Tumor Biology

, Volume 37, Issue 7, pp 9045–9057 | Cite as

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures

  • Elizabete Bagordakis
  • Iris Sawazaki-Calone
  • Carolina Carneiro Soares Macedo
  • Carolina M. Carnielli
  • Carine Ervolino de Oliveira
  • Priscila Campioni Rodrigues
  • Ana Lucia C. A. Rangel
  • Jean Nunes dos Santos
  • Juha Risteli
  • Edgard Graner
  • Tuula Salo
  • Adriana Franco Paes Leme
  • Ricardo D. Coletta
Original Article


An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-β1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.


Cancer-associated fibroblasts Secretome Extracellular matrix Type I collagen FNDC1 SERPINE1 STC2 



This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP, São Paulo, Brazil; Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Brasília, Brazil, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, Brasília, Brazil (AUXPE-PVES-570/2013).

Compliance with ethical standard

Conflicts of interest


Supplementary material

13277_2015_4629_Fig9_ESM.gif (46 kb)
Supplementary Figure 1

TGF-β1 induces transdifferentiation of NOFs to CAFs. NOFs were cultured with 10 ng/ml of TGF-β1 in culture media containing 0.1% of calf serum for 2 days. Following treatment, cells were collected and subjected RNA purification and quantitative PCR analysis. The levels of the CAF marker α-SMA were markedly increased after incubation with TGF-β1. (GIF 45 kb)

13277_2015_4629_MOESM1_ESM.tif (15.2 mb)
(TIF 15526 kb)
13277_2015_4629_MOESM2_ESM.docx (17 kb)
Supplementary Table 1 Primers used in the quantitative PCR. (DOCX 17 kb)
13277_2015_4629_MOESM3_ESM.docx (84 kb)
Supplementary Table 2 Proteins identified in NOF-1 and CAF-1 cell lines by LC-MS/MS. (DOCX 84 kb)
13277_2015_4629_MOESM4_ESM.docx (59 kb)
Supplementary Table 3 Overrepresented GO terms for the dataset of differentially expressed proteins between CAF-1 and NOF-1 cell lines. (DOCX 58 kb)
13277_2015_4629_MOESM5_ESM.docx (71 kb)
ESM 1 (DOCX 71 kb)


