysis based on CD133 expression upon chemotherapy. (E, F) Sphere-forming capacity of bulk cells (XhCRC or SW620 cells) pre-treated by chemotherapeutic agents or DMSO (Ctrl). **<i>P</i>< 0.01, ***<i>P</i>< 0.001.</p

CAFs prime XhCRC CSCs to be more drug resistance through paracrine pathway.

<p>(A) CAFs derived from patient specimen were validated by positive immunostaining for CAF markers (α-SMA,Vimentin and FAP) and negative immunostaining for an epithelial marker (EpCAM). Scale bars, 30μm. (B) XhCRC CSCs were validated by positive immunostaining for epithelial marker (EpCAM) and CSC marker (CD133), Scale bars, 10μm. (C) Sphere-forming capacity of XhCRC CSCs in CAFs-derived conditioned media (e.g., 5-FU, OXA, DMSO-treated CAFs). (D) Sphere-forming capacity of XhCRC CSCs treated with CAF-derived CM during chemotherapy (5-Fu or OXA). Representative microscopic images are shown. Scale bars, 50μm. (E, F) CAF-derived CM affected on tumor growth of XhCRC CSCs in female NOD/SCID mice treated with OXA. Tumor growth curves are shown in E, Shown in F are tumor weights and images at the end of experiments. Data are presented as mean ± SD; *<i>P</i>< 0.1; **<i>P</i>< 0.05. (G)CAF-derived CM affected on tumor growth of XhCRC CSCs transplanted in female NOD/SCID mice upon treatment with 5-Fu.</p

18Co cells prime SW620 CSCs to be more drug resistance via paracrine pathway.

<p>(A) Sphere-forming capacity of SW620 CSCs treated with 18Co-derived CM during chemotherapy (5-Fu or OXA). Representative microscopic images are shown. Scale bars, 100μm. (B, C) 18Co-derived conditioned medium affected tumor growth of SW620 CSCs in female Balb/c-nu mice treated with OXA. Tumor growth curves are shown in C, Shown in D are tumor weights and images at the end of experiments. Data are presented as mean ± SD; *<i>P</i>< 0.1; **<i>P</i>< 0.05; ***<i>P</i>< 0.001.</p

Exosomes prime CSCs through mediating Wnt activity.

<p>(A) TOP–GFP levels are associated with CSC maker expression. (B) Sphere-forming efficiency and representative spheres (insert) of TOP-GFP⁺ and TOP-GFP^-/lo SW620 cells. ^***<i>P</i><0.001. (C)Representative flow cytometry and quantification (insert) of TOP-GFP expression of CSCs treated with 18Co-derived exosomes (green line).Gray line represents the control group.(D, E) Representative flow cytometry and quantification (insert) of TOP-GFP expression of CSCs treated with 18Co-derived exosomes (magenta line) or GW4869 (black line)/DMSO (orange line)-pretreated 18Co-CM upon chemotherapy (5-Fu or OXA). Gray line represents the control group. (F) Fibroblast-derived exosomes were purified by ultracentrifugation. The pellet, supernatant and fibroblast cell lysate were subjected to western blotting for exosome maker CD81 and Wnt3a.</p