Journal: bioRxiv

Article Title: Invadopodia-Mediated Remodeling of the Lymphatic Endothelium Drives Cancer Cell Lymphatic Dissemination and is Regulated by a CCR7-Vav2-Rac3 Signaling axis

doi: 10.1101/2025.10.15.682688

Figure Lengend Snippet: (A) The lymph node– homing MDA-MB-231 cells (231LN) was used to generate stable cell lines expressing either a non-targeting control or CCR7-targeting sequence. Western blot of CCR7 expression in control and knockdown cell lines. (B) Control and CCR7 knockdown cell lines were seeding on 568-labeled gelatin and rates of invadopodia formation were quantified. N=3. One-way ANOVA. (C) CCR7 tyrosine phosphorylation. CCR7 was immunoprecipitated from MDA-MB-231LN cells and analyzed by western blot for tyrosine phosphorylation. (D) Src-dependent tyrosine phosphorylation of CCR7 is required for Vav2 binding. MDA-MB-231LN cells were treated with control or Src inhibitor (saracatinib) prior to CCR7 immunoprecipitation. Western blot of co-immunoprecipitated Vav2 and CCR7 are shown. (E-F) Transient knockdown of Vav2 reduces invadopodia activity. Control and Vav2 siRNA cells were plated on 568-labeled gelatin and either left untreated or treated with control or CCL19. Rates of invadopodia formation were quantified. One-Way ANOVA. (G) Pan Rac inhibitor ETH1864 was added to MDA-MB-231LN cells plated on568-labeled gelatin and rates of invadopodia formation were quantified. One-way ANOVA. (H , I) Rac3 expression in MDA-MB-231LN cells compared with parental MDA-MB-231 cell. (H) Representative western blot of Rac3 expression and (I) quantification of Rac3 expression. One Sample T-test. (J , K , L) Rac3 activity in MDA-MB-231LN cells as measured by Pak1-PBD pulldown assays. (J) Rac3 activity in control and Vav2 knockdown cells. (K) Rac3 activity in the presence of CCL19 or control. (L) Rac3 activity in the presence of CCR7 ligand (CCL19), CCR7 antagonist, or control. Mean ± SEM. Asterisk denotes significance.

Article Snippet: Antibodies specific for following proteins were purchased from the suppliers indicated: Tks5 (for western blot: Cell Signaling 16619S; 1:250), Tks5 (for Immunofluorescence: Millipore Sigma 09-403), β-actin (for western blot: Sigma A5441; 1:5000), ZO-1 (Thermo Fisher Scientific 33-9100; 1:50), Vinculin (for western blot: Abcam, ab91495, 1:3000), VE-Cadherin (Abcam ab33168; 1:50), CCR7 (for Immunofluorescence (1:500) and Immunoprecipitation (1ug): Novus Biologicals NBP2-67324), CCR7 (for western blot: Abcam ab 191575, 1:500), Vav2 (Proteintech 21924-1-AP,1:500), Rac3 (Invitrogen MA5-53241,1:500), Phosphotyrosine (for western blot: Invitrogen 14-5001-82, 1:1500) and Rabbit Isotype control (Biolegend, Cat. No. 910801).

Techniques: Stable Transfection, Expressing, Control, Sequencing, Western Blot, Knockdown, Labeling, Phospho-proteomics, Immunoprecipitation, Binding Assay, Activity Assay