  1. 1.
    Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–86.CrossRefPubMedGoogle Scholar
  2. 2.
    Warnakulasuriya S. Living with oral cancer: epidemiology with particular reference to prevalence and life-style changes that influence survival. Oral Oncol. 2010;46:407–10.CrossRefPubMedGoogle Scholar
  3. 3.
    Shah FD, Begum R, Vajaria BN, Patel KR, Patel JB, Shukla SN, et al. A review on salivary genomics and proteomics biomarkers in oral cancer. Indian J Clin Biochem. 2011;26:326–34.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell. 2012;21:309–22.CrossRefPubMedGoogle Scholar
  5. 5.
    Koontongkaew S. The tumor microenvironment contribution to development, growth, invasion and metastasis of head and neck squamous cell carcinomas. J Cancer. 2013;4:66–83.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Curry JM, Sprandio J, Cognetti D, Luginbuhl A, Bar-ad V, Pribitkin E, et al. Tumor microenvironment in head and neck squamous cell carcinoma. Semin Oncol. 2014;41:217–34.CrossRefPubMedGoogle Scholar
  7. 7.
    Gandellini P, Andriani F, Merlino G, D’Aiuto F, Roz L, Callari M. Complexity in the tumour microenvironment: cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types. Semin Cancer Biol. 2015.Google Scholar
  8. 8.
    Sobral LM, Bufalino A, Lopes MA, Graner E, Salo T, Coletta RD. Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of activin A. Oral Oncol. 2011;47:840–6.CrossRefPubMedGoogle Scholar
  9. 9.
    Hinsley EE, Kumar S, Hunter KD, Whawell SA, Lambert DW. Endothelin-1 stimulates oral fibroblasts to promote oral cancer invasion. Life Sci. 2012;91:557–61.CrossRefPubMedGoogle Scholar
  10. 10.
    Tommelein J, Verset L, Boterberg T, Demetter P, Bracke M, De Wever O. Cancer-associated fibroblasts connect metastasis-promoting communication in colorectal cancer. Front Oncol. 2015;5:63.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kellermann MG, Sobral LM, da Silva SD, Zecchin KG, Graner E, Lopes MA, et al. Myofibroblasts in the stroma of oral squamous cell carcinoma are associated with poor prognosis. Histopathology. 2007;51:849–53.CrossRefPubMedGoogle Scholar
  12. 12.
    Kawashiri S, Tanaka A, Noguchi N, Hase T, Nakaya H, Ohara T, et al. Significance of stromal desmoplasia and myofibroblast appearance at the invasive front in squamous cell carcinoma of the oral cavity. Head Neck. 2009;31:1346–53.CrossRefPubMedGoogle Scholar
  13. 13.
    Vered M, Dobriyan A, Dayan D, Yahalom R, Talmi YP, Bedrin L, et al. Tumor-host histopathologic variables, stromal myofibroblasts and risk score, are significantly associated with recurrent disease in tongue cancer. Cancer Sci. 2010;101:274–80.CrossRefPubMedGoogle Scholar
  14. 14.
    Bello IO, Vered M, Dayan D, Dobriyan A, Yahalom R, Alanen K, et al. Cancer-associated fibroblasts, a parameter of the tumor microenvironment, overcomes carcinoma-associated parameters in the prognosis of patients with mobile tongue cancer. Oral Oncol. 2011;47:33–8.CrossRefPubMedGoogle Scholar
  15. 15.
    Marsh D, Suchak K, Moutasim KA, Vallath S, Hopper C, Jerjes W, et al. Stromal features are predictive of disease mortality in oral cancer patients. J Pathol. 2011;223:470–81.CrossRefPubMedGoogle Scholar
  16. 16.
    Fujii N, Shomori K, Shiomi T, Nakabayashi M, Takeda C, Ryoke K, et al. Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance. J Oral Pathol Med. 2012;41:444–51.CrossRefPubMedGoogle Scholar
  17. 17.
    Ding L, Zhang Z, Shang D, Cheng J, Yuan H, Wu Y, et al. α-Smooth muscle actin-positive myofibroblasts, in association with epithelial-mesenchymal transition and lymphogenesis, is a critical prognostic parameter in patients with oral tongue squamous cell carcinoma. J Oral Pathol Med. 2014;43:335–43.CrossRefPubMedGoogle Scholar
  18. 18.
    De Boeck A, Hendrix A, Maynard D, Van Bockstal M, Daniëls A, Pauwels P, et al. Differential secretome analysis of cancer-associated fibroblasts and bone marrow-derived precursors to identify microenvironmental regulators of colon cancer progression. Proteomics. 2013;13:379–88.CrossRefPubMedGoogle Scholar
  19. 19.
    Chen SX, Xu XE, Wang XQ, Cui SJ, Xu LL, Jiang YH, et al. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation. J Proteomics. 2014;110:155–71.CrossRefPubMedGoogle Scholar
  20. 20.
