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

Laboratory investigation

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Object

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.

Methods

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.

Results

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.

Conclusions

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: cpcifarelli@virginia.edu.

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.

Headings

Figures

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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.

References

1

Abramoff MDMagelhaes PJRam SJ: Image processing with Image. J Biophotonics Int 11:36422004

2

American Cancer Society: Cancer Facts & Figures 2008 http://www.cancer.org/docroot/stt/content/stt_1x_cancer_facts_and_figures_2008.asp) [Accessed March 25 2010]

3

Asano KDuntsch CDZhou QWeimar JDBordelon DRobertson JH: Correlation of N-cadherin expression in high grade gliomas with tissue invasion. J Neurooncol 70:3152004

4

Baumgart ECohen MSSilva Neto BJacobs MAWotkowicz CRieger-Christ KM: Identification and prognostic significance of an epithelial-mesenchymal transition expression profile in human bladder tumors. Clin Cancer Res 13:168516942007

5

Boggon TJMurray JChappuis-Flament SWong EGumbiner BMShapiro L: C-cadherin ectodomain structure and implications for cell adhesion mechanisms. Science 296:130813132002

6

Burden-Gulley SMPayne HRLemmon V: Growth cones are actively influenced by substrate-bound adhesion molecules. J Neurosci 15:437043811995

7

Central Brain Tumor Registry of the United States: CBTRUS Statistical Report: Primary Brain Tumors in the United States 2000–2004 Central Brain Tumor Registry of the United States2008

8

Chappuis-Flament SWong EHicks LDKay CMGumbiner BM: Multiple cadherin extracellular repeats mediate homophilic binding and adhesion. J Cell Biol 154:2312432001

9

Couldwell WTde Tribolet NAntel JPGauthier TKuppner MC: Adhesion molecules and malignant gliomas: implications for tumorigenesis. J Neurosurg 76:7827911992

10

de Groot JMilano V: Improving the prognosis for patients with glioblastoma: the rationale for targeting Src. J Neurooncol 95:1511632009

11

Gadducci AFerdeghini MCosio SAnnicchiarico CCiampi BBianchi R: Preoperative serum E-cadherin assay in patients with ovarian carcinoma. Anticancer Res 19:1B7697721999

12

Giese ABjerkvig RBerens MEWestphal M: Cost of migration: invasion of malignant gliomas and implications for treatment. J Clin Oncol 21:162416362003

13

Giese AKucinski TKnopp UGoldbrunner RHamel WMehdorn HM: Pattern of recurrence following local chemotherapy with biodegradable carmustine (BCNU) implants in patients with glioblastoma. J Neurooncol 66:3513602004

14

Hazan RBPhillips GRQiao RFNorton LAaronson SA: Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis. J Cell Biol 148:7797902000

15

Hirohashi S: Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am J Pathol 153:3333391998

16

Kashibuchi KTomita KSchalken JAKume HTakeuchi TKitamura T: The prognostic value of E-cadherin, alpha-, betaand gamma-catenin in bladder cancer patients who underwent radical cystectomy. Int J Urol 14:7897942007

17

Kleinman HKJacob K: Invasion assays. Curr Protoc Cell Biol Chapter 12 Unit 12.22001

18

Lagenaur CLemmon V: An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc Natl Acad Sci U S A 84:775377571987

19

Lu KVZhu SCvrljevic AHuang TTSarkaria SAhkavan D: Fyn and SRC are effectors of oncogenic epidermal growth factor receptor signaling in glioblastoma patients. Cancer Res 69:688968982009

20

Maretzky TReiss KLudwig ABuchholz JScholz FProksch E: ADAM10 mediates E-cadherin shedding and regulates epithelial cell-cell adhesion, migration, and beta-catenin translocation. Proc Natl Acad Sci U S A 102:918291872005

21

Mohammadizadeh FGhasemibasir HRajabi PNaimi AEftekhari AMesbah A: Correlation of E-cadherin expression and routine immunohistochemistry panel in breast invasive ductal carcinoma. Cancer Biomark 5:182009

22

Nakada MAnderson EMDemuth TNakada SReavie LBDrake KL: The phosphorylation of ephrin-B2 ligand promotes glioma cell migration and invasion. Int J Cancer 126:115511652010

23

Paradies NEGrunwald GB: Purification and characterization of NCAD90, a soluble endogenous form of N-cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite-promoting function. J Neurosci Res 36:33451993

24

Perego CVanoni CMassari SRaimondi APola SCattaneo MG: Invasive behaviour of glioblastoma cell lines is associated with altered organisation of the cadherin-catenin adhesion system. J Cell Sci 115:333133402002

25

Perl AKWilgenbus PDahl USemb HChristofori G: A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392:1901931998

26

Reiss KMaretzky TLudwig ATousseyn Tde Strooper BHartmann D: ADAM10 cleavage of N-cadherin and regulation of cell-cell adhesion and beta-catenin nuclear signalling. EMBO J 24:7427522005

27

Riehl RJohnson KBradley RGrunwald GBCornel ELilienbaum A: Cadherin function is required for axon outgrowth in retinal ganglion cells in vivo. Neuron 17:8378481996

28

Rønn LCRalets IHartz BPBech MBerezin ABerezin V: A simple procedure for quantification of neurite outgrowth based on stereological principles. J Neurosci Methods 100:25322000

29

Shapiro LLove JColman DR: Adhesion molecules in the nervous system: structural insights into function and diversity. Annu Rev Neurosci 30:4514742007

30

Shinoura NParadies NEWarnick REChen HLarson JJTew JJ: Expression of N-cadherin and alpha-catenin in astrocytomas and glioblastomas. Br J Cancer 72:6276331995

31

Stupp RHegi MEGilbert MRChakravarti A: Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25:412741362007

32

Stupp RHegi MEvan den Bent MJMason WPWeller MMirimanoff RO: Changing paradigms—an update on the multidisciplinary management of malignant glioma. Oncologist 11:1651802006

33

Takeichi M: The cadherin superfamily in neuronal connections and interactions. Nat Rev Neurosci 8:11202007

34

Tysnes BBMahesparan R: Biological mechanisms of glioma invasion and potential therapeutic targets. J Neurooncol 53:1291472001

35

Uemura KKihara TKuzuya AOkawa KNishimoto TNinomiya H: Characterization of sequential N-cadherin cleavage by ADAM10 and PS1. Neurosci Lett 402:2782832006

36

Wang LFFokas EJuricko JYou ARose FPagenstecher A: Increased expression of EphA7 correlates with adverse outcome in primary and recurrent glioblastoma multiforme patients. BMC Cancer 8:792008

37

Wanner IBGuerra NKMahoney JKumar AWood PMMirsky R: Role of N-cadherin in Schwann cell precursors of growing nerves. Glia 54:4394592006

38

Wanner IBWood PM: N-cadherin mediates axon-aligned process growth and cell-cell interaction in rat Schwann cells. J Neurosci 22:406640792002

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