Skip to main content
SpringerLink
Log in

Three-dimensional and co-culture models for preclinical evaluation of metal-based anticancer drugs

  • PRECLINICAL STUDIES
  • Published:
Investigational New Drugs Aims and scope Submit manuscript

Summary

Background Hypoxic and necrotic regions that accrue within solid tumors in vivo are known to be associated with metastasis formation, radio- and chemotherapy resistance, and drug metabolism. Therefore, integration of these tumor characteristics into in vitro drug screening models is advantageous for any reliable investigation of the anticancer activity of novel drug candidates. In general, usage of cell culture models with in vivo like characteristics has become essential in preclinical drug studies and allows evaluation of complex problems such as tumor selectivity and anti-invasive properties of the drug candidates. Materials and Methods In this study, we investigated the anticancer activity of clinically approved, investigational and experimental drugs based on platinum (cisplatin, oxaliplatin and KP1537), gallium (KP46), ruthenium (KP1339) and lanthanum (KP772) in different cell culture models such as monolayers, multicellular spheroids, as well as invasion and metastasis models. Results Application of the Alamar Blue assay to multicellular spheroids and a spheroid-based invasion assay resulted in an altered rating of compounds with regard to their cytotoxicity and ability to inhibit invasion when compared with monolayer-based cytotoxicity and transwell assays. For example, the gallium-based drug candidate KP46 showed in spheroid cultures significantly enhanced properties to inhibit protrusion formation and fibroblast mediated invasiveness, and improved cancer cell selectivity. Conclusion Taken together, our results demonstrate the advantages of spheroid-based assays and underline the necessity of using different experimental models for reliable preclinical investigations assessing and better predicting the anticancer potential of new compounds.

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
Fig. 7

Similar content being viewed by others

Abbreviations

CAFs:

Carcinoma associated fibroblasts

CC3:

Caspase 3

CDDP:

Cisplatin

CLSM:

Confocal laser scanning microscopy

DAPI:

4′,6-diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

EDTA:

Ethylenediaminetetraacetic acid

FCS:

Fetal calf serum

FGM:

Fibroblast growth medium

GAPDH:

Glycerinaldehyd-3-phosphat-dehydrogenase

HFS:

Hypotonic fluorochrome solution

HIF1α:

Hypoxia-inducible factor 1-alpha

HRP:

Horseradish peroxidase

IC50:

Half maximal inhibitory concentration

I-OHP:

Oxaliplatin

MCR:

Multicellular resistance

MEM:

Minimum essential medium

PBS:

Phosphate buffered saline

PBST:

Phosphate buffered saline with triton X-100

PI:

Propidium iodide

α-SMA:

Alpha-smooth muscle actin

STR:

Short tandem repeat analysis

References

  1. Unger C, Kramer N, Walzl A, Scherzer M, Hengstschläger M, Dolznig H (2014) Modeling human carcinomas: physiologically relevant 3D models to improve anti-cancer drug development. Adv Drug Deliv Rev 79–80:50–67

    Article  PubMed  Google Scholar 

  2. Sutherland RM (1988) Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240(4849):177–184

    Article  CAS  PubMed  Google Scholar 

  3. Friedrich J, Ebner R, Kunz-Schughart LA (2007) Experimental anti-tumor therapy in 3-D: spheroids-old hat or new challenge? Int J Radiat Biol 83(11–12):849–871

    Article  CAS  PubMed  Google Scholar 

  4. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148(1):3–15

    Article  CAS  PubMed  Google Scholar 

  5. Kim H, Phung Y, Ho M (2012) Changes in global gene expression associated with 3D structure of tumors: an ex vivo matrix-free mesothelioma spheroid model. PLoS One 7(6), e39556

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Mehta G, Hsiao AY, Ingram M, Luker GD, Takayama S (2012) Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release 164(2):192–204

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Durand RE, Olive PL (2001) Resistance of tumor cells to chemo- and radiotherapy modulated by the three-dimensional architecture of solid tumors and spheroids. Methods Cell Biol 64:211–233

    Article  CAS  PubMed  Google Scholar 

  8. Desoize B, Jardillier JK (2000) Multicellular resistance: a paradigm for clinical resistance? Crit Rev Oncol Hematol 36(2–3):193–207

    Article  CAS  PubMed  Google Scholar 

  9. Shannon AM, Bouchier-Hayes DJ, Condron CM, Toomey D (2003) Tumour hypoxia, chemotherapeutic resistance and hypoxia-related therapies. Cancer Treat Rev 29(4):297–307

    Article  CAS  PubMed  Google Scholar 

  10. Bertout JA, Patel SA, Simon MC (2008) The impact of O2 availability on human cancer. Nat Rev Cancer 8(12):967–975

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Brown JM (1999) The hypoxic cell: a target for selective cancer therapy—eighteenth Bruce F. Cain Memorial award lecture. Cancer Res 59:5863–5870