    Rasanen K, Sriswasdi S, Valiga A, Tang HY, Zhang G, Perego M, et al. Comparative secretome analysis of epithelial and mesenchymal subpopulations of head and neck squamous cell carcinoma identifies S100A4 as a potential therapeutic target. Mol Cell Proteomics. 2013;12:3778–92.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ge S, Mao Y, Yi Y, Xie D, Chen Z, Xiao Z. Comparative proteomic analysis of secreted proteins from nasopharyngeal carcinoma-associated stromal fibroblasts and normal fibroblasts. Exp Ther Med. 2012;3:857–60.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Salo S, Bitu C, Merkku K, Nyberg P, Bello IO, Vuoristo J, et al. Human bone marrow mesenchymal stem cells induce collagen production and tongue cancer invasion. PLoS One. 2013;8:e77692.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Sobral LM, Zecchin KG, de Nascimento Aquino S, Lopes MA, Graner E, Coletta RD. Isolation and characterization of myofibroblast cell lines from oral squamous cell carcinoma. Oncol Rep. 2011;25:1013–20.PubMedGoogle Scholar
  24. 24.
    Sobral LM, Montan PF, Zecchin KG, Martelli-Junior H, Vargas PA, Graner E, et al. Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor. J Periodontol. 2011;82:642–51.CrossRefPubMedGoogle Scholar
  25. 25.
    Sawazaki-Calone I, Rangel A, Bueno AG, Morais CF, Nagai HM, Kunz RP, et al. The prognostic value of histopathological grading systems in oral squamous cell carcinomas. Oral Dis. 2015;21:755–61.CrossRefPubMedGoogle Scholar
  26. 26.
    Risteli L, Risteli J. Biochemical markers of bone metabolism. Ann Med. 1993;25:385–93.CrossRefPubMedGoogle Scholar
  27. 27.
    Kellermann MG, Sobral LM, da Silva SD, Zecchin KG, Graner E, Lopes MA, et al. Mutual paracrine effects of oral squamous cell carcinoma cells and normal oral fibroblasts: induction of fibroblast to myofibroblast transdifferentiation and modulation of tumor cell proliferation. Oral Oncol. 2008;44:509–17.CrossRefPubMedGoogle Scholar
  28. 28.
    Han Y, Zhang Y, Jia T, Sun Y. Molecular mechanism underlying the tumor-promoting functions of carcinoma-associated fibroblasts. Tumour Biol. 2015;36:1385–94.CrossRefPubMedGoogle Scholar
  29. 29.
    Erickson HP. Irisin and FNDC5 in retrospect: an exercise hormone or a transmembrane receptor? Adipocyte. 2013;2:289–93.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Fain JN, Company JM, Booth FW, Laughlin MH, Padilla J, Jenkins NT, et al. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs. Metabolism. 2013;62:1503–11.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Sung CO, Kim SC, Karnan S, Karube K, Shin HJ, Nam DH, et al. Genomic profiling combined with gene expression profiling in primary central nervous system lymphoma. Blood. 2011;117:1291–300.CrossRefPubMedGoogle Scholar
  32. 32.
    Itoh T, Hayashi Y, Kanamaru T, Morita Y, Suzuki S, Wang W, et al. Clinical significance of urokinase-type plasminogen activator activity in hepatocellular carcinoma. J Gastroenterol Hepatol. 2000;15:422–30.CrossRefPubMedGoogle Scholar
  33. 33.
    Zheng Q, Tang ZY, Xue Q, Shi DR, Song HY, Tang HB. Invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor. J Cancer Res Clin Oncol. 2000;126:641–6.CrossRefPubMedGoogle Scholar
  34. 34.
    Schmitt M, Harbeck N, Brünner N, Jänicke F, Meisner C, Mühlenweg B, et al. Cancer therapy trials employing level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1. Expert Rev Mol Diagn. 2011;11:617–34.CrossRefPubMedGoogle Scholar
  35. 35.
    Duffy MJ, McGowan PM, Harbeck N, Thomssen C, Schmitt M. uPA and PAI-1 as biomarkers in breast cancer: validated for clinical use in level-of-evidence-1 studies. Breast Cancer Res. 2014;16:428.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Look MP, van Putten WL, Duffy MJ, Harbeck N, Christensen IJ, Thomssen C, et al. Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients. J Natl Cancer Inst. 2002;94:116–28.CrossRefPubMedGoogle Scholar
  37. 37.
    Harris L, Fritsche H, Mennel R, Norton L, Ravdin P, Taube S, et al. American society of clinical oncology update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol. 2007;25:5287–312.CrossRefPubMedGoogle Scholar
  38. 38.
    Roca C, Primo L, Valdembri D, Cividalli A, Declerck P, Carmeliet P, et al. Hyperthermia inhibits angiogenesis by a plasminogen activator inhibitor 1-dependent mechanism. Cancer Res. 2003;63:1500–7.PubMedGoogle Scholar
  39. 39.
