Abstract
Glioblastomas are deadly neoplasms resistant to current treatment modalities. Fibroblast activation protein (FAP) is a protease which is not expressed in most of the normal adult tissues but is characteristically present in the stroma of extracranial malignancies. FAP is considered a potential therapeutic target and is associated with a worse patient outcome in some cancers. The FAP localization in the glioma microenvironment and its relation to patient survival are unknown. By analyzing 56 gliomas and 15 non-tumorous brain samples, we demonstrate increased FAP expression in a subgroup of high-grade gliomas, in particular on the protein level. FAP expression was most elevated in the mesenchymal subtype of glioblastoma. It was neither associated with glioblastoma patient survival in our patient cohort nor in publicly available datasets. FAP was expressed in both transformed and stromal cells; the latter were frequently localized around dysplastic blood vessels and commonly expressed mesenchymal markers. In a mouse xenotransplantation model, FAP was expressed in glioma cells in a subgroup of tumors that typically did not express the astrocytic marker GFAP. Endogenous FAP was frequently upregulated and part of the FAP+ host cells coexpressed the CXCR4 chemokine receptor. In summary, FAP is expressed by several constituents of the glioblastoma microenvironment, including stromal non-malignant mesenchymal cells recruited to and/or activated in response to glioma growth. The limited expression of FAP in healthy tissues together with its presence in both transformed and stromal cells suggests that FAP may be a candidate target for specific delivery of therapeutic agents in glioblastoma.
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References
Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor microenvironment. Glia. 2012;59(8):1169–80.
Rettig WJ, Garin-Chesa P, Beresford HR, Oettgen HF, Melamed MR, Old LJ. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells. Proc Natl Acad Sci U S A. 1988;85(9):3110–4.
Bae S, Park CW, Son HK, Ju HK, Paik D, Jeon CJ, et al. Fibroblast activation protein alpha identifies mesenchymal stromal cells from human bone marrow. Br J Haematol. 2008;142(5):827–30. doi:10.1111/j.1365-2141.2008.07241.x.
Busek P, Hrabal P, Fric P, Sedo A. Co-expression of the homologous proteases fibroblast activation protein and dipeptidyl peptidase-IV in the adult human Langerhans islets. Histochem Cell Biol. 2015;143(5):497–504. doi:10.1007/s00418-014-1292-0.
Kelly T, Huang Y, Simms AE, Mazur A. Fibroblast activation protein-alpha: a key modulator of the microenvironment in multiple pathologies. International review of cell and molecular biology. 2012;297:83–116.
Jacob M, Chang L, Pure E. Fibroblast activation protein in remodeling tissues. Current molecular medicine. 2012;12(10):1220–43.
Koczorowska MM, Tholen S, Bucher F, Lutz L, Kizhakkedathu JN, De Wever O, et al. Fibroblast activation protein-alpha, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations. Mol Oncol. 2016;10(1):40–58. doi:10.1016/j.molonc.2015.08.001.
Lee HO, Mullins SR, Franco-Barraza J, Valianou M, Cukierman E, Cheng JD. FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells. BMC Cancer. 2011;11:245.
Lo A, Wang LC, Scholler J, Monslow J, Avery D, Newick K, et al. Tumor-promoting desmoplasia is disrupted by depleting FAP-expressing stromal cells. Cancer Res. 2015;75(14):2800–10. doi:10.1158/0008-5472.CAN-14-3041.
Bhati R, Patterson C, Livasy CA, Fan C, Ketelsen D, Hu Z, et al. Molecular characterization of human breast tumor vascular cells. Am J Pathol. 2008;172(5):1381–90. doi:10.2353/ajpath.2008.070988.
Aimes RT, Zijlstra A, Hooper JD, Ogbourne SM, Sit ML, Fuchs S, et al. Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis. Thromb Haemost. 2003;89(3):561–72.
