Fig 2: IKKε knockdown or inhibition suppressed colorectal cancer cells metastasis and invadopodia formation in vivo.(A) H&E staining of the cecum derived from mice orthotopically microinjected with HCT116 cells stably expressing shNC or shIKKε. Scale bar, 50 μm. (B) Immunofluorescent analysis of Tks5 and cortactin co-localization in cecum tumor sections from shNC and shIKKε mice (left). Quantification of cells with invadopodia (right). Scale bars represent 200 μm. (N>300 cells/sample, **P<0.01,). (C) The gross view of tumor nodules established in liver and the bioluminescence images of liver from mice orthotopic microinjected with HCT116 cells stably expressing shNC or shIKKε (left). Yellow arrowheads indicate tumor nodules in the liver. Quantitative analysis of photon flux in shNC (n=4) and shIKKε (n=4) mice (*P<0.05) (right). (D) H&E staining of the liver derived from mice which were orthotopic microinjected with HCT116 cells stably expressing shNC or shIKKε (left). Scale bar, 500 μm (left) and 50 μm (middle). Images for IHC staining of CK18 in in metastatic sites in liver (right) Scale bar, 50 μm. (E) Bioluminescence images of shNC and shIKKε mice (left). Quantitative analysis of photon flux in shNC (n=6) and shIKKε (n=8) mice (***P<0.001) (right). (F) Bioluminescence images of lungs, liver and kidneys isolated from shNC and shIKKε mice (left). Quantification of photon flux in lungs, livers and kidneys isolated from shNC (n=6) and shIKKε (n=8) mice (***P <0.001) (right). (G) H&E staining sections of the lung and kidney sections from shNC and shIKKε mice (left). Scale bar, 500 μm. Images for IHC staining of CK18 and EpCAM in metastatic sites in lung and kidney (middle and right). Scale bar, 50 μm. (H) Bioluminescence images of vehicle-treated or amlexanox-treated mice (left). Quantitative analysis of photon flux in vehicle-treated (n=6) and amlexanox-treated (50 mg/kg, n=8) mice (***P <0.001) (right). (I) Bioluminescence images of lungs, liver and kidneys isolated from vehicle-treated or amlexanox-treated mice (left). Quantification of photon flux in lungs, livers and kidneys isolated from vehicle-treated (n=6) and amlexanox-treated (n=8) mice (*P<0.05) (right). (J) H&E staining sections of the lung and kidney sections from vehicle-treated or amlexanox-treated mice (left). Scale bar, 500 μm. Images for IHC staining of CK18 and EpCAM in in metastatic sites in lung and kidney (middle and right). Scale bar, 50 μm.

Fig 3: IKKe co-localizes with F-actin and Tks5 in active invadopodia. (A) F-actin (red), Tks5 (purple) and IKKe (blue) co-localization and gelatin (green) degradation in HCT116 cells. Inset highlight area of gelatin degradation where F-actin, Tks5 and IKKe are co-localized in the box. (B) Z-stack acquisition was performed and orthogonal views of the x-z plane and y-z plane are presented. Black arrows indicate areas of gelatin degradation where IKKe, Tks5 and F-actin co-localize. Scale bar, 10µm.

Fig 4: Survival.A, Overall survival of 110 gastric cancer patients based on Tks5 expression in peritoneal mesothelial cells. No significant difference was found between the Tks5-positive and -negative groups (p = 0.093). B, Peritoneal recurrence-free survival of 98 gastric cancer patients based on Tks5 expression in peritoneal mesothelial cells. The 3-year PRFS of the Tks5-positive group was significantly worse than that in Tks5-negative group (p = 0.024).

Fig 5: Representative images of peritoneal mesothelial cells of patients with gastric cancer.Tks5 expression was found in the cytoplasm of the PMCs forming a monolayer on the greater omentum, lesser omentum, or retroperitoneum in a GC patient. Intensity score: 0; no staining, 1+; weak staining, 2+; strong staining. Positive staining was defined as 1+ or 2+. Arrow, PMC: peritoneal mesothelial cell.