title: Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity.
abstract: Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].
authors.affiliation: Division of Respiratory Surgery, Kobe University Hospital, Kobe University Graduate School of Medicine, Kobe, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Takata
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Maniwa
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
authors.forename: Yugo
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Kenji
authors.initials: K
authors.lastname: Okada
authors.forename: Wataru
authors.initials: W
authors.lastname: Nishio
authors.forename: Chiho
authors.initials: C
authors.lastname: Ohbayashi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yoshimura
authors.forename: Yoshitake
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Okita
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D001681:Biological Assay; D015820:Cadherins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D002465:Cell Movement; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005782:Gels / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000473:pathology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D017154:Stromal Cells / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D015820:Cadherins; D005782:Gels; D018960:Hyaluronan Receptors; D061965:Matrix Metalloproteinase Inhibitors; D034741:RNA, Small Interfering; D003094:Collagen; D020782:Matrix Metalloproteinases