Tchou J, Zhang PJ, Bi Y, Satija C, Marjumdar R, Stephen TL, et al. Fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer. Hum Pathol. 2013;44(11):2549–57. doi:10.1016/j.humpath.2013.06.016.
Arnold JN, Magiera L, Kraman M, Fearon DT. Tumoral immune suppression by macrophages expressing fibroblast activation protein-alpha and heme oxygenase-1. Cancer immunology research. 2014;2(2):121–6. doi:10.1158/2326-6066.CIR-13-0150.
Hamson EJ, Keane FM, Tholen S, Schilling O, Gorrell MD. Understanding fibroblast activation protein (FAP): substrates, activities, expression and targeting for cancer therapy. PROTEOMICS-Clinical Applications. 2014;8(5–6):454–63. doi:10.1002/prca.201300095.
Huang Y, Simms AE, Mazur A, Wang S, Leon NR, Jones B, et al. Fibroblast activation protein-alpha promotes tumor growth and invasion of breast cancer cells through non-enzymatic functions. Clin Exp Metastasis. 2011;28(6):567–79.
Huang Y, Wang S, Kelly T. Seprase promotes rapid tumor growth and increased microvessel density in a mouse model of human breast cancer. Cancer Res. 2004;64(8):2712–6.
Yang W, Han W, Ye S, Liu D, Wu J, Liu H, et al. Fibroblast activation protein-alpha promotes ovarian cancer cell proliferation and invasion via extracellular and intracellular signaling mechanisms. Exp Mol Pathol. 2013;95(1):105–10. doi:10.1016/j.yexmp.2013.06.007.
Wang H, Wu Q, Liu Z, Luo X, Fan Y, Liu Y, et al. Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-ERK signaling in oral squamous cell carcinoma. Cell Death Dis. 2014;5:e1155. doi:10.1038/cddis.2014.122.
Liu F, Qi L, Liu B, Liu J, Zhang H, Che D, et al. Fibroblast activation protein overexpression and clinical implications in solid tumors: a meta-analysis. PLoS One. 2015;10(3):e0116683. doi:10.1371/journal.pone.0116683.
Bellail AC, Hunter SB, Brat DJ, Tan C, Van Meir EG. Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. Int J Biochem Cell Biol. 2004;36(6):1046–69.
Clavreul A, Guette C, Faguer R, Tetaud C, Boissard A, Lemaire L, et al. Glioblastoma-associated stromal cells (GASCs) from histologically normal surgical margins have a myofibroblast phenotype and angiogenic properties. J Pathol. 2014;233(1):74–88. doi:10.1002/path.4332.
Trylcova J, Busek P, Smetana Jr K, Balaziova E, Dvorankova B, Mifkova A, et al. Effect of cancer-associated fibroblasts on the migration of glioma cells in vitro. Tumour Biol. 2015;36(8):5873–9. doi:10.1007/s13277-015-3259-8.
Stremenova J, Krepela E, Mares V, Trim J, Dbaly V, Marek J, et al. Expression and enzymatic activity of dipeptidyl peptidase-IV in human astrocytic tumours are associated with tumour grade. Int J Oncol. 2007;31(4):785–92.
Mentlein R, Hattermann K, Hemion C, Jungbluth AA, Held-Feindt J. Expression and role of the cell surface protease seprase/fibroblast activation protein-alpha (FAP-alpha) in astroglial tumors. Biol Chem. 2011;392(3):199–207. doi:10.1515/BC.2010.119.
Mikheeva SA, Mikheev AM, Petit A, Beyer R, RG O, Khorasani L, et al. TWIST1 promotes invasion through mesenchymal change in human glioblastoma. Mol Cancer. 2010;9:194.
Busek P, Stremenova J, Sromova L, Hilser M, Balaziova E, Kosek D, et al. Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity. Int J Biochem Cell Biol. 2012;44(5):738–47. doi:10.1016/j.biocel.2012.01.011.
Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, Diserens AC, et al. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol. 1999;9(3):469–79.
Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell. 2009;4(6):568–80.
Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 2010;17(1):98–110.
da Huang W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57.
McFaden D. Conditional logit analysis of qualitative choice behavior. In: Zarembka P, editor. Frontiers in econometrics. New York: Academic Press; 1973. p. 105–42.
Gravendeel LA, Kouwenhoven MC, Gevaert O, de Rooi JJ, Stubbs AP, Duijm JE, et al. Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology. Cancer Res. 2009;69(23):9065–72. doi:10.1158/0008-5472.CAN-09-2307.
Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol. 2007;170(5):1445–53.
Busek P, Stremenova J, Sedo A. Dipeptidyl peptidase-IV enzymatic activity bearing molecules in human brain tumors—good or evil? Front Biosci. 2008;13:2319–26.
Annovazzi L, Mellai M, Caldera V, Valente G, Schiffer D. SOX2 expression and amplification in gliomas and glioma cell lines. Cancer genomics & proteomics. 2011;8(3):139–47.
Karsy M, Gelbman M, Shah P, Balumbu O, Moy F, Arslan E. Established and emerging variants of glioblastoma multiforme: review of morphological and molecular features. Folia Neuropathol. 2012;50(4):301–21.
Phillips HS, Kharbanda S, Chen R, Forrest WF, Soriano RH, Wu TD, et al. Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis. Cancer Cell. 2006;9(3):157–73.
Appaix F, Nissou MF, van der Sanden B, Dreyfus M, Berger F, Issartel JP, et al. Brain mesenchymal stem cells: the other stem cells of the brain? World journal of stem cells. 2014;6(2):134–43. doi:10.4252/wjsc.v6.i2.134.
Reilkoff RA, Bucala R, Herzog EL. Fibrocytes: emerging effector cells in chronic inflammation. Nat Rev Immunol. 2011;11(6):427–35. doi:10.1038/nri2990.
Kraman M, Bambrough PJ, Arnold JN, Roberts EW, Magiera L, Jones JO, et al. Suppression of antitumor immunity by stromal cells expressing fibroblast activation protein-alpha. Science. 2010;330(6005):827–30.
Lee KN, Jackson KW, Christiansen VJ, Dolence EK, McKee PA. Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor alpha2-antiplasmin. J Thromb Haemost. 2011;9(5):987–96.
Brat DJ, Van Meir EG. Vaso-occlusive and prothrombotic mechanisms associated with tumor hypoxia, necrosis, and accelerated growth in glioblastoma. Lab Investig. 2004;84(4):397–405. doi:10.1038/labinvest.3700070.
Tremblay P, Beaudet MJ, Tremblay E, Rueda N, Thomas T, Vallieres L. Matrix metalloproteinase 2 attenuates brain tumour growth, while promoting macrophage recruitment and vascular repair. J Pathol. 2011;224(2):222–33. doi:10.1002/path.2854.
Paulus W, Huettner C, Tonn JC. Collagens, integrins and the mesenchymal drift in glioblastomas: a comparison of biopsy specimens, spheroid and early monolayer cultures. Int J Cancer. 1994;58(6):841–6.
Santos AM, Jung J, Aziz N, Kissil JL, Pure E. Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice. J Clin Invest. 2009;119(12):3613–25.
Cai F, Li Z, Wang C, Xian S, Xu G, Peng F, et al. Short hairpin RNA targeting of fibroblast activation protein inhibits tumor growth and improves the tumor microenvironment in a mouse model. BMB Rep. 2013;46(5):252–7.
Narra K, Mullins SR, Lee HO, Strzemkowski-Brun B, Magalong K, Christiansen VJ, et al. Phase II trial of single agent Val-boroPro (Talabostat) inhibiting fibroblast activation protein in patients with metastatic colorectal cancer. Cancer Biol Ther. 2007;6(11):1691–9.
