Two Phases of Pseudopod Protrusion in Tumor Cells Revealed by a Micropipette

• Published in: Microvascular research Vol. 47; no. 1; pp. 55 - 67
• Main Authors: Dong, Cheng, Aznavoorian, Sadie, Liotta, Lance A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 1994; Elsevier
• Subjects: Biological and medical sciences; Calcium - physiology; Chemotaxis - drug effects; Collagen - pharmacology; Cytological Techniques - instrumentation; Dissemination; Egtazic Acid - analogs & derivatives; Egtazic Acid - pharmacology; GTP-Binding Proteins - physiology; Humans; Image Processing, Computer-Assisted; Medical sciences; Melanoma - pathology; Micromanipulation; Neoplastic Stem Cells - drug effects; Neoplastic Stem Cells - ultrastructure; Tumor cell; Tumor Cells, Cultured; Tumors; Human; Cell line; Melanoma; Pseudopode; Kinetics; Metastasis; Chemotaxis; Tumor cell
• ISSN: 0026-2862; 1095-9319
• DOI: 10.1006/mvre.1994.1005

Pseudopod protrusion at the leading edge is a characteristic of migrating tumor cells as they traverse vascular subendothelial basement membranes to establish metastases. A micropipette system has been developed to study the dynamics of pseudopod protrusion in individual human melanoma cells in response to type IV collagen, a component of basement membranes. Soluble type IV collagen stimulates chemotaxis of A2058 melanoma cells through a G protein-coupled receptor, and induces an early burst of intracellular calcium (Savarese et al., 1992, J. Biol. Chem. 267, 21928-21935). A micropipette filled with type IV collagen solution (100 μg/ml) was positioned so that the tip was adjacent to a cell suspended in Dulbeceo's modified Eagle's medium. Within 10 min, tumor cells generated a pseudopod which entered the micropipette with an average velocity of 0.24 μm/min and proceeded to lengthen for 40 min. Pseudopods from individual cells ranged from 7.5-10 μm at this time and were characterized by an irregular shape which did not fill the lumen of the micropipette. Pretreatment of cells with pertussis toxin (0.5 μg/ml), which inhibits cell migration by ∼90% (but not the calcium burst), blocked formation of the irregular, extended pseudopod, while allowing a much smaller outpouching, or bleb, to form. Lengths of such blebs from individual PT-treated cells reached a plateau at ∼20 min and ranged from 2.2-4.0 μm at the end point. Treatment of cells with bis-(amino-phenoxy)ethane tetraacetic acid (75 μ M), an intracellular Ca 2+ chelator, blocked initial bleb formation and prevented extension. From these observations we hypothesize that tumor cell pseudopod protrusion induced by soluble type IV collagen takes place in distinct, separable phases: an initial convex, symmetrical outpouching, caused by localized Ca +2-activated actin depolymerization and osmotic flux, followed by an extension with an irregular shape, which requires G protein-mediated actin polymerization.