Skip to main content
SpringerLink
Log in

Three-Dimensional Spheroids for Cancer Research

  • Protocol
  • First Online:
Cancer Cell Culture

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2645))

Abstract

In vitro cell culture is one of the most widely used tools used today for increasing our understanding of various things such as protein production, mechanisms of drug action, tissue engineering, and overall cellular biology. For the past decades, however, cancer researchers have relied heavily on conventional two-dimensional (2D) monolayer culture techniques to test a variety of aspects of cancer research ranging from the cytotoxic effects of antitumor drugs to the toxicity of diagnostic dyes and contact tracers. However, many promising cancer therapies have either weak or no efficacy in real-life conditions, therefore delaying or stopping altogether their translating to the clinic. This is, in part, due to the reductionist 2D cultures used to test these materials, which lack appropriate cell-cell contacts, have altered signaling, do not represent the natural tumor microenvironment, and have different drug responses, due to their reduced malignant phenotype when compared to real in vivo tumors. With the most recent advances, cancer research has moved into 3D biological investigation. Three-dimensional (3D) cultures of cancer cells not only recapitulate the in vivo environment better than their 2D counterparts, but they have, in recent years, emerged as a relatively low-cost and scientifically accurate methodology for studying cancer. In this chapter, we highlight the importance of 3D culture, specifically 3D spheroid culture, reviewing some key methodologies for forming 3D spheroids, discussing the experimental tools that can be used in conjunction with 3D spheroids and finally their applications in cancer research.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kapalczynska M, Kolenda T, Przybyla W, Zajaczkowska M, Teresiak A, Filas V et al (2018) 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci 14(4):910–919. https://doi.org/10.5114/aoms.2016.63743

    Article  CAS  PubMed  Google Scholar 

  2. Duval K, Grover H, Han L-H, Mou Y, Pegoraro AF, Fredberg J et al (2017) Modeling physiological events in 2D vs. 3D cell culture. Physiology (Bethesda) 32(4):266–277. https://doi.org/10.1152/physiol.00036.2016

    Article  CAS  PubMed  Google Scholar 

  3. Quail DF, Joyce JA (2013) Microenvironmental regulation of tumor progression and metastasis. Nat Med 19(11):1423–1437. https://doi.org/10.1038/nm.3394

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Belli C, Trapani D, Viale G, D’Amico P, Duso BA, Della Vigna P et al (2018) Targeting the microenvironment in solid tumors. Cancer Treat Rev 65:22–32. https://doi.org/10.1016/j.ctrv.2018.02.004

    Article  CAS  PubMed  Google Scholar 

  5. Pinto B, Henriques AC, Silva PMA, Bousbaa H (2020) Three-dimensional spheroids as in vitro preclinical models for cancer research. Pharmaceutics 12(12):1186. https://doi.org/10.3390/pharmaceutics12121186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hamburger AW, Salmon SE (1977) Primary bioassay of human tumor stem cells. Science (New York, NY) 197(4302):461–463. https://doi.org/10.1126/science.560061

    Article  CAS  Google Scholar 

  7. Labani-Motlagh A, Ashja-Mahdavi M, Loskog A (2020) The tumor microenvironment: a milieu hindering and obstructing antitumor immune responses. Front Immunol 11:940. https://doi.org/10.3389/fimmu.2020.00940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang JJ, Lei KF, Han F (2018) Tumor microenvironment: recent advances in various cancer treatments. Eur Rev Med Pharmacol Sci 22(12):3855–3864. https://doi.org/10.26355/eurrev_201806_15270

    Article  PubMed  Google Scholar 

  9. Jing X, Yang F, Shao C, Wei K, Xie M, Shen H et al (2019) Role of hypoxia in cancer therapy by regulating the tumor microenvironment. Mol Cancer 18(1):157. https://doi.org/10.1186/s12943-019-1089-9

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wu T, Dai Y (2017) Tumor microenvironment and therapeutic response. Cancer Lett 387:61–68. https://doi.org/10.1016/j.canlet.2016.01.043

    Article  CAS  PubMed  Google Scholar 

  11. White KA, Grillo-Hill BK, Barber DL (2017) Cancer cell behaviors mediated by dysregulated pH dynamics at a glance. J Cell Sci 130(4):663–669. https://doi.org/10.1242/jcs.195297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hannon G, Tansi FL, Hilger I, Prina-Mello A (2021) The effects of localized heat on the hallmarks of cancer. Adv Ther 4(7):2000267. https://doi.org/10.1002/adtp.202000267

