Cadherin-dependent adhesion of human U373MG glioblastoma cells promotes neurite outgrowth and increases migratory capacity

Laboratory investigation

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The current management of primary CNS tumors involves a multimodal approach, incorporating cytoreductive techniques including resection, radiotherapy, and antiproliferative chemotherapeutic agents. Despite these attempts, the majority of patients with a diagnosis of a high-grade glioma have a dismal prognosis, with the leading cause of treatment failure and tumor recurrence attributable to local invasion of adjacent brain parenchyma. The current study examines the capacity of glioma tumor cells to undergo neurite outgrowth and local migration, specifically focusing on the role of the cadherin cell adhesion system.


Using a recombinant cadherin ectodomain protein, U373MG human glioblastoma cells were assessed for their ability to adhere and migrate in a cadherin-dependent manner in culture. Adhesion was evaluated via growth assessment and neurite length at 72 hours growth on an immobilized cadherin substrate and compared with other matrix adhesion proteins, such as Type IV collagen and vitronectin. Migratory capacity was measured via modified transwell assays, also using recombinant cadherin ectodomain in comparison with collagen and vitronectin.


Cadherin adherent cells adopt a fasciculated morphology, with a significant increase in neurite extension, measuring 104 ± 13.3 μm in length, compared with background adhesion on bovine serum albumin and nonfunctional cadherin ectodomain controls measuring 55 ± 4.4 and 47 ± 3.84 μm, respectively (p = 0.029). Significant increases in neurite length compared with controls were also observed in the vitronectin (81 ± 4.69 μm) and Type IV collagen (91 ± 7.7 μm) groups (p = 0.017 and 0.025, respectively). With respect to migration, U373 cells demonstrate increased invasion in response to cadherin ectodomain exposure, whereas vitronectin and Type IV collagen were not potent initiators of migration through the transwell barrier. Both adhesion and migration outcomes were noted in the absence of any relative changes in cell proliferation, indicating a primary role for the cadherin-based adhesion system in tumor invasion.


Cadherin-based adhesion promotes increased adhesion, neurite outgrowth, and migration in human U373MG glioblastoma cells, providing a novel area of research for the development of therapeutic targets addressing local tumor invasion.

Abbreviations used in this paper: BSA = bovine serum albumin; DMEM = Dulbecco modified eagle medium; PBS = phosphate-buffered saline.

Article Information

Address correspondence to: Christopher P. Cifarelli, M.D., Ph.D., Department of Neurological Surgery, University of Virginia Health System, P.O Box 800212, Charlottesville, Virginia 22908-0212. email:

Please include this information when citing this paper: published online April 23, 2010; DOI: 10.3171/2010.3.JNS091451.

© AANS, except where prohibited by US copyright law.



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    Cadherin expression, neurite extension, and morphological analysis of U373 cells. A: Western blot showing cadherin expression in U87 and U373 cell lines. A 120–130 kDa pancadherin reactive band (arrow) is present in both U87 and U373 radioimmunoprecipitation assay extracted fractions, with significantly higher reactivity for the full-length protein in the U373 cell extract. The relative increased proportion of the 60–80 kDa fragment in the U87 cells may represent increased baseline proteolysis compared with the U373 cells. B: Phase-contrast microscopic image showing immobilized cadherin-dependent adhesion and neurite extension. The U373 cells cultured for 3 days on nitrocellulose bound recombinant EC1–5 cadherin fusion protein (right) demonstrate qualitative increases in cell spreading and neurite length compared with cells grown on BSA-coated surface (left) as demarcated by the dashed line. C: Cell morphology analysis of cadherin-based adhesion compared with controls. The U373 cells grown in adhesion culture on either recombinant EC1–5 or BSA were categorized based on growth patterns as cell clusters (≥ 2 cells) versus single cells, with a significant (*p = 0.0005) increase in single cell growth in the cadherin-adherent cells. D: Proliferation of cadherin-adherent cells. The U373 cells grown on immobilized cadherin ectodomain do not demonstrate a significant increase in cell proliferation compared with controls grown on BSA-coated culture dishes (p = 0.118).

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    Microscopic image showing U373 cells grown on cadherin ectodomain substrate adopt a fasciculated appearance. At 14 days in culture, the U373 cells grown on the recombinant ectodomain fragment appear densely packed with parallel alignment, compared with the BSA adherent controls than maintain a loosely defasciculated growth pattern. Original magnification × 40.

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    Graph showing immobilized recombinant cadherin ectodomain promotes neurite extension. The U373 cells grown for 3 days on immobilized EC1–5, as well as the nonadhesive EC1–5 mutant control, were analyzed for neurite growth in comparison with Type IV collagen, vitronectin, and BSA controls. Significant increases in neurite length were observed in Type IV collagen, vitronectin, and EC1–5 (cadherin) based adhesion (p < 0.05 in all groups) in comparison with BSA and the EC1–5 negative control. *p = 0.017, **p = 0.029, ***p = 0.025.

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    Analysis of the ability of U373 cells to traverse an artificial membrane. A: Transwell invasion assay. The U373 cells appearance as grown on cadherin-coated transwell membranes with an 8.0-μm pore size display similar growth patterns to those grown on cadherin-coated culture dishes at 3 days of culture. Crystal violet, original magnification × 40. B: Graph of U373 cell migration. The cadherin ectodomain coated transwell membranes promote a significant increase in migratory capacity as measured by the number of cells passing through into the lower chamber. Cadherin-adherent cells demonstrate nearly a 3-fold increase in migration through the transwell membrane compared with both the controls and the Type IV collagen substrate (*p = 0.00023). C: Transwell migration is not a function of cell proliferation in U373 cells. Analysis of the number of cells remaining bound to the transwell membrane following 3 days of culture does demonstrate a significant relationship between migratory capacity and proliferation with respect to cadherin-based adhesion (p = 0.088), but did reveal a significant increase in Type IV collagen-mediated proliferation (**p = 0.002). *p = 0.02.


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