Cancer invasion is a hallmark of metastasis. as well as invadopodia formation and matrix degradation were impaired upon Lamellipodin depletion. Mechanistically we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex which stimulates actin branching. In contrast Lamellipodin interaction with Scar/WAVE but not Ena/VASP is required for random 2D cell Panulisib migration. We identify a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP while Src-dependent phosphorylation enhances binding to Scar/WAVE but not Ena/VASP. Through these selective regulated interactions Lamellipodin mediates directional sensing of EGF gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis. we implanted fluorescently labeled MDA-MB-231 breast cancer cells into the perivitelline cavities of zebrafish embryos. In this assay the injected cancer cells intravasate and then infiltrate the trunk of the fish27 28 Overexpression of GFP-Lpd in MDA-MB-231 cells enhanced the frequency of seeding of these breast cancer cells compared to GFP expressing control cells (Figure 2J K). We then performed tail vein injections of the Lpd knockdown and control LM2 cell lines into immunocompromised mice and quantified lung metastases after 28 days to test whether Lpd influenced the later stages of the metastatic cascade. Lpd depletion did not reduce the number of metastatic foci in the lungs of the mice compared to controls (Supplemental Figure 2D E). Taken together our results reveal that Panulisib Lpd promotes local tumor invasion intravasation and metastasis but is not required for extravasation. Breast cancer cell migration towards blood vessels is guided by cues from the tumor microenvironment such as EGF29. We reasoned that the effect of Lpd depletion on EGF-induced 3D invasion might arise from defects in lamellipodial dynamics. Depletion of Lpd in MDA-MB-231 breast cancer cells decreased lamellipodia size (Supplemental Figure 3A) similar to B16-F1 Ctsk mouse melanoma cells in which Lpd-depletion also reduces protrusion speed under steady state conditions17. EGF-stimulated MDA-MB-231 Lpd knockdown cells displayed reduced protrusion persistence and distance without affecting protrusion speed (Supplemental Figure 3B). We chose MTLn3 cells a mammary adenocarcinoma cancer cell line in which protrusion responses to EGF have been extensively characterized to examine EGF-elicited protrusion in more detail since lamellipodial size is least affected by Lpd knockdown in this cell line (Supplemental Figure 3A). In agreement with our findings in MDA-MB-231 cells in EGF-stimulated MTLn3 cells reduced Lpd levels significantly decreased protrusion persistence Panulisib and distance (Figure 3A B) but did not affect protrusion speed (Supplemental Figure 3D) compared to controls. Lpd was diffusely distributed throughout the Panulisib cytoplasm of serum-starved cells but was rapidly recruited to the cell edge following bath Panulisib application of EGF (Figure 3C D). Lamellipodial initiation was detected 30 seconds after EGF stimulation in Ctrl-shRNA-expressing cells but was delayed significantly when Lpd levels were reduced (Figure 3B E F; Supplemental Figure 3E; Supplemental Videos 3 4 Taken together our data suggest that in breast cancer cells Lpd depletion reduces EGF-elicited lamellipodial protrusion formation and persistence but not speed. Figure 3 Lpd is required for EGF-induced membrane-protrusion Membrane extension during lamellipodial protrusion is driven by actin polymerization4-6. To determine how Lpd depletion influences actin polymerization we used a G-actin incorporation assay30 to measure the abundance and distribution of polymerization-competent free (uncapped) F-actin barbed-ends in lamellipodia of living cells. Silencing Lpd significantly reduced free barbed-end formation 1 minute after EGF stimulation relative to Ctrl-shRNA-expressing cells (Figure 4A B). Collectively these data indicate that Lpd promotes lamellipodial protrusion by increasing actin polymerization downstream of EGFR activation. Figure 4 Lpd is required for chemosensing EGF-dependent membrane protrusion in MTLn3.