3-D Cell Culture: Cancer Cell Lines

Cancer Cells

The Extracel-X™ Hydrogel Kit provides a basic scaffold for 3-D cell growth suitable for cancer cell lines. It is composed of Glycosil™ (thiol-modified hyaluronan), Gelin-S™ (thiol-modified gelatin, denatured collagens), and Extralink™ (thiol-reactive crosslinking agent).  Extracel-X has been used extensively for in vivo tumor xenograft studies and for companion in vitro experimentation1. 

For in vitro cell culture, cells can be encapsulated during crosslinking where they attach and grow within the hydrogel matrix, or they can be plated on top of the hydrogel for pseudo three-dimensional growth2,3,4. Some cell types form spheroids if encapsulated. For in vivo cell implantation in tumor xenograft experiments, cells are encapsulated in Extracel-X and then injected subcutaneously (see Tumor xenograft protocol) 1. All Extracel-X Hydrogel Kits are tested for bacterial growth, endotoxins, and lactate dehydrogenase-elevating virus (LDEV).

Hyaluronan and Cancer

Glycosil is thiol-modified hyaluronic acid (HA). HA is the simplest glycosaminoglycan (a class of negatively charged polysaccharides) and a major constituent of the extracellular matrix (ECM)5, a scaffold secreted by cells and which surrounds them in vivo6. HA is a linear, non-sulfated polysaccharide with a chain length from ~0.200 to 10 MDa, with the most common sizes ranging from 2-5 MDa7-9.

To date, fourteen types of carcinoma have been shown to have elevated levels of hyaluronan in the tumor cells or the surrounding stroma10,11. For ovarian cancer,  the correlation between the accumulation of HA and cancer is so strong that it is being considered as a prognostic marker11. Low molecular weight (MW) HA can stimulate the migration of tumor cells, but high MW HA cannot11. Additionally, HA expression has also been shown to affect several signaling pathways (Erb2, Ras, MAPK, and PI3 kinase/Akt) that promote tumor growth and survival10.

As a case in point, inhibition of endogenous HA synthesis (by transfection of antisense-hyaluronan synthase) has been shown to dramatically reduce PC3M-LN4 (human prostate carcinoma) tumor growth in vivo when injected subcutaneously in immuno-compromised mice. These same tumors showed 70-80% lower blood vessel densities. Tumor growth was restored by injecting the tumors with exogenous, high molecular weight HA10.

Hyaluronan and Angiogenesis

HA plays both structural and biochemical roles within the ECM. It is broken down by hyaluronidase enzymes (Hyal-1, Hyal-2, Hyal-3, Hyal-4, and PH-20) that degrade the long HA polymer into short oligosaccharides. Hyal-1 is responsible for the catabolism of intracellular HA12,13 and it is the enzyme that creates angiogenic HA fragments13. Hyal-1 is expressed in most tissues as well as being detected in plasma and urine. It is upregulated in bladder and prostrate cancers13.

HA oligosaccharides (small molecular weight HA fragments) produced by the degradation of HA by Hyal-1 promote angiogenesis, while high molecular weight HA retards it10,13-15. Besides HA fragments and polymers affecting angiogenesis, hyaluronidase levels and HA degradation correlate to tumor growth and angiogenesis. It is inferred that degradation of tumor HA generates HA oligosaccharides, which in turn stimulates angiogenesis and tumor growth. There is some contradictory information to this theory, indicating that more research is required to fully understand the complicated relationship between HA and vascularization14.

Cancer Cells Grown

The following cancer cell lines have been cultured in Extracel-based hydrogels11,12:

  • A549 (lung)16
  • MDA-MB-231 (breast)16
  • MDA-MB-468 (breast)1
  • MCF-7 (breast)1
  • OVCAR-3 (ovarian)1
  • SK-OV-3 (ovarian)1
  • HCT-116 (colon)16
  • Caco-2 (colon)1
  • MiaPaCa-2 (pancreatic)17

References

  1. Liu, X. Z. Shu, and G. D. Prestwich, “Tumor Engineering: Orthotopic Cancer Models in Mice Using Cell-Loaded, Injectable, Crosslinked Hyaluronan-Derived Hydrogels,” Tissue Engineering 13(5), 1091-1101(2007).
  2. G. D. Prestwich, Y. Liu, M. Serban, B. Yu, X. Z. Shu, and A. Scott, “3-D Culture in Synthetic Extracellular Matrices: New Tissue Models for Drug Toxicology and Cancer Drug Discovery,” invited, Adv. Enz. Res., in press (2007).
  3. X. Z. Shu, Y. Liu, F. Palumbo, Y. Luo, G. D. Prestwich, “In Situ Crosslinkable Hyaluronan Hydrogels for Tissue Engineering,” Biomaterials, 25, 1339-1348 (2004).
  4. G. D. Prestwich, X. Z. Shu, Y. Liu, S. Cai, J. F. Walsh, C. W. Hughes, K. R. Kirker, R. R. Orlandi, A. H. Park, S. L. Thibeault, M. E. Smith, “Injectable Synthetic Extracellular Matrices for Tissue Engineering and Repair,” Adv. Exp. Med. Biol., 585, 125-133 (2006).
  5. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 25: The Hyaluronan Synthases (P.H. Weige)
  6. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 1: Solution Properties of Hyaluronan (T. Hardingham)
  7. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 4: Biodegradation of Hyaluronan (G. Lepperdinger, C. Fehrer, S. Reitinger)
  8. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 8: Structural and Functional Diversity of Hyaluronan-Binding Proteins (C.D. Blundell, N.T. Seyfried, A.J. Day)
  9. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 6: The Role of Hyaluornan Receptor RHAMM in Wound Repair and Tumorigenesis (C. Tolg, S.R. Hamilton, E.A. Turley)
  10. B.P. Toole and V.C. Hascall, “Hyaluronan and Tumor Growth,” American Journal of Pathology 161(3):745-747 (2002).
  11. Chemistry and Biology of Hyaluronan H.G. Garg and C.A. Hales (editors) 2004 Elsevier; Chapter 13: The Role of Hyaluronan in Cancer (S. Patel and M.J. Page)
  12. Ingber, D. “Mechanical control of tissue morphogenesis during embryological development,” Int. J. Dev. Biol. 50: 255-266 (2006).
  13. S. Gerecht, J.A. Brudick, L.S. Ferreira, S.A. Townsend, R. Langer, G. Vunjak-Novakovic, “Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells,“ PNAs 104(27): 11298-11303.
  14. W.S. Turner, E. Scmelzer, R. McClelland, E. Wauthier, W. Chen, L.M. Reid, “Human Hepatoblast Phenotype Maintained by Hyaluronan Hydrogels”, J. Biomed Mater Res Part B, 82B: 156-168 (2006).
  15. P.Rooney, M.Wang, P.Kumar, S.Kumar, “Angiogenic oligosaccharides of hyaluronan enhance the productions of collagens by endothelial cells,” Journal of Cell Science 105:213-28 (1993).
  16. Unpublished data from G. D. Prestwich, et al, University of Utah.

  17. Unpublished data from C. Scaife, et al, University of Utah.