Lobopodia-based migration is distinct from cancer cell motility. (A) Amoeboid and mesenchymal HT1080 cells in the CDM. Phase-contrast image showing amoeboid (rounded) and mesenchymal (elongated) HT1080 cells in the CDM. (B) The percentage of amoeboid and mesenchymal HT1080 cells in the CDM (n = 1,001). (C and D) HT1080 cells do not form lobopodia in the 3D CDM. Round amoeboid cells lack matrix adhesions, whereas elongated mesenchymal cells have prominent lamellipodia and matrix adhesions. Images show maximally projected confocal stacks of amoeboid (C) and mesenchymal (D) HT1080 cells, expressing GFP-actin or YFP-paxillin, migrating inside the CDM (Alexa Fluor 633, grayscale). Arrowheads indicate matrix adhesions. (E) Matrix adhesions are present during both lamellipodia- and lobopodia-based 3D migration of elongated normal fibroblasts. Maximally projected confocal stacks of HFFs expressing YFP-paxillin or vinculin–tension sensor (TS) migrating inside 3D collagen or the 3D CDM. (F) Matrix adhesions contribute to both lamellipodia- and lobopodia-based migration of normal fibroblasts. Blocking α_vβ₃ and β₁ integrins significantly decreased the velocity and directionality of HFFs migrating in 3D collagen and CDM, indicating that integrin-mediated adhesion contributed to the efficient directional migration of both cell populations. Quantification of the velocity (top) and directionality (bottom) of HFFs migrating on glass, CDM, or 3D collagen, either in media or in media with 100 µM cyclic RGD (cRGD; an α_Vβ₃-blocking peptide) plus 500 µg/ml β₁ integrin–blocking antibody (mAb13). *, P < 0.05 versus the untreated control. All cells are oriented with the leading edge toward the right of the figure. Bars: (A) 50 µm; (C–E) 5 µm.