    CAS  PubMed  Google Scholar 

  12. Ma HL, Jiang Q, Han S, Wu Y, Cui Tomshine J, Wang D, Gan Y, Zou G, Liang XJ (2012) Multicellular tumor spheroids as an in vivo–like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellular penetration. Mol Imaging 11(6):487–498

    CAS  PubMed  Google Scholar 

  13. Trédan O, Galmarini CM, Patel K, Tannock IF (2006) Drug resistance and the solid tumor microenvironment. Nat Rev Cancer 6(8):583–592

    Article  Google Scholar 

  14. Phillips RM, Loadman PM, Cronin BP (1998) Evaluation of a novel in vitro assay for assessing drug penetration into avascular regions of tumours. Br J Cancer 77(12):2112–2119

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Wilson WR, Hay MP (2011) Targeting hypoxia in cancer therapy. Nat Rev Cancer 11(6):393–410

    Article  CAS  PubMed  Google Scholar 

  16. Semenza GL (2012) Molecular mechanisms mediating metastasis of hypoxic breast cancer cells. Trends Mol Med 18(9):534–543

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Chang Q, Jurisica I, Do T, Hedley DW (2011) Hypoxia predicts aggressive growth and spontaneous metastasis formation from orthotopically grown primary xenografts of human pancreatic cancer. Cancer Res 71(8):3110–3120

    Article  CAS  PubMed  Google Scholar 

  18. Vaupel P (2009) Prognostic potential of the pre-therapeutic tumor oxygenation status. Adv Exp Med Biol 645:241–246

    Article  PubMed  Google Scholar 

  19. Nagelkerke A, Bussink J, Mujcic H, Wouters BG, Lehmann S, Sweep FC, Span PN (2013) Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response. Breast Cancer Res 15(1):R2

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Tian X, Wang W, Zhang Q, Zhao L, Wei J, Xing H, Song Y, Wang S, Ma D, Meng L, Chen G (2010) Hypoxia-inducible factor-1α enhances the malignant phenotype of multicellular spheroid HeLa cells in vitro. Oncol Lett 1(5):893–897

    CAS  PubMed Central  PubMed  Google Scholar 

  21. Korch C, Spillman MA, Jackson TA, Jacobsen BM, Murphy SK, Lessey BA, Jordan VC, Bradfrod AP (2012) DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecol Oncol 127:241–248

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Hart FA, Laming FP (1964) Complexes of 1,10-phenanthroline with lanthanide chlorides and thiocyanates. J Inorg Nucl Chem 26:579–585

    Article  CAS  Google Scholar 

  23. Peti W, Pieper T, Sommer M, Keppler BK, Giester G (1999) Synthesis of tumor-inhibiting complex salts containing the anion trans-tetrachlorobis(indazole)ruthenate(III) and crystal structure of the tetraphenylphosphonium salt. Eur J Inorg Chem 1551–1555

  24. Collery P, Jakupec MA, Kynast B, Keppler BK (2006) Preclinical and early clinical development of the antitumor gallium complex KP46 (FFC11). In: Alpoim MC, Morais PC, Santos MA, Cristóvão AJ, Centeno JA, Collery P (eds.) Metal ions in biology and medicine 9: 521–524

  25. Abramkin SA, Jungwirth U, Valiahdi SM, Dworak C, Habala L, Meelich K, Berger W, Jakupec MA, Hartinger CG, Nazarov AA, Galanski M, Keppler BK (2010) {(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine}oxalatoplatinum(II): a novel enantiomerically pure oxaliplatin derivative showing improved anticancer activity in vivo. J Med Chem 53:7356–7364

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Dhara SC (1970) Rapid method for the synthesis of cis-[Pt(NH3)2Cl2]. Indian J Chem 8:193–194

    Google Scholar 

  27. Kidani Y, Inagaki K, Iigo M, Hoshi A, Kuretani K (1978) Antitumor activity of 1,2-diaminocyclohexane–platinum complexes against sarcoma-180 ascites form. J Med Chem 21:1315–1318

    Article  CAS  PubMed  Google Scholar 

  28. Albini A, Iwamoto Y, Kleinman HK (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47(12):3239–3245

    CAS  PubMed  Google Scholar 

  29. Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W, Lomas C et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10(1):29

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Liu WD, Zhang T, Wang CL, Meng HM, Song YW, Zhao Z, Li ZM (2012) Sphere-forming tumor cells possess stem-like properties in human fibrosarcoma primary tumors and cell lines. Oncol Lett 4(6):1315–1320

    CAS  PubMed Central  PubMed  Google Scholar 

  31. Bjørge L, Junnikkala S, Kristoffersen EK, Hakulinen J, Matre R, Meri S (1997) Resistance of ovarian teratocarcinoma cell spheroids to complement-mediated lysis. Br J Cancer 75(9):1247–1255

    Article  PubMed  Google Scholar 

  32. Laurent J, Frongia C, Cazales M, Mondesert O, Ducommun B, Lobjois V (2013) Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D. BMC Cancer 13:73

    Article  PubMed Central  PubMed  Google Scholar 

  33. Ke Q, Costa M (2006) Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70(5):1469–1480

    Article  CAS  PubMed  Google Scholar 

  34. Maxwell PH, Dachs GU, Gleadle JM, Nicholls LG, Harris AL, Stratford IJ, Hankinson O, Pugh CW, Ratcliffe PJ (1997) Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci U S A 94:8104–8109

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Hofheinz RD, Dittrich C, Jakupec MA, Drescher A, Jaehde U, Gneist M, Graf von Keyserlingk N, Keppler BK, Hochhaus A (2005) Early results from a phase I study on orally administered tris(8-quinolinolato)gallium(III) (FFC11, KP46) in patients with solid tumors—a CESAR study (Central European Society for Anticancer Drug Research – EWIV). Int J Clin Pharmacol Ther 43(12):590–591

    Article  CAS  PubMed  Google Scholar 

  36. Collery P, Domingo JL, Keppler BK (1996) Preclinical toxicology and tissue gallium distribution of a novel antitumour gallium compound: tris(8-quinolinolato)gallium (III). Anticancer Res 16:687–692

    CAS  PubMed  Google Scholar 

  37. Thompson DS, Weiss GJ, Jones SF, Burris HA, Ramanathan RK, Infante RJ, Bendell JC, Ogden A, Von Hoff DD (2012) NKP-1339: maximum tolerated dose defined for first-in-human GRP78 targeted agent. J Clin Oncol 30, Suppl., abstr. 3033

  38. Bergamo A, Masi A, Jakupec MA, Keppler BK, Sava G (2009) Inhibitory effects of the ruthenium complex KP1019 in models of mammary cancer cell migration and invasion. Met-Based Drugs 681270

  39. Dolznig H, Rupp C, Puri C, Haslinger C, Schweifer N, Kerjaschki D, Garin-Chesa P (2011) Modeling adenocarcinomas in vitro: a novel 3D co-culture system induces cancer relevant pathways upon tumour-stroma interaction. Am J Path 179(1):487–501

    Article  PubMed Central  PubMed  Google Scholar 

  40. 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  CAS  PubMed Central  PubMed  Google Scholar 

  41. Christian MM, Moy RL, Wagner RF, Yen-Moore A (2001) A correlation of alpha-smooth muscle actin and invasion in micronodular basal cell carcinoma. Dermatol Surg 27(5):441–445

    CAS  PubMed  Google Scholar 

  42. de Wever O, Demetter P, Mareel M, Brack M (2008) Stromal myofibroblasts are drivers of invasive cancer growth Int. J Cancer 123:2229–2238

    Google Scholar 

  43. Valiahdi SM, Heffeter P, Jakupec MA, Marculescu R, Berger W, Rappersberger K, Keppler BK (2009) The gallium complex KP46 exerts strong activity against primary explanted melanoma cells and induces apoptosis in melanoma cell lines. Melanoma Res 19(5):283–293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Desoize B, Collery P, Akéli JC, Keppler B (2000) Tris(8-quinolinolato)Ga(III) is active against unicellular and multicellular resistance. Met Ions Biol Med 6:573–576

    Google Scholar 

  45. Carlsson J, Nederman T (1989) Tumour spheroid technology in cancer therapy research. Eur J Cancer 25(8):1127–1133

    Article  CAS  Google Scholar 

  46. Hazlehurst LA, Dalton WS (2001) Mechanisms associated with cell adhesion mediated drug resistance (CAM-DR) in hematopoietic malignancies. Cancer Metastasis Rev 20(1–2):43–50

  47. Erler JT, Cawthorne CJ, Williams KJ, Koritzinsky M, Wouters BG, Wilson C, Miller C, Demonacos C, Stratford IJ, Dive C (2004) Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and –independent mechanisms and contributes to drug resistance. Mol Cell Biol 24:2875–2889

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Enyedi ÉA, Dömötör O, Bali K, Hetényi A, Tuccinardi T, Keppler BK (2015) Interaction of the anticancer gallium(III) complexes of 8-hydroxyquinoline and maltol with human serum proteins. J Biol Inorg Chem 20:77–88

    Article  Google Scholar 

  49. Rainaldi G, Calcabrini A, Arancia G, Santini MT (1999) Differential expression of adhesion molecules (CD44, ICAM-1 and LFA-3) in cancer cells grown in monolayer or as multicellular spheroids. Anticancer Res 19(3A):1769–1778

