Tumor Biology

, Volume 37, Issue 10, pp 13961–13971 | Cite as

Fibroblast activation protein alpha is expressed by transformed and stromal cells and is associated with mesenchymal features in glioblastoma

  • Petr Busek
  • Eva Balaziova
  • Ivana Matrasova
  • Marek Hilser
  • Robert Tomas
  • Martin Syrucek
  • Zuzana Zemanova
  • Evzen Krepela
  • Jaromir Belacek
  • Aleksi Sedo
Original Article


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.


Fibroblast activation protein α Seprase Glioma Serine protease Stromal cells 

Supplementary material

13277_2016_5274_MOESM1_ESM.rtf (151 kb)
ESM 1(RTF 151 kb)
13277_2016_5274_MOESM2_ESM.pptx (786 kb)
Supplementary Figure S1Characteristics 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)
13277_2016_5274_MOESM3_ESM.pptx (146 kb)
Supplementary Figure S2Fibroblast 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)
13277_2016_5274_MOESM4_ESM.xlsx (37 kb)
Table S1(XLSX 36 kb)


  1. 1.
    Charles NA, Holland EC, Gilbertson R, Glass R, Kettenmann H. The brain tumor microenvironment. Glia. 2012;59(8):1169–80.CrossRefGoogle Scholar
  2. 2.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    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. CrossRefPubMedGoogle Scholar
  4. 4.
    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. CrossRefPubMedGoogle Scholar
  5. 5.
    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.CrossRefPubMedGoogle Scholar
  6. 6.
    Jacob M, Chang L, Pure E. Fibroblast activation protein in remodeling tissues. Current molecular medicine. 2012;12(10):1220–43.CrossRefPubMedGoogle Scholar
  7. 7.
    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. CrossRefPubMedGoogle Scholar
  8. 8.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    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.PubMedGoogle Scholar
  12. 12.
    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. CrossRefPubMedGoogle Scholar
  13. 13.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    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. CrossRefPubMedGoogle Scholar
  15. 15.
    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.CrossRefPubMedGoogle Scholar
  16. 16.
    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.CrossRefPubMedGoogle Scholar
  17. 17.
    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. CrossRefPubMedGoogle Scholar
  18. 18.
    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. CrossRefPubMedGoogle Scholar
  19. 19.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    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.CrossRefPubMedGoogle Scholar
  21. 21.
    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. CrossRefPubMedGoogle Scholar
  22. 22.
    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. CrossRefPubMedGoogle Scholar
  23. 23.
    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.PubMedGoogle Scholar
  24. 24.
    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. CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    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. CrossRefPubMedGoogle Scholar
  27. 27.
    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.CrossRefPubMedGoogle Scholar
  28. 28.
    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.CrossRefPubMedGoogle Scholar
  29. 29.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    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.CrossRefGoogle Scholar
  31. 31.
    McFaden D. Conditional logit analysis of qualitative choice behavior. In: Zarembka P, editor. Frontiers in econometrics. New York: Academic Press; 1973. p. 105–42.Google Scholar
  32. 32.
    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. CrossRefPubMedGoogle Scholar
  33. 33.
    Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol. 2007;170(5):1445–53.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    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.CrossRefPubMedGoogle Scholar
  35. 35.
    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.Google Scholar
  36. 36.
    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.CrossRefPubMedGoogle Scholar
  37. 37.
    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.CrossRefPubMedGoogle Scholar
  38. 38.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    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. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    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.CrossRefPubMedGoogle Scholar
  41. 41.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    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.CrossRefPubMedGoogle Scholar
  43. 43.
    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. CrossRefPubMedGoogle Scholar
  44. 44.
    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.CrossRefPubMedGoogle Scholar
  45. 45.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    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.CrossRefPubMedGoogle Scholar
  48. 48.
    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.PubMedGoogle Scholar
  49. 49.
    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. Google Scholar
  50. 50.
    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.Google Scholar
  51. 51.
    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. PubMedGoogle Scholar
  52. 52.
    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. CrossRefPubMedGoogle Scholar
  53. 53.
    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.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Petr Busek
    • 1
  • Eva Balaziova
    • 1
  • Ivana Matrasova
    • 1
  • Marek Hilser
    • 1
  • Robert Tomas
    • 2
  • Martin Syrucek
    • 3
  • Zuzana Zemanova
    • 4
  • Evzen Krepela
    • 1
  • Jaromir Belacek
    • 5
  • Aleksi Sedo
    • 1
  1. 1.Institute of Biochemistry and Experimental Oncology, First Faculty of MedicineCharles University in PraguePrague 2Czech Republic
  2. 2.Department of NeurosurgeryNa Homolce HospitalPrague 5Czech Republic
  3. 3.Department of PathologyNa Homolce HospitalPrague 5Czech Republic
  4. 4.Institute of Clinical Biochemistry and Laboratory Diagnostics of the First Faculty of MedicineCharles University in Prague and General University Hospital in PraguePrague 2Czech Republic
  5. 5.Institute of Biophysics and Bioinformatics, First Faculty of MedicineCharles University in PraguePrague 2Czech Republic

Personalised recommendations