Exosomal L1CAM Stimulates Glioblastoma Cell Motility, Proliferation, and Invasiveness.

Immunoglobulin superfamily protein L1CAM (L1, CD171) normally facilitates neuronal migration, differentiation, and axon guidance during development. Many types of cancers, including glioblastoma (GBM), also abnormally express L1, and this has been associated with poor prognosis due to increased cell proliferation, invasiveness, or metastasis. We showed previously that the soluble L1 ectodomain, which is proteolyzed from the transmembrane form, can stimulate proliferation and motility of GBM cells in vitro by acting through integrins and fibroblast growth factor receptors (FGFRs). Minute L1-decorated exosomal vesicles also are released by GBM cells and potentially could stimulate cell motility, proliferation, and invasiveness, but this needed to be demonstrated. In the present study, we aimed to determine if minute L1-decorated extracellular vesicles (exosomes) were capable of stimulating GBM cell motility, proliferation, and invasiveness. L1-decorated exosomes were isolated from the conditioned media of the human T98G GBM cell line and were evaluated for their effects on the behavior of glioma cell lines and primary tumor cells. L1-decorated exosomes significantly increased cell velocity in the three human glioma cells tested (T98G/shL1, U-118 MG, and primary GBM cells) in a highly quantitative SuperScratch assay compared to L1-reduced exosomes from L1-attenuated T98G/shL1 cells. They also caused a marked increase in cell proliferation as determined by DNA cell cycle analysis and cell counting. In addition, L1-decorated exosomes facilitated initial GBM cell invasion when mixed with non-invasive T98G/shL1 cells in our chick embryo brain tumor model, whereas mixing with L1-reduced exosomes did not. Chemical inhibitors against focal adhesion kinase (FAK) and fibroblast growth factor receptor (FGFR) decreased L1-mediated motility and proliferation to varying degrees. These novel data show that L1-decoratred exosomes stimulate motility, proliferation and invasion to influence GBM cell behavior, which adds to the complexity of how L1 stimulates cancer cells through not only soluble ectodomain but also through exosomes.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells.

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and ß3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers ß3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.