Fig 2: MDA-MB-231 cells in high plasticity matrices extend invadopodia protrusions. a RFP-LifeAct MDA-MB-231 cells were imaged for 12 h using time-lapse confocal fluorescence microscopy. Transient actin-rich spots were observed in all IPNs (yellow arrows, top), but in HP IPNs, these spots often elongated into oscillatory, actin-rich protrusions (yellow arrows, bottom). Scale bar is 10 µm. b Probability of a cell extending one or more protrusions in LP, MP, and HP IPNs, with either vehicle alone or protease inhibitor (10 µM GM6001) added to the medium. Graph displays the number of cells per condition, taken from R = 3–5 biological replicate experiments. Bars indicate mean probabilities and error bars indicate 95% confidence intervals. Differences in protrusivity as indicated are statistically significant (****P < 0.0001, Fisher’s exact). Probability of extending a protrusion also trends with plasticity (####P < 0.0001, ?2 tests for trend). c and d Histograms of extensions and widths of cell protrusions in LP and HP IPNs. Includes number of cells indicated per condition, pooled from R = 3 biological replicate experiments each. e Tracings of protrusion lengths, for one cell each in LP and HP IPNs, over time during a 6 h timeframe, plotted alongside a sinusoidal fit. f–h Confocal immunofluorescence imaging was used to investigate localization of indicated proteins. Main panel scale bar is 10 µm. f and g Imaging of indicated staining on cryosections of MDA-MB-231 cells encapsulated in IPNs for 1 day. In f, main image shows merged maximum intensity projection (MIP) and inset (1.5× zoom for LP and 4× zoom for HP) shows merged image on one z-plane. In g, inset is 2× zoom. h Live imaging of actin (RFP-LifeAct) and TKS5 (TKS5-EGFP). Inset is 1.5× zoom. i Probability of a cell extending one or more protrusions, for cells transfected with control siRNA, siRNA targeting cortactin (siCTTN), or siRNA targeting TKS5 (siTKS5)

Fig 3: IKKe 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 shIKKe. Scale bar, 50 µm. (B) Immunofluorescent analysis of Tks5 and cortactin co-localization in cecum tumor sections from shNC and shIKKe 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 shIKKe (left). Yellow arrowheads indicate tumor nodules in the liver. Quantitative analysis of photon flux in shNC (n=4) and shIKKe (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 shIKKe (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 shIKKe mice (left). Quantitative analysis of photon flux in shNC (n=6) and shIKKe (n=8) mice (***P<0.001) (right). (F) Bioluminescence images of lungs, liver and kidneys isolated from shNC and shIKKe mice (left). Quantification of photon flux in lungs, livers and kidneys isolated from shNC (n=6) and shIKKe (n=8) mice (***P <0.001) (right). (G) H&E staining sections of the lung and kidney sections from shNC and shIKKe 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 4: IKKε-induced invadopodia formation depends on kindlin-2 S159 phosphorylation. (A) HCT116 cells were transfected with GFP-tagged IKKε and stained with antibodies against kindlin-2, Tks5 and F-actin (phalloidin). Confocal images were acquired to visualize the localization of IKKε, kindlin-2, Tks5 and F-actin. White arrowhead point to the co-localization of IKKε, kindlin-2, Tks5 and F-actin. Scale bar, 10 μm. (B) Co-localization of Tks5 and F-actin in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). Inset shows magnified image of invadopodia in the box. Quantification of cells with invadopodia (right). (C) Gelatin degradation in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). The area of degraded gelatin per cell (right). All data represent the means ± S.D. (N>400 cells/sample, *P<0.05, **P< 0.01, and ***P < 0.001).

Fig 5: IKKε co-localizes with F-actin and Tks5 in active invadopodia. (A) F-actin (red), Tks5 (purple) and IKKε (blue) co-localization and gelatin (green) degradation in HCT116 cells. Inset highlight area of gelatin degradation where F-actin, Tks5 and IKKε 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 IKKε, Tks5 and F-actin co-localize. Scale bar, 10μm.