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CD90 Expression Controls Migration and Predicts Dasatinib Response In Published OnlineFirst September 22, 2017; DOI: 10.1158/1078-0432.CCR-17-1549 Biology of Human Tumors Clinical Cancer Research CD90 Expression Controls Migration and Predicts Dasatinib Response in Glioblastoma Tony Avril1,2, Amandine Etcheverry3, Raphael€ Pineau1, Joanna Obacz1, Gwena ele€ Jegou1,2, Florence Jouan1, Pierre-Jean Le Reste1,4, Masumeh Hatami5, Rivka R. Colen5,6, Brett L. Carlson7, Paul A. Decker7, Jann N. Sarkaria7, Elodie Vauleon 1,2,3, Dan Cristian Chiforeanu8, Anne Clavreul9, Jean Mosser3, Eric Chevet1,2, and Veronique Quillien1,2,3 Abstract Purpose: CD90 (Thy-1) is a glycophosphatidylinositol- migration gene signature and with invasive tumor features. Mod- anchored glycoprotein considered as a surrogate marker for a ulation of CD90 expression in GBM cells dramatically affected variety of stem cells, including glioblastoma (GBM) stem cells their adhesion and migration properties. Moreover, orthotopic (GSC). However, the molecular and cellular functions of CD90 xenografts revealed that CD90 expression induced invasive phe- remain unclear. notypes in vivo. Indeed, CD90 expression led to enhanced SRC and Experimental Design: The function of CD90 in GBM was FAK signaling in our GBM cellular models and GBM patients' addressed using cellular models from immortalized and primary specimens. Pharmacologic inhibition of these signaling nodes GBM lines, in vivo orthotopic mouse models, and GBM specimens' blunted adhesion and migration in CD90-positive cells. Remark- transcriptome associated with MRI features from GBM patients. ably, dasatinib blunted CD90-dependent GBM cell invasion CD90 expression was silenced in U251 and GBM primary cells in vivo and killed CD90high primary GSC lines. and complemented in CD90-negative U87 cells. Conclusions: Our data demonstrate that CD90 is an actor Results: We showed that CD90 is not only expressed on GSCs of GBM invasiveness through SRC-dependent mechanisms and but also on more differentiated GBM cancer cells. In GBM could be used as a predictive factor for dasatinib response in patients, CD90 expression was associated with an adhesion/ CD90high GBM patients. Clin Cancer Res; 23(23); 1–15. Ó2017 AACR. Introduction radio- and chemotherapy; (ii) diffuse features due to the inva- siveness properties of tumor cells throughout the surrounding Glioblastoma (GBM) is one of the deadliest human cancers brain parenchyma, and (iii) tumor intra- and inter-heterogeneity with an incidence of about 3.5/100,000 per year worldwide (1). (1, 3). Despite the aggressive standard of care currently used including CD90 (Thy-1) is a marker for mesenchymal stromal/stem cells surgery, chemo-, and radiotherapy, the prognosis remains very (4) that has been earlier described in glioma/GBM specimens (5) poor with approximately 15 months overall survival (2). The and immortalized glioma/GBM cell lines (6–10). In the past inevitable recurrence of GBM is associated with: (i) resistance to years, CD90 has been described as a human GBM stem cell (GSC) marker (11–15). CD90 is also expressed in GBM-associated stromal cells (GASC; ref. 16) and mesenchymal stem cell–like 1Proteostasis and Cancer Team, INSERM U1242, Chemistry, Oncogenesis Stress pericytes (17), thereby reflecting GBM cellular heterogeneity. Signaling, Universite de Rennes 1, Rennes, France. 2Centre Eugene Marquis, Rennes, France. 3Integrated Functional Genomics and Biomarkers Team, CD90 is a N-glycosylated, glycophosphatidylinositol (GPI)- UMR6290 CNRS, Universite de Rennes 1, Rennes, France. 4Departement de anchored cell surface protein, originally described in murine Neuro-Chirurgie, CHU Pontchaillou, Rennes, France. 5Department of Cancer thymocytes (18). CD90 is also expressed in many other cell types Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, including endothelial cells, fibroblasts, and neurons (4, 19–21). 6 Texas. Department of Diagnostic Radiology, University of Texas MD Anderson CD90 has been involved in neurite outgrowth inhibition, T-cell Cancer Center, Houston, Texas. 7Department of Radiation Oncology, Mayo 8 activation and apoptosis, leukocytes and melanoma cell adhesion Clinic, Rochester, Minnesota. Departement d'Anatomo-pathologie, CHU fi Pontchaillou, Rennes, France. 