    CAS  PubMed  Google Scholar 

  50. Jungwirth U, Gojo J, Tuder T, Walko G, Holcmann M, Schöfl T, Nowikovsky K, Wilfinger N, Schoonhoven S, Kowol CR, Lemmens-Gruber R, Heffeter P, Keppler BK, Berger W (2014) Calpain-mediated integrin deregulation as a novel mode of action for the anticancer gallium compound KP46. Mol Cancer Ther 13(10):2436–2449

    Article  CAS  PubMed  Google Scholar 

  51. Heffeter P, Jakupec MA, Körner W, Wild S, von Keyserlingk NG, Elbling L, Zorbas H, Korynevska A, Knasmüller S, Sutterlüty H, Micksche M, Keppler BK, Berger W (2006) Anticancer activity of the lanthanum compound [tris(1,10-phenanthroline)lanthanum(III)]trithiocyanate (KP772; FFC24). Biochem Pharmacol 71(4):426–440

    Article  CAS  PubMed  Google Scholar 

  52. Trondl R, Heffeter P, Kowol CR, Jakupec MA, Berger W, Keppler BK (2014) NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem Sci 5:2925–2932

    Article  CAS  Google Scholar 

  53. Heffeter P, Atil B, Kryeziu K, Groza D, Koellensperger G, Körner W, Jungwirth U, Mohr T, Keppler BK, Berger W (2013) The ruthenium compound KP1339 potentiates the anticancer activity of sorafenib in vitro and in vivo. Eur J Cancer 49(15):3366–3375

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  54. Bytzek AK, Boeck K, Hermann G, Hann S, Keppler BK, Hartinger CG, Koellensperger G (2011) LC- and CZE-ICP-MS approaches for the in vivo analysis of the anticancer drug candidate sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339) in mouse plasma. Metallomics 3(10):1049–1055

    Article  CAS  PubMed  Google Scholar 

  55. Dömötör O, Hartinger CG, Bytzek AK, Kiss T, Keppler BK, Enyedy EA (2013) Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies. J Biol Inorg Chem 18(1):9–17

    Article  PubMed  Google Scholar 

  56. Roberts DL, Williams KJ, Cowen RL, Barathova M, Eustace AJ, Brittain-Dissont S, Tilby MJ, Pearson DG, Ottley CJ, Stratford IJ, Dive C (2009) Contribution of HIF-1 and drug penetrance to oxaliplatin resistance in hypoxic colorectal cancer cells. Br J Cancer 101(8):1290–1297

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Conflict of interest

The authors declare that they have no conflict of interest.

Authorship contributions

Participated in research design: Ekaterina Schreiber-Brynzak, Simone Göschl, Robert Trondl, Helmut Dolznig, Michael A. Jakupec, Bernhard K. Keppler

Conducted experiments: Ekaterina Schreiber-Brynzak, Erik Klapproth, Christine Unger, Irene Lichtscheidl-Schultz, Sarah Schweighofer

Performed data analysis: Ekaterina Schreiber-Brynzak, Erik Klapproth, Simone Göschl, Christine Unger

Wrote or contributed to the writing of the manuscript: Ekaterina Schreiber-Brynzak, Christine Unger, Simone Göschl, Michael A. Jakupec

Author information

Authors and Affiliations

  1. University of Vienna, Faculty of Chemistry, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090, Vienna, Austria

    Ekaterina Schreiber-Brynzak, Erik Klapproth, Simone Göschl, Robert Trondl, Michael A. Jakupec & Bernhard K. Keppler

  2. University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Vienna, Austria

    Irene Lichtscheidl-Schultz

  3. Medical University of Vienna, Institute of Medical Genetics, Vienna, Austria

    Christine Unger, Sarah Schweighofer & Helmut Dolznig

  4. University of Vienna, Research Platform “Translational Cancer Therapy Research”, Waehringer Str. 42, A-1090, Vienna, Austria

    Robert Trondl, Michael A. Jakupec & Bernhard K. Keppler

Authors
  1. Ekaterina Schreiber-Brynzak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erik Klapproth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christine Unger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Irene Lichtscheidl-Schultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simone Göschl
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sarah Schweighofer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Robert Trondl
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Helmut Dolznig
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael A. Jakupec
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Bernhard K. Keppler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael A. Jakupec.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 2207 kb)

(AVI 14816 kb)

(MPG 6140 kb)

(AVI 13689 kb)

(MPG 12274 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schreiber-Brynzak, E., Klapproth, E., Unger, C. et al. Three-dimensional and co-culture models for preclinical evaluation of metal-based anticancer drugs. Invest New Drugs 33, 835–847 (2015). https://doi.org/10.1007/s10637-015-0260-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10637-015-0260-4

Keywords

Search

Navigation