Skip to main content

Advertisement

SpringerLink
Log in

The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts

  • Original Article
  • Published:
Cancer Microenvironment

Abstract

Within the tumour stroma, a heterogeneous population of cell types reciprocally regulates cell proliferation, which considerably affects the progression of the disease. In this study, using tumour conditioned medium (TCM) derived from breast tumour cell lines – MCF7 and MDA MB 231, we have demonstrated the differentiation of adipose-derived mesenchymal stem cells (ADSCs) into tumour-associated fibroblasts (TAFs). Since the Wnt signalling pathway is a key signalling pathway driving breast tumour growth, the effect of the Wnt antagonist secreted frizzled-related protein 4 (sFRP4) was also examined. The response of ADSCs to TCM and sFRP4 treatments was determined by using cell viability assay to determine the changes in ADSC viability, immunofluorescence for mesenchymal markers, glucose uptake assay, and glycolysis stress test using the Seahorse Extracellular Flux analyser to determine the glycolytic activity of ADSCs. ADSCs have been shown to acquire a hyper-proliferative state, significantly increasing their number upon short-term and long-term exposure to TCM. Changes have also been observed in the expression of key mesenchymal markers as well as in the metabolic state of ADSCs. SFRP4 significantly inhibited the differentiation of ADSCs into TAFs by reducing cell growth as well as mesenchymal marker expression (cell line-dependent). However, sFRP4 did not induce further significant changes to the altered metabolic phenotype of ADSCs following TCM exposure. Altogether, this study suggests that the breast tumour milieu may transform ADSCs into a tumour-supportive phenotype, which can be altered by Wnt antagonism, but is independent of metabolic changes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ADSCs:

Adipose-derived mesenchymal stem cells

TAFs:

Tumour-associated fibroblasts

sFRP4:

Secreted frizzled-related protein 4

TCM:

Tumour conditioned medium

TGF-β:

Transforming growth factor-beta

LPA:

Lysophosphatidic acid

ECAR:

Extracellular acidification rates

OCR:

Oxygen consumption rates

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

References

  1. Junttila MR, de Sauvage FJ (2013) Influence of tumour micro-environment heterogeneity on therapeutic response. Nature 501(7467):346–354. https://doi.org/10.1038/nature12626

    Article  PubMed  CAS  Google Scholar 

  2. Jotzu C, Alt E, Welte G, Li J, Hennessy BT, Devarajan E, Krishnappa S, Pinilla S, Droll L, Song YH (2011) Adipose tissue derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor derived factors. Cell Oncol 34(1):55–67

    Article  Google Scholar 

  3. Cho JA, Park H, Lim EH, Lee KW (2012) Exosomes from breast cancer cells can convert adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells Int J Oncol:40

  4. Cho JA, Park H, Lim EH, Kim KH, Choi JS, Lee JH (2011) Exosomes from ovarian cancer cells induce adipose tissue-derived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts. Gynecol Oncol 123:379–386. https://doi.org/10.1016/j.ygyno.2011.08.005

    Article  PubMed  CAS  Google Scholar 

  5. Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B, Andreeff M, Marini F (2009) Mesenchymal stem cell transition to tumor-associated fibroblasts contributes to fibrovascular network expansion and tumor progression. PLoS One 4(4):7

    Article  CAS  Google Scholar 

  6. Mishra P, Humeniuk R, Medina D, Alexe G, Mesirov J, Ganesan S, Glod J, Banerjee D (2008) Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res 11:4331–4339

    Article  CAS  Google Scholar 

  7. Paunescu V, Bojin FM, Tatu CA, Gavriliuc OI, Rosca A, Gruia AT (2011) Tumour-associated fibroblasts and mesenchymal stem cells: more similarities than differences. J Cell Mol Med 15:635–646. https://doi.org/10.1111/j.1582-4934.2010.01044.x

    Article  PubMed  CAS  Google Scholar 

  8. Matushansky I, Hernando E, Socci ND, Mills JE, Matos TA, Edgar MA, Singer S, Maki RG, Cordon-Cardo C (2007) Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway. J Clin Invest 117(11):3248–3257

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Jian H, Shen X, Liu I, Semenov M, He X, Wang XF (2006) Smad3-dependent nuclear translocation of beta-catenin is required for TGF-beta1-induced proliferation of bone marrow-derived adult human mesenchymal stem cells. Genes Dev 20(6):666–674. https://doi.org/10.1101/gad.1388806

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Sato M (2006) Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids. Acta Derm Venereol 86(4):300–307. https://doi.org/10.2340/00015555-0101

    Article  PubMed  CAS  Google Scholar 

  11. DiRenzo DM, Chaudhary MA, Shi X, Franco SR, Zent J, Wang K, Guo L-W, Kent KC (2016) A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation. Cell Signal 28(5):498–505. https://doi.org/10.1016/j.cellsig.2016.02.011

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Zhou S (2011) TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells. J Cell Biochem 112(6):1651–1660. https://doi.org/10.1002/jcb.23079

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Akhmetshina A, Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, Horn A, Kireva T, Beyer C, Zwerina J, Schneider H, Sadowski A, Riener M-O, MacDougald OA, Distler O, Schett G, Distler JHW (2012) Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis. Nat Commun 3:735. https://doi.org/10.1038/ncomms1734

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. Saran U, Arfuso F, Zeps N, Dharmarajan A (2012) Secreted frizzled-related protein 4 expression is positively associated with responsiveness to cisplatin of ovarian cancer cell lines in vitro and with lower tumour grade in mucinous ovarian cancers. BMC Cell Biol 13(1):25

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Muley A, Majumder S, Kolluru GK, Parkinson S, Viola H, Hool L, Arfuso F, Ganss R, Dharmarajan A, Chatterjee S (2010) Secreted frizzled-related protein 4: an angiogenesis inhibitor. Am J Pathol 176(3):1505–1516

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Wolf V, Ke G, Dharmarajan AM, Bielke W, Artuso L, Saurer S, Friis R (1997) DDC-4, an apoptosis-associated gene, is a secreted frizzled relative. FEBS Letters 417 (3):385–389. doi:doi:https://doi.org/10.1016/S0014-5793(97)01324-0

  17. Warrier S, Balu SK, Kumar AP, Millward M, Dharmarajan A (2013) Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), increases chemotherapeutic response of glioma stem-like cells. Oncol Res 21(2):93–102. https://doi.org/10.3727/096504013x13786659070154

    Article  PubMed  Google Scholar 

  18. Perumal V, Pohl S, Keane KN, Arfuso F, Newsholme P, Fox S, Dharmarajan A (2016) Therapeutic approach to target mesothelioma cancer cells using the Wnt antagonist, secreted frizzled-related protein 4: metabolic state of cancer cells. Exp Cell Res 341(2):218–224. https://doi.org/10.1016/j.yexcr.2016.02.008

    Article  PubMed  CAS  Google Scholar 

  19. Anderberg C, Pietras K (2009) On the origin of cancer-associated fibroblasts. Cell Cycle 8(10):1461–1462. https://doi.org/10.4161/cc.8.10.8560

    Article  PubMed  CAS  Google Scholar 

  20. Hanley CJ, Noble F, Ward M, Bullock M, Drifka C, Mellone M, Manousopoulou A, Johnston HE, Hayden A, Thirdborough S, Liu Y, Smith DM, Mellows T, Kao WJ, Garbis SD, Mirnezami A, Underwood TJ, Eliceiri KW, Thomas GJ (2016) A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers. Oncotarget 7 (5):6159–6174. doi:https://doi.org/10.18632/oncotarget.6740

  21. Luo H, Tu G, Liu Z, Liu M (2015) Cancer-associated fibroblasts: a multifaceted driver of breast cancer progression. Cancer Lett 361(2):155–163. https://doi.org/10.1016/j.canlet.2015.02.018

    Article  PubMed  CAS  Google Scholar 

  22. Subramaniam KS, Tham ST, Mohamed Z, Woo YL, Mat Adenan NA, Chung I (2013) Cancer-associated fibroblasts promote proliferation of endometrial cancer cells. PLoS One 8(7):e68923. https://doi.org/10.1371/journal.pone.0068923

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Tyan SW, Kuo WH, Huang CK, Pan CC, Shew JY, Chang KJ, Lee EY, Lee WH (2011) Breast cancer cells induce cancer-associated fibroblasts to secrete hepatocyte growth factor to enhance breast tumorigenesis. PLoS One 6(1):e15313. https://doi.org/10.1371/journal.pone.0015313

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Noma K, Smalley KS, Lioni M, Naomoto Y, Tanaka N, El-Deiry W, King AJ, Nakagawa H, Herlyn M (2008) The essential role of fibroblasts in esophageal squamous cell carcinoma-induced angiogenesis. Gastroenterology 134(7):1981–1993. https://doi.org/10.1053/j.gastro.2008.02.061

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kellermann MG, Sobral LM, da Silva SD, Zecchin KG, Graner E, Lopes MA, Kowalski LP, Coletta RD (2008) 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 44(5):509–517. https://doi.org/10.1016/j.oraloncology.2007.07.001

    Article  PubMed  CAS  Google Scholar 

  26. Jeon ES, Lee IH, Heo SC, Shin SH, Choi YJ, Park JH, Park DY, Kim JH (2010) Mesenchymal stem cells stimulate angiogenesis in a murine xenograft model of A549 human adenocarcinoma through an LPA1 receptor-dependent mechanism. Biochim Biophys Acta 1801(11):1205–1213. https://doi.org/10.1016/j.bbalip.2010.08.003

    Article  PubMed  CAS  Google Scholar 

  27. Gabbiani G, Ryan GB, Majne G (1971) Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27(5):549–550

    Article  PubMed  CAS  Google Scholar 

  28. Katoh K, Kano Y, Masuda M, Onishi H, Fujiwara K (1998) Isolation and contraction of the stress Fiber. Mol Biol Cell 9(7):1919–1938

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Ronnov-Jessen L, Petersen OW (1993) Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Laboratory investigation; a journal of technical methods and pathology 68(6):696–707

    PubMed  CAS  Google Scholar 

  30. Jeon ES, Moon HJ, Lee MJ, Song HY, Kim YM, Cho M, Suh DS, Yoon MS, Chang CL, Jung JS, Kim JH (2008) Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells. Stem Cells 26(3):789–797. https://doi.org/10.1634/stemcells.2007-0742

    Article  PubMed  CAS  Google Scholar 

  31. Mazzocca A, Dituri F, Lupo L, Quaranta M, Antonaci S, Giannelli G (2011) Tumor-secreted lysophostatidic acid accelerates hepatocellular carcinoma progression by promoting differentiation of peritumoral fibroblasts in myofibroblasts. Hepatology 54(3):920–930. https://doi.org/10.1002/hep.24485

    Article  PubMed  CAS  Google Scholar 

  32. Gottschling S, Granzow M, Kuner R, Jauch A, Herpel E, Xu EC (2013) Mesenchymal stem cells in non-small cell lung cancer–different from others? Insights from comparative molecular and functional analyses Lung Cancer 80:19–29. https://doi.org/10.1016/j.lungcan.2012.12.015

    Article  PubMed  Google Scholar 

  33. Xu X, Zhang X, Wang S, Qian H, Zhu W, Cao H (2011) Isolation and comparison of mesenchymal stem-like cells from human gastric cancer and adjacent non-cancerous tissues. J Cancer Res Clin Oncol 137:495–504. https://doi.org/10.1007/s00432-010-0908-6

    Article  PubMed  CAS  Google Scholar 

  34. Ding G, Shao J, Ding Q, Fang Z, Wu Z, Xu J (2012) Comparison of the characteristics of mesenchymal stem cells obtained from prostate tumors and from bone marrow cultured in conditioned medium Exp Ther Med:4

  35. Xu X, Zhang X, Wang S, Qian H, Zhu W, Cao H, Wang M, Chen Y, Xu W (2011) Isolation and comparison of mesenchymal stem-like cells from human gastric cancer and adjacent non-cancerous tissues. J Cancer Res Clin Oncol 137(3):495–504. https://doi.org/10.1007/s00432-010-0908-6

    Article  PubMed  CAS  Google Scholar 

  36. Lacher MD, Siegenthaler A, Jager R, Yan X, Hett S, Xuan L, Saurer S, Lareu RR, Dharmarajan AM, Friis R (2003) Role of DDC-4//sFRP-4, a secreted frizzled-related protein, at the onset of apoptosis in mammary involution. Cell Death Differ 10(5):528–538

    Article  PubMed  CAS  Google Scholar 

  37. Drake JM, Friis RR, Dharmarajan AM (2003) The role of sFRP4, a secreted frizzled-related protein, in ovulation. Apoptosis 8(4):389–397

    Article  PubMed  CAS  Google Scholar 

  38. Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12(9):2730–2741

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Mar PK, Roy P, Yin HL, Cavanagh HD, Jester JV (2001) Stress Fiber Formation is Required for Matrix Reorganization in a Corneal Myofibroblast Cell Line. Experimental Eye Research 72 (4):455–466. doi:doi:https://doi.org/10.1006/exer.2000.0967

  40. Ford CE, Jary E, Ma SS, Nixdorf S, Heinzelmann-Schwarz VA, Ward RL (2013) The Wnt gatekeeper SFRP4 modulates EMT, cell migration and downstream Wnt signalling in serous ovarian cancer cells. PLoS One 8(1):e54362. https://doi.org/10.1371/journal.pone.0054362