9Departement de Neuro-Chirurgie, CHU Angers, and migration, tumor suppression in ovarian cancers, and bro- fi France. blast proliferation and migration in wound healing and brosis (19, 21). Although the exact mechanisms of action of CD90 Note: Supplementary data for this article are available at Clinical Cancer fi – Research Online (http://clincancerres.aacrjournals.org/). remain ill-de ned, a role in cell cell/matrix interactions has been proposed (19, 21). E. Chevet and V. Quillien contributed equally to this article. We show that CD90 expression is not only restricted to GBM Corresponding Author: Tony Avril, Centre Eugene Marquis, Rue de la bataille stem–like cells but is also observed in more differentiated GBM Flandres Dunkerque, CS44229, Rennes 35042, France. Phone: 332-9925-3169; cells (primary adherent lines) and in freshly dissociated GBM Fax: 332-9925-3170; E-mail: [email protected] specimens. In GBM patients, CD90 is associated with a cell doi: 10.1158/1078-0432.CCR-17-1549 adhesion/migration gene signature and with multifocal/multi- Ó2017 American Association for Cancer Research. centric MRI features. Using in vitro and in vivo approaches, we www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst September 22, 2017; DOI: 10.1158/1078-0432.CCR-17-1549 Avril et al. extent of surgery was evaluated with an enhanced MRI performed Translational Relevance within 24 hours after the resection. Adherent (RADH) and neuro- CD90/Thy-1 is considered a surrogate marker for stem spheres (RNS; enriched in stem cells) GBM primary cell lines were cells, including glioblastoma (GBM) stem cells (GSC). In this obtained from GBM samples as described in refs. 22, 23. Briefly, study, we demonstrated that CD90 is expressed not only on fresh tumor tissues were mechanically dissociated using gentle- GSCs, but also on more differentiated GBM cells. In GBM MACS dissociator following the manufacturer's instructions patients, CD90 expression was associated with an adhesion/ (Miltenyi Biotec). Cells were directly cultured and frozen before migration gene signature and invasive tumor features, as further use such as flow cytometry analysis. RADH cells were observed by MRI. In GBM cells, modulation of CD90 expres- grown in DMEM (Lonza) supplemented with 10% FBS (Lonza). sion dramatically affected adhesion/migration properties. RNS cells were grown in DMEM/Ham:F12 (Lonza) supplemented Moreover, orthotopic xenografts revealed that CD90 over- with B27 and N2 additives (Invitrogen), EGF (20 ng/mL), and expression induced invasive phenotypes in vivo. Furthermore, basic fibroblast growth factor (20 ng/mL; PeproTech, Tebu-Bio). we showed that CD90 signals through SRC and FAK in All GBM RNS and RADH cells were used between the 5th and 15th both cellular models and GBM specimens. Remarkably, the passages for the experiments. Human immortalized U251 and SRC inhibitor dasatinib blunted CD90-dependent invasion U87 GBM cell lines were cultured in DMEM 10% FBS. in vitro and in vivo, thereby suggesting its potential use for CD90high tumors. Hence, we demonstrate that CD90 plays a Preparation of CD90 knocked-down U251 and CD90- key role in GBM invasiveness through SRC-dependent expressing U87 GBM cell lines mechanisms and could be used as a predictive factor for U251 cells were transfected with pLKO.1-puro plasmids con- 0 dasatinib response in CD90high GBM patients. taining shRNA constructs #24 (5 -ccggcgaaccaacttcaccagcaaactc- gagtttgctggtgaagttggttcgtttttg-30) and #25 (50-ccgggctcagaga- caaactggtcaactcgagttgaccagtttgtctctgagctttttg-30) targeting CD90 mRNA and #CTR (50-ccggcaacaagatgaagagcaccaactcgagttggtgctctt- catcttgttgttttt-30) targeting nonmammalian mRNA (Sigma- demonstrate the critical role of CD90 in GBM migration/invasion. Aldrich) using the Lipofectamine 2000 reagent (Life Technolo- We show that CD90 signaling through SRC, FAK, and RhoA gies) according to the manufacturer's instructions. After one week promotes cell migration and importantly, that high CD90 expres- of culture under the selective antibiotic puromycin used at 10 mg/ sion affects the cell response to the SRC inhibitor dasatinib. We mL, transfected U251 cells were amplified and then cloned in 96- propose a model in which CD90 expression represents a novel well plates at 0.1 cell/well. CD90 knocked-down U251 cell lines fi strati cation tool for selecting patients to be treated with dasati- were expanded and selected for their decreased expression of nib. Moreover, in this model, dasatinib could not only impair the high CD90. U87 cells were transfected with CD90 cDNA (GeneWiz, adhesion/migration of CD90 -differentiated tumor cells, but Sigma-Aldrich) cloned into the pLKO.1-puro plasmid with EcoRI also the proliferation of CD90high GSCs, thereby increasing its high and BamHI enzymes using the Lipofectamine 2000 reagent. therapeutic potential for CD90 patients. CD90-expressing U87 cell lines were obtained as described above with U251 and were selected for their high expression of CD90. Materials and Methods Preparation of CD90 knocked-down RNS GBM Reagents and antibodies primary cell lines All reagents not specified below were purchased from Sigma- RNS cells were infected with lentiviral particles generated
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