    Article  Google Scholar 

  13. Jarosz-Biej M, Smolarczyk R, Cichoń T, Kułach N (2019) Tumor microenvironment as a “game changer” in cancer radiotherapy. Int J Mol Sci 20(13):3212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Riera-Domingo C, Audigé A, Granja S, Cheng W-C, Ho P-C, Baltazar F et al (2020) Immunity, hypoxia, and metabolism–the Ménage à Trois of cancer: implications for immunotherapy. Physiol Rev 100(1):1–102. https://doi.org/10.1152/physrev.00018.2019

    Article  CAS  PubMed  Google Scholar 

  15. Pitt JM, Marabelle A, Eggermont A, Soria JC, Kroemer G, Zitvogel L (2016) Targeting the tumor microenvironment: removing obstruction to anticancer immune responses and immunotherapy. Ann Oncol 27(8):1482–1492. https://doi.org/10.1093/annonc/mdw168

    Article  CAS  PubMed  Google Scholar 

  16. Najafi M, Farhood B, Mortezaee K (2019) Extracellular matrix (ECM) stiffness and degradation as cancer drivers. J Cell Biochem 120(3):2782–2790. https://doi.org/10.1002/jcb.27681

    Article  CAS  PubMed  Google Scholar 

  17. Kapałczyńska M, Kolenda T, Przybyła W, Zajączkowska M, Teresiak A, Filas V et al (2018) 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci 14(4):910–919. https://doi.org/10.5114/aoms.2016.63743

    Article  CAS  PubMed  Google Scholar 

  18. Kim JB (2005) Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol 15(5):365–377. https://doi.org/10.1016/j.semcancer.2005.05.002

    Article  PubMed  Google Scholar 

  19. Gurski LA, Petrelli NJ, Jia X, Farach-Carson MC (2010) 3D matrices for anti-cancer drug testing and development. Oncol Issues 25(1):20–25. https://doi.org/10.1080/10463356.2010.11883480

    Article  Google Scholar 

  20. Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30(1):215–224. https://doi.org/10.1016/0092-8674(82)90027-7

    Article  CAS  PubMed  Google Scholar 

  21. Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12(4):207–218. https://doi.org/10.1089/adt.2014.573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Llewellyn SV, Conway GE, Shah U-K, Evans SJ, Jenkins GJS, Clift MJD et al (2020) Advanced 3D liver models for in vitro genotoxicity testing following long-term nanomaterial exposure. J Vis Exp 160:e61141. https://doi.org/10.3791/61141

    Article  CAS  Google Scholar 

  23. Bell CC, Dankers ACA, Lauschke VM, Sison-Young R, Jenkins R, Rowe C et al (2018) Comparison of hepatic 2D Sandwich cultures and 3D spheroids for long-term toxicity applications: a multicenter study. Toxicol Sci 162(2):655–666. https://doi.org/10.1093/toxsci/kfx289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chang TT, Hughes-Fulford M (2009) Monolayer and spheroid culture of human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene expression patterns and functional phenotypes. Tissue Eng Part A 15(3):559–567. https://doi.org/10.1089/ten.tea.2007.0434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gaskell H, Sharma P, Colley HE, Murdoch C, Williams DP, Webb SD (2016) Characterization of a functional C3A liver spheroid model. Toxicol Res 5(4):1053–1065. https://doi.org/10.1039/c6tx00101g

    Article  CAS  Google Scholar 

  26. Kizawa H, Nagao E, Shimamura M, Zhang G, Torii H (2017) Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery. Biochem Biophys Rep 10:186–191. https://doi.org/10.1016/j.bbrep.2017.04.004

    Article  PubMed  PubMed Central  Google Scholar 

  27. Fessart D, Begueret H, Delom F (2013) Three-dimensional culture model to distinguish normal from malignant human bronchial epithelial cells. Eur Respir J 42(5):1345–1356. https://doi.org/10.1183/09031936.00118812

    Article  PubMed  Google Scholar 

  28. Shabalina EY, Skorova EY, Chudakova DA, Anikin VB, Reshetov IV, Mynbaev OA et al (2021) The matrix-dependent 3D spheroid model of the migration of non-small cell lung cancer: a step towards a rapid automated screening. Front Mol Biosci 8:610407. https://doi.org/10.3389/fmolb.2021.610407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Xu WH, Han M, Dong Q, Fu ZX, Diao YY, Liu H et al (2012) Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation. Int J Nanomedicine 7:2661–2671. https://doi.org/10.2147/ijn.s30445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Boylan KLM, Manion RD, Shah H, Skubitz KM, Skubitz APN (2020) Inhibition of ovarian cancer cell spheroid formation by synthetic peptides derived from Nectin-4. Int J Mol Sci 21(13):4637. https://doi.org/10.3390/ijms21134637