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Bhuvanalakshmi G, Arfuso F, Millward M, Dharmarajan A, Warrier S (2015) Secreted frizzled-related protein 4 inhibits glioma stem-like cells by reversing epithelial to mesenchymal transition, inducing apoptosis and decreasing cancer stem cell properties. PLoS One 10(6):e0127517. https://doi.org/10.1371/journal.pone.0127517

    Article  PubMed  CAS  Google Scholar 

  42. Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314

    Article  PubMed  CAS  Google Scholar 

  43. Sotgia F, Martinez-Outschoorn UE, Pavlides S, Howell A, Pestell RG, Lisanti MP (2011) Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment. Breast cancer research: BCR 13(4):213–213. https://doi.org/10.1186/bcr2892

    Article  PubMed  PubMed Central  Google Scholar 

  44. Martinez-Outschoorn UE, Sotgia F, Lisanti MP (2012) Power surge: supporting cells "fuel" cancer cell mitochondria. Cell Metab 15(1):4–5. https://doi.org/10.1016/j.cmet.2011.12.011

    Article  PubMed  CAS  Google Scholar 

  45. Guido C, Whitaker-Menezes D, Capparelli C, Balliet R, Lin Z, Pestell RG, Howell A, Aquila S, Ando S, Martinez-Outschoorn U, Sotgia F, Lisanti MP (2012) Metabolic reprogramming of cancer-associated fibroblasts by TGF-beta drives tumor growth: connecting TGF-beta signaling with "Warburg-like" cancer metabolism and L-lactate production. Cell Cycle 11(16):3019–3035. https://doi.org/10.4161/cc.21384

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Zhang D, Wang Y, Shi Z, Liu J, Sun P, Hou X, Zhang J, Zhao S, Zhou BP, Mi J (2015) Metabolic reprogramming of cancer-associated fibroblasts by IDH3alpha downregulation. Cell Rep 10(8):1335–1348. https://doi.org/10.1016/j.celrep.2015.02.006

    Article  PubMed  CAS  Google Scholar 

  47. G B AF, Millward M, Dharmarajan A, Warrier S (2015) Secreted frizzled-related protein 4 inhibits glioma stem-like cells by reversing epithelial to mesenchymal transition, inducing apoptosis and decreasing Cancer stem cell properties. PLoS One 10(6):e0127517. https://doi.org/10.1371/journal.pone.0127517

    Article  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

We acknowledge the research and technical support from the School of Biomedical Sciences and Curtin Health Innovation Research Institute, Curtin University, where the work was carried out.

Funding

MV is supported by scholarship from the Office of Research and Development, Faculty of Health sciences, Curtin University. MV would also like to acknowledge the contribution of an Australian Government Research Training Program Scholarship in supporting this research. AD is supported by strategic research funds from the School of Biomedical Sciences (Curtin University), Commercialisation Advisory Board of Curtin University, Cancer Council of Western Australia, and Actinogen Ltd., Perth, Western Australia.

Author information

Authors and Affiliations

  1. Stem Cell and Cancer Biology Laboratory, School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, WA, 6102, Australia

    Malini Visweswaran, Frank Arfuso & Arun Dharmarajan

  2. School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia

    Kevin N. Keane & Philip Newsholme

  3. Ear Sciences Centre, University of Western Australia, Perth, Australia

    Rodney J. Dilley

Authors
  1. Malini Visweswaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kevin N. Keane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frank Arfuso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rodney J. Dilley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philip Newsholme
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arun Dharmarajan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MV conceptualised, performed all experiments, analysed data, and drafted the manuscript. KK assisted the experiments performed using Seahorse flux analyser, its data analysis, interpretation and critical revision of the manuscript. FA was involved with conceptualisation and critical revision of manuscript. RD, PN were involved with critical revision of the manuscript. AD was involved with conceptualisation, critical revision of the manuscript, and funding of the experiments. All authors have read and approved the final version of this manuscript.

Corresponding author

Correspondence to Arun Dharmarajan.

Ethics declarations

Not applicable.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Consent for Publication

Not applicable.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Visweswaran, M., Keane, K.N., Arfuso, F. et al. The Influence of Breast Tumour-Derived Factors and Wnt Antagonism on the Transformation of Adipose-Derived Mesenchymal Stem Cells into Tumour-Associated Fibroblasts. Cancer Microenvironment 11, 71–84 (2018). https://doi.org/10.1007/s12307-018-0210-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12307-018-0210-8

Keywords

Search

Navigation