    Bajou K, Masson V, Gerard RD, Schmitt PM, Albert V, Praus M, et al. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies. J Cell Biol. 2001;152:777–84.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Czekay RP, Loskutoff DJ. Plasminogen activator inhibitors regulate cell adhesion through a uPAR-dependent mechanism. J Cell Physiol. 2009;220:655–63.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Dhanda J, Triantafyllou A, Liloglou T, Kalirai H, Lloyd B, Hanlon R, et al. SERPINE1 and SMA expression at the invasive front predict extracapsular spread and survival in oral squamous cell carcinoma. Br J Cancer. 2014;111:2114–21.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Pedroza M, Le TT, Lewis K, Karmouty-Quintana H, To S, George AT, et al. STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation. FASEB J. 2015 Aug 31.Google Scholar
  43. 43.
    Na SS, Aldonza MB, Sung HJ, Kim YI, Son YS, Cho S, et al. Stanniocalcin-2 (STC2): a potential lung cancer biomarker promotes lung cancer metastasis and progression. Biochim Biophys Acta. 1854;2015:668–76.Google Scholar
  44. 44.
    Wang Y, Gao Y, Cheng H, Yang G, Tan W. Stanniocalcin 2 promotes cell proliferation and cisplatin resistance in cervical cancer. Biochem Biophys Res Commun. 2015;466:362–8.CrossRefPubMedGoogle Scholar
  45. 45.
    Hou J, Wang Z, Xu H, Yang L, Yu X, Yang Z, et al. Stanniocalicin 2 suppresses breast cancer cell migration and invasion via the PKC/claudin-1-mediated signaling. PLoS One. 2015;10:e0122179.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Bouras T, Southey MC, Chang AC, Reddel RR, Willhite D, Glynne R, et al. Stanniocalcin 2 is an estrogen-responsive gene coexpressed with the estrogen receptor in human breast cancer. Cancer Res. 2002;62:1289–95.PubMedGoogle Scholar
  47. 47.
    Ieta K, Tanaka F, Yokobori T, Kita Y, Haraguchi N, Mimori K, et al. Clinicopathological significance of stanniocalcin 2 gene expression in colorectal cancer. Int J Cancer. 2009;125:926–31.CrossRefPubMedGoogle Scholar
  48. 48.
    Tamura K, Furihata M, Chung SY, Uemura M, Yoshioka H, Iiyama T, et al. Stanniocalcin 2 overexpression in castration-resistant prostate cancer and aggressive prostate cancer. Cancer Sci. 2009;100:914–9.CrossRefPubMedGoogle Scholar
  49. 49.
    Law AY, Lai KP, Ip CK, Wong AS, Wagner GF, Wong CK. Epigenetic and HIF-1 regulation of stanniocalcin-2 expression in human cancer cells. Exp Cell Res. 2008;314:1823–30.CrossRefPubMedGoogle Scholar
  50. 50.
    Law AY, Wong CK. Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia. Exp Cell Res. 2010;316:466–76.CrossRefPubMedGoogle Scholar
  51. 51.
    Chang AC, Jellinek DA, Reddel RR. Mammalian stanniocalcins and cancer. Endocrinol Relat Cancer. 2003;10:359–73.CrossRefGoogle Scholar
  52. 52.
    Van Bockstal M, Lambein K, Van Gele M, De Vlieghere E, Limame R, Braems G, et al. Differential regulation of extracellular matrix protein expression in carcinoma-associated fibroblasts by TGF-β1 regulates cancer cell spreading but not adhesion. Oncoscience. 2014;1:634–48.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Kenny HA, Krausz T, Yamada SD, Lengyel E. Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum. Int J Cancer. 2007;121:1463–72.CrossRefPubMedGoogle Scholar
  54. 54.
    Tang D, Gao J, Wang S, Ye N, Chong Y, Huang Y, et al. Cancer-associated fibroblasts promote angiogenesis in gastric cancer through galectin-1 expression. Tumour Biol. 2015. doi: 10.1007/s13277-015-3942-9.Google Scholar
  55. 55.
    Liu J, Liao S, Diop-Frimpong B, Chen W, Goel S, Naxerova K, et al. TGF-β blockade improves the distribution and efficacy of therapeutics in breast carcinoma by normalizing the tumor stroma. Proc Natl Acad Sci U S A. 2012;109:16618–23.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Elizabete Bagordakis
    • 1
  • Iris Sawazaki-Calone
    • 2
  • Carolina Carneiro Soares Macedo
    • 1
  • Carolina M. Carnielli
    • 3
  • Carine Ervolino de Oliveira
    • 1
  • Priscila Campioni Rodrigues
    • 1
  • Ana Lucia C. A. Rangel
    • 2
  • Jean Nunes dos Santos
    • 4
  • Juha Risteli
    • 5
    • 6
  • Edgard Graner
    • 1
  • Tuula Salo
    • 5
    • 6
    • 7
    • 8
  • Adriana Franco Paes Leme
    • 3
  • Ricardo D. Coletta
    • 1
  1. 1.Department of Oral Diagnosis, School of DentistryState University of CampinasPiracicabaBrazil
  2. 2.Oral Pathology and Oral Medicine, Dentistry SchoolWestern Paraná State UniversityCascavelBrazil
  3. 3.Brazilian Biociences National Laboratory-CNPEMCampinasBrazil
  4. 4.Laboratory of Surgical Pathology, Dental SchoolFederal University of Bahia-UFBASalvadorBrazil
  5. 5.Cancer and Translational Medicine Research UnitUniversity of OuluOuluFinland
  6. 6.Medical Research CenterOulu University HospitalOuluFinland
  7. 7.Oral and Maxillofacial Diseases UnitUniversity of HelsinkiHelsinkiFinland
  8. 8.Helsinki University HospitalHelsinkiFinland

Personalised recommendations