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rodríguez-Dorantes M, Cruz-Hernandez CD, Cortés-Ramírez SA, Cruz-Burgos JM, Reyes-Grajeda JP, Peralta-Zaragoza O et al (2021) Prostate cancer spheroids: a three-dimensional model for studying tumor heterogeneity. In: Robles-Flores M (ed) Cancer cell signaling: methods and protocols. Springer US, New York, pp 13–17

    Chapter  Google Scholar 

  32. Omer D, Pleniceanu O, Gnatek Y, Namestnikov M, Cohen-Zontag O, Goldberg S et al (2021) Human kidney spheroids and monolayers provide insights into SARS-CoV-2 renal interactions. J Am Soc Nephrol 32(9):2242–2254. https://doi.org/10.1681/asn.2020111546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Huang YL, Shiau C, Wu C, Segall JE, Wu M (2020) The architecture of co-culture spheroids regulates tumor invasion within a 3D extracellular matrix. Biophys Rev Lett 15(3):131–141. https://doi.org/10.1142/s1793048020500034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nath S, Devi GR (2016) Three-dimensional culture systems in cancer research: focus on tumor spheroid model. Pharmacol Ther 163:94–108. https://doi.org/10.1016/j.pharmthera.2016.03.013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4(3):309–324. https://doi.org/10.1038/nprot.2008.226

    Article  CAS  PubMed  Google Scholar 

  36. Groebe K, Mueller-Klieser W (1991) Distributions of oxygen, nutrient, and metabolic waste concentrations in multicellular spheroids and their dependence on spheroid parameters. Eur Biophys J 19(4):169–181. https://doi.org/10.1007/BF00196343

    Article  CAS  PubMed  Google Scholar 

  37. Alvarez-Pérez J, Ballesteros P, Cerdán S (2005) Microscopic images of intraspheroidal pH by 1H magnetic resonance chemical shift imaging of pH sensitive indicators. Magn Reson Mater Phys Biol Med 18(6):293–301. https://doi.org/10.1007/s10334-005-0013-z

    Article  CAS  Google Scholar 

  38. Bell HS, Whittle IR, Walker M, Leaver HA, Wharton SB (2001) The development of necrosis and apoptosis in glioma: experimental findings using spheroid culture systems. Neuropathol Appl Neurobiol 27(4):291–304. https://doi.org/10.1046/j.0305-1846.2001.00319.x

    Article  CAS  PubMed  Google Scholar 

  39. Vermeulen PB, van Golen KL, Dirix LY (2010) Angiogenesis, lymphangiogenesis, growth pattern, and tumor emboli in inflammatory breast cancer. Cancer 116(S11):2748–2754. https://doi.org/10.1002/cncr.25169

    Article  CAS  PubMed  Google Scholar 

  40. Lehman HL, Dashner EJ, Lucey M, Vermeulen P, Dirix L, Laere SV et al (2013) Modeling and characterization of inflammatory breast cancer emboli grown in vitro. Int J Cancer 132(10):2283–2294. https://doi.org/10.1002/ijc.27928

    Article  CAS  PubMed  Google Scholar 

  41. Chandrasekaran S, Marshall JR, Messing JA, Hsu JW, King MR (2014) TRAIL-mediated apoptosis in breast cancer cells cultured as 3D spheroids. PLoS One 9(10):e111487. https://doi.org/10.1371/journal.pone.0111487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tong JG, Valdes YR, Barrett JW, Bell JC, Stojdl D, McFadden G et al (2015) Evidence for differential viral oncolytic efficacy in an in vitro model of epithelial ovarian cancer metastasis. Mol Ther Oncol 2:15013. https://doi.org/10.1038/mto.2015.13

    Article  Google Scholar 

  43. Peshwa MV, Wu FJ, Sharp HL, Cerra FB, Hu WS (1996) Mechanistics of formation and ultrastructural evaluation of hepatocyte spheroids. In Vitro Cell Dev Biol Anim 32(4):197–203. https://doi.org/10.1007/bf02722946