Hofheinz RD, al-Batran SE, Hartmann F, Hartung G, Jager D, Renner C, et al. Stromal antigen targeting by a humanised monoclonal antibody: an early phase II trial of sibrotuzumab in patients with metastatic colorectal cancer. Onkologie. 2003;26(1):44–8.
Fang J, Xiao L, Joo KI, Liu Y, Zhang C, Liu S, et al. A potent immunotoxin targeting fibroblast activation protein for treatment of breast cancer in mice. Int J Cancer. 2015. doi:10.1002/ijc.29831.
Fischer E, Chaitanya K, Wuest T, Wadle A, Scott, AM, van den Broek M et al. Radioimmunotherapy of fibroblast activation protein positive tumors by rapidly internalizing antibodies. Clin Cancer Res. 2012.
Akinboye ES, Brennen WN, Rosen DM, Bakare O, Denmeade SR. Iterative design of emetine-based prodrug targeting fibroblast activation protein (FAP) and dipeptidyl peptidase IV DPPIV using a tandem enzymatic activation strategy. Prostate. 2016. doi:10.1002/pros.23162.
Wang LC, Lo A, Scholler J, Sun J, Majumdar RS, Kapoor V, et al. Targeting fibroblast activation protein in tumor stroma with chimeric antigen receptor T cells can inhibit tumor growth and augment host immunity without severe toxicity. Cancer immunology research. 2014;2(2):154–66. doi:10.1158/2326-6066.CIR-13-0027.
Lee J, Fassnacht M, Nair S, Boczkowski D, Gilboa E. Tumor immunotherapy targeting fibroblast activation protein, a product expressed in tumor-associated fibroblasts. Cancer Res. 2005;65(23):11156–63.
Acknowledgments
The work was supported by the grant from the Ministry of Health of the Czech Republic, Internal grant agency IGA 12237-5/2011. The authors thank Igor Romanko for assistance with CXCR4 immunohistochemistry and Zdislava Vanickova for the help with RNA isolation. The excellent technical assistance of Kvetoslava Vlasicova and Karin Roubickova is acknowledged. Special thanks go to Martin Prevorovsky for the help with the TCGA expression data.
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This study was funded by the Ministry of Health of the Czech Republic, Internal grant agency IGA 12237–5/2011.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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Supplementary Figure S1
Characteristics of the glioma stem-like cell cultures a) GSC57 and b) GSC48. Expression of Sox2 (Sox2-PerCP, RD Systems) and CD133 (CD133-APC, Miltenyi Biotech) was determined by flow cytometry, detection of the differentiation markers glial fibrillary acidic protein (GFAP) and beta III tubulin was performed in cells grown on laminin in defined stem-cell serum free media (undifferentiated) or in the presence of 10 % fetal calf serum (differentiated). (PPTX 785 kb)
Supplementary Figure S2
Fibroblast activation protein (FAP) and survival in gliomas. The Kaplan-Meier survival plots for glioma patients and the proportion of individual tumor grades in the subgroups based on FAP expression in studies comprising grade I-IV tumors. A) Greavendeel [9] dataset, b) Data from REMBRANDT. Patients were divided into groups based on FAP mRNA expression: ∆ low expression = 1st quartile, □ medium expression = 2nd + 3rd quartile, ○ high expression = 4th quartile. + = censored data. Log rank test p < 0.05 for all intergroup comparisons in A), and p < 0.05 for downregulated vs. intermediate and downregulated vs. upregulated in B). (PPTX 146 kb)
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Busek, P., Balaziova, E., Matrasova, I. et al. Fibroblast activation protein alpha is expressed by transformed and stromal cells and is associated with mesenchymal features in glioblastoma. Tumor Biol. 37, 13961–13971 (2016). https://doi.org/10.1007/s13277-016-5274-9
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DOI: https://doi.org/10.1007/s13277-016-5274-9