Abstract
Neuroblastoma is characterized by biological and genetic heterogeneity that leads to diverse, often unpredictable, clinical behavior. Differential expression of the Trk family of neurotrophin receptors strongly correlates with clinical behavior; TrkA expression is associated with favorable outcome, whereas TrkB with unfavorable outcome. Neuroblastoma cells cultured in a microgravity rotary bioreactor spontaneously aggregate into tumor-like structures, called organoids. We wanted to determine if the clinical heterogeneity of TrkA- or TrkB-expressing neuroblastomas was reflected in aggregation kinetics and organoid morphology. Trk-null SY5Y cells were stably transfected to express either TrkA or TrkB. Short-term aggregation kinetics were determined by counting the number of single (non-aggregated) viable cells in the supernatant over time. Organoids were harvested after 8 d of bioreactor culture, stained, and analyzed morphometrically. SY5Y-TrkA cells aggregated significantly slower than SY5Y and SY5Y-TrkB cells, as quantified by several measures of aggregation. SY5Y and TrkB cell lines formed irregularly shaped organoids, featuring stellate projections. In contrast, TrkA cells formed smooth (non-stellate) organoids. SY5Y organoids were slightly smaller on average, but had significantly larger average perimeter than TrkA or TrkB organoids. TrkA expression alone is sufficient to dramatically alter the behavior of neuroblastoma cells in three-dimensional, in vitro rotary bioreactor culture. This pattern is consistent with both clinical behavior and in vivo tumorigenicity, in that SY5Y-TrkA represents a more differentiated, less aggressive phenotype. The microgravity bioreactor is a useful in vitro tool to rapidly investigate the biological characteristics of neuroblastoma and potentially to assess the effect of cytotoxic as well as biologically targeted drugs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arien-Zakay H.; Nagler A.; Galski H.; Lazarovici P. Neuronal conditioning medium and nerve growth factor induce neuronal differentiation of collagen-adherent progenitors derived from human umbilical cord blood. J. Mol. Neurosci. 32: 179–191; 2007.
Barbacid M. Structural and functional properties of the TRK family of neurotrophin receptors. Ann. N. Y. Acad. Sci. 766: 442–458; 1995.
Biedler J. L.; Roffler-Tarlov S.; Schachner M.; Freedman L. S. Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones. Cancer Res. 38: 3751–3757; 1978.
Brodeur G. M. Neuroblastoma: biological insights into a clinical enigma. Nat. Rev. Cancer 3: 203–216; 2003.
Brodeur G. M.; Minturn J. E.; Ho R.; Simpson A. M.; Iyer R.; Varela C. R.; Light J. E.; Kolla V.; Evans A. E. Trk receptor expression and inhibition in neuroblastomas. Clin. Cancer Res. 15: 3244–3250; 2009.
Bunone G.; Mariotti A.; Compagni A.; Morandi E.; Della Valle G. Induction of apoptosis by p75 neurotrophin receptor in human neuroblastoma cells. Oncogene 14: 1463–1470; 1997.
Eggert A.; Grotzer M. A.; Ikegaki N.; X-g L.; Evans A. E.; Brodeur G. M. Expression of the neurotrophin receptor TRKA downregulates expression and function of angiogenic stimulators in sh-sy5y neuroblastoma cells. Cancer Res. 62: 1802–1808; 2002.
Eggert A.; Ikegaki N.; Liu X.; Chou T. T.; Lee V. M.; Trojanowski J. Q.; Brodeur G. M. Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells. Oncogene 19: 2043–2051; 2000a.
Eggert A.; Sieverts H.; Ikegaki N.; Brodeur G. M. p75 mediated apoptosis in neuroblastoma cells is inhibited by expression of TrkA. Med. Pediatr. Oncol. 35: 573–576; 2000b.
Freed L. E.; Vunjak-Novakovic G. Microgravity tissue engineering. In Vitro Cell. Dev. Biol. Anim. 33: 381–385; 1997.
Ho R.; Eggert A.; Hishiki T.; Minturn J. E.; Ikegaki N.; Foster P.; Camoratto A. M.; Evans A. E.; Brodeur G. M. Resistance to chemotherapy mediated by TrkB in neuroblastomas. Cancer Res. 62: 6462–6466; 2002.
Ingram M.; Techy G.; Saroufeem R.; Yazan O.; Narayan K.; Goodwin T.; Spaulding G. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. In Vitro Cell. Dev. Biol. Anim. 33: 459–466; 1997.
Jessup JM, Goodwin TJ, Spaulding G (1993) Prospects for use of microgravity-based bioreactors to study three-dimensional host-tumor interactions in human neoplasia. 1993 51:290-300
Lammens T.; Swerts K.; Derycke L.; De Craemer A.; De Brouwer S.; De Preter K.; Van Roy N.; Vandesompele J.; Speleman F.; Philippé J.; Benoit Y.; Beiske K.; Bracke M.; Laureys G. N-Cadherin in Neuroblastoma Disease: expression and clinical significance. PLoS ONE 7: e31206; 2012.
Lavenius E.; Gestblom C.; Johansson I.; Nanberg E.; Pahlman S. Transfection of TRK-A into human neuroblastoma cells restores their ability to differentiate in response to nerve growth factor. Cell Growth Differ. 6: 727–736; 1995.
Lee S.; Qiao J.; Paul P.; O’Connor K. L.; Evers M.; Chung D. H. FAK is a critical regulator of neuroblastoma liver metastasis. Oncotarget 3: 1576–1587; 2012.
Licato L. L.; Prieto V. G.; Grimm E. A. A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels. In Vitro Cell Dev Biol Animal 37: 121–126; 2009.
Light J. E.; Koyama H.; Minturn J. E.; Ho R.; Simpson A. M.; Iyer R.; Mangino J. L.; Kolla V.; London W. B.; Brodeur G. M. Clinical significance of NTRK family gene expression in neuroblastomas. Pediatric blood & cancer 59: 226–232; 2012.
Maris J. M. Recent advances in neuroblastoma. New Eng J Med 362: 2202–2211; 2010.
Nakagawara A.; Arima-Nakagawara M.; Scavarda N. J.; Azar C. G.; Cantor A. B.; Brodeur G. M. Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N. Engl. J. Med. 328: 847–854; 1993.
Nakagawara A.; Azar C. G.; Scavarda N. J.; Brodeur G. M. Expression and function of TRK-B and BDNF in human neuroblastomas. Molecular and cellular biology 14: 759–767; 1994a.
Nakagawara A.; Azar C. G.; Scavarda N. J.; Brodeur G. M. Expression and function of TRK-B and BDNF in human neuroblastomas. Mol. Cell. Biol. 14: 759–767; 1994b.
O’Connor K. C. Three-dimensional cultures of prostatic cells: tissue models for the development of novel anti-cancer therapies. Pharm. Res. 16: 486–493; 1999.
Redden R. A.; Doolin E. J. Microgravity assay of neuroblastoma: in vitro aggregation kinetics and organoid morphology correlate with MYCN expression. In Vitro Cell. Dev. Biol. Anim. 47: 312–317; 2011.
Rhee H. W.; Zhau H. E.; Pathak S.; Multani A. S.; Pennanen S.; Visakorpi T.; Chung L. W. K. Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel. In Vitro Cell. Dev. Biol. Anim. 37: 127–140; 2001.
Suzuki T.; Bogenmann E.; Shimada H.; Stram D.; Seeger R. C. Lack of high-affinity nerve growth factor receptors in aggressive neuroblastomas. J. Natl. Cancer Inst. 85: 377–384; 1993.
Thiele C. J.; Li Z.; McKee A. E. On Trk—the TRKB signal transduction pathway is an increasingly important target in cancer biology. Clin. Cancer Res. 15: 5962–5967; 2009.
Vamvakidou A. P.; Mondrinos M. J.; Petushi S. P.; Garcia F. U.; Lelkes P. I.; Tozeren A. Heterogeneous breast tumoroids: an in vitro assay for investigating cellular heterogeneity and drug delivery. J. Biomol. Screen. 12: 13–20; 2007.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: T Okamoto
Rights and permissions
About this article
Cite this article
Redden, R.A., Iyer, R., Brodeur, G.M. et al. Rotary bioreactor culture can discern specific behavior phenotypes in Trk-null and Trk-expressing neuroblastoma cell lines. In Vitro Cell.Dev.Biol.-Animal 50, 188–193 (2014). https://doi.org/10.1007/s11626-013-9716-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-013-9716-z