    Article  CAS  PubMed  Google Scholar 

  44. Wong SF, No da Y, Choi YY, Kim DS, Chung BG, Lee SH (2011) Concave microwell based size-controllable hepatosphere as a three-dimensional liver tissue model. Biomaterials 32(32):8087–8096. https://doi.org/10.1016/j.biomaterials.2011.07.028

    Article  CAS  PubMed  Google Scholar 

  45. Riccalton-Banks L, Liew C, Bhandari R, Fry J, Shakesheff K (2003) Long-term culture of functional liver tissue: three-dimensional coculture of primary hepatocytes and stellate cells. Tissue Eng 9(3):401–410. https://doi.org/10.1089/107632703322066589

    Article  CAS  PubMed  Google Scholar 

  46. Messner S, Agarkova I, Moritz W, Kelm JM (2013) Multi-cell type human liver microtissues for hepatotoxicity testing. Arch Toxicol 87(1):209–213. https://doi.org/10.1007/s00204-012-0968-2

    Article  CAS  PubMed  Google Scholar 

  47. Lee J, Lilly GD, Doty RC, Podsiadlo P, Kotov NA (2009) In vitro toxicity testing of nanoparticles in 3D cell culture. Small 5(10):1213–1221. https://doi.org/10.1002/smll.200801788

    Article  CAS  PubMed  Google Scholar 

  48. Elje E, Mariussen E, Moriones OH, Bastus NG, Puntes V, Kohl Y et al (2020) Hepato(Geno)Toxicity assessment of nanoparticles in a HepG2 liver spheroid model. Nanomaterials (Basel) 10(3):545. https://doi.org/10.3390/nano10030545

    Article  CAS  PubMed  Google Scholar 

  49. Dubiak-Szepietowska M, Karczmarczyk A, Jonsson-Niedziolka M, Winckler T, Feller KH (2016) Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro. Toxicol Appl Pharmacol 294:78–85. https://doi.org/10.1016/j.taap.2016.01.016

    Article  CAS  PubMed  Google Scholar 

  50. Vorrink SU, Zhou Y, Ingelman-Sundberg M, Lauschke VM (2018) Prediction of drug-induced hepatotoxicity using long-term stable primary hepatic 3D spheroid cultures in chemically defined conditions. Toxicol Sci 163(2):655–665. https://doi.org/10.1093/toxsci/kfy058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Mikhail AS, Eetezadi S, Allen C (2013) Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomedicines in vitro: a comparison of docetaxel-loaded block copolymer micelles and Taxotere(R). PLoS One 8(4):e62630. https://doi.org/10.1371/journal.pone.0062630

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ramaiahgari SC, den Braver MW, Herpers B, Terpstra V, Commandeur JN, van de Water B et al (2014) A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol 88(5):1083–1095. https://doi.org/10.1007/s00204-014-1215-9

    Article  CAS  PubMed  Google Scholar 

  53. Kunz-Schughart LA, Kreutz M, Knuechel R (1998) Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int J Exp Pathol 79(1):1–23. https://doi.org/10.1046/j.1365-2613.1998.00051.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Fang Y, Eglen RM (2017) Three-dimensional cell cultures in drug discovery and development. SLAS Discov 22(5):456–472. https://doi.org/10.1177/1087057117696795

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:29. https://doi.org/10.1186/1741-7007-10-29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ekert JE, Johnson K, Strake B, Pardinas J, Jarantow S, Perkinson R et al (2014) Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro – implication for drug development. PLoS One 9(3):e92248. https://doi.org/10.1371/journal.pone.0092248

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Achilli T-M, Meyer J, Morgan JR (2012) Advances in the formation, use and understanding of multi-cellular spheroids. Expert Opin Biol Ther 12(10):1347–1360. https://doi.org/10.1517/14712598.2012.707181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Cho CY, Chiang TH, Hsieh LH, Yang WY, Hsu HH, Yeh CK et al (2020) Development of a novel hanging drop platform for engineering controllable 3D microenvironments. Front Cell Dev Biol 8:327. https://doi.org/10.3389/fcell.2020.00327

    Article  PubMed  PubMed Central  Google Scholar 

  59. Wu HW, Hsiao YH, Chen CC, Yet SF, Hsu CH (2016) A PDMS-based microfluidic hanging drop Chip for Embryoid body formation. Molecules 21(7):882. https://doi.org/10.3390/molecules21070882

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zhao L, Xiu J, Liu Y, Zhang T, Pan W, Zheng X et al (2019) A 3D printed hanging drop dripper for tumor spheroids analysis without recovery. Sci Rep 9(1):19717. https://doