Title : The effects of exosomal l1cam on glioblastoma migration and proliferation
Abstract:
L1CAM (L1, CD171) is a cell surface immunoglobulin superfamily protein normally involved in axon guidance, differentiation, and cell migration during development. LI also is expressed abnormally in many types of cancers including breast, pancreatic, colon, melanoma and glioma, and has been associated with poor prognosis due to increased proliferation, invasiveness or metastasis. We have shown previously that the soluble L1 ectodomain, which can be proteolyzed from the normal transmembrane form, can stimulate proliferation and motility of glioblastoma (GBM) cells in vitro by acting through integrins and fibroblast growth factor receptors (FGFRs). We also previously showed that minute exosomal membrane vesicles are released by glioma cells and are decorated with L1, and these potentially could stimulate motility, proliferation, and invasiveness into brain tissue. Here, L1-decorated exosomes were isolated from T98G glioma cell media and evaluated for their effects on glioma cell behavior. The hypothesis being tested was that L1-decorated exosomes increase the proliferation, motility and invasiveness of glioma cells through integrins and FGFRs. This study tested the effect of L1-decorated exosomes on several glioma cell lines and primary tumor cell cultures. The velocity of migrating cells was assessed in a highly quantitative Super Scratch assay using time-lapse microscopy. Effects on proliferation were determined by quantitation of DNA and cell cycle analysis. L1-decorated exosomes significantly increased cell velocity in the three glioma cell lines tested (T98G/shL1, U-118 MG, primary GBM cells). There also was a marked increase in cell proliferation. Chemical inhibitors against focal adhesion kinase (FAK) and FGFR activity decreased this augmented motility and proliferation to various degrees. L1-decorated exosomes also facilitated cell invasion in our chick embryo brain tumor model. Taken together, these data show that L1-decoratred exosomes stimulate motility, proliferation and invasion through both integrins and FGFRS, which adds to the complexity of how L1CAM stimulates cancer cells. This information may help to find novel approaches to combat glioblastoma.
Background : Tumors of the central nervous system (CNS) are a devastating disease. Of the 17,000 primary brain tumors diagnosed each year, approximately 60% are gliomas (1), which arise in the brain and spine from the supporting glial cells or their precursors (2). Gliomas in the brain are typically dangerous whether malignant or benign because of their location and may cause dysfunction (2). Unfortunately, due to the lack of symptoms, gliomas are usually detected in the late stages (3). The World Health Organization (WHO) has graded gliomas from grades I-IV based on cell morphology, malignancy, and pathogenicity (4). Low grade gliomas are characterized by cellular morphology and cause local effects that do not spread in the brain. High grade gliomas are malignant and can spread throughout the brain tissue. Glioblastoma multiforme (GBM; grade IV) arise from account for 15% of all brain tumors and are classified as such due to their extensive differentiation, high invasiveness, and thus high malignancy of the tumor (2,3). The 4-year mean survival rate of a patient from initial GBM diagnosis is approximately 11 months (5) due to their highly invasive nature and late diagnosis after a patient experiences headaches, seizures, memory loss, vision changes, and personality changes. Current treatments for GBM include surgery, radiation, and chemotherapy (6), but these are practically ineffective. GBM patients will lose brain tissue in their treatment which can be detrimental to their health. One of the factors that increases glioma cell proliferation, motility, and invasiveness is autocrine/paracrine stimulation by cell adhesion molecule L1CAM (L1, CD171), which normally is located on the membrane of developing neuronal cells but is also expressed in gliomas (6-7). L1 has an extracellular ectodomain that has five fibronectin domains and 6 immunoglobin-like repeat domains which is often excised and released in extracellular fluid where it can bind with other receptors in cancers. L1 has an Arginine, Guanine and Aspartic acid (RGD) domain that binds to integrin receptors and has a molecular weight of 200-220 kDa (9). L1 interacts with various binding partners including itself, cell surface integrins, and other cellular surface components (8). Two of L1 binding partners that are particularly important for gliomas are integrins, which activate Focal Adhesion Kinase (FAK), and fibroblast growth factor receptor (FGFR) (9-10). Previously, our lab and others explored the interaction between L1 with these molecules along with the source of L1 expression (11-13). It has been shown that L1-containing media can increase migration of glioma cells (14). L1 also is present decorating the surface of minute exosomal vesicles released by glioma cells. Exosomes are 40-100 nm membranous vesicles. Exosomes, formed by inward budding of the late endosomal membrane, are produced by exocytosis from the multivesicular body (MVB) (14). Exosomes were initially isolated from blood samples. They were believed to be secreted by reticulocytes during differentiation (15). Tumor samples have been found to be rich in exosomes. Tumor exosomes induce tumor invasion and support tumor cell survival by promoting their invasive properties (16-17).We previously showed that glioma cells released exosomes decorated with L1, which raised the possibility that this, too, may be a source of L1 that can autocrine/paracrine stimulate glioma cell proliferation, motility, and invasion. In this study, tumor exosomes, including those decorated with L1, were isolated from GBM cell culture media and the effects of L1-decorated exosomes on glioma cell migration and proliferation were explored. We evaluated the ability of L1-decorated exosomes vs soluble L1to stimulate glioma cell motility and proliferation in vitro and invasion into embryonic chick brain tissue in vivo. We found that L1-decorated exosomes can increase the migration and proliferation rates of gliomas. The interaction of L1 with integrins and FGFR will be explored to determine their effects on migration and proliferation (14-15). We compared the extent by which the integrin and FGFR pathways are stimulated by L1-decorated exosomes versus soluble L1. The hypothesis explored was that L1-decorated exosomes promote motility, proliferation, and invasion of glioma cells through FAK and FGFR pathways. This study explored the relationship between L1, Focal Adhesion Kinase (FAK), and Fibroblast Growth Factor Receptor (FGFR) by using inhibitors of the molecules. Any disruption of the normal migration and proliferation due to inhibitors was determined to be critical in that pathway. The interaction between L1 with these molecules showed a reduction in the initial stimulation of migration and proliferation rates back to below basal levels. And finally, we explored the ability of L1-decorated exosomes to promote invasiveness of glioma cells in vivo. There was an increased invasiveness of gliomas when the L1-decorated exosomes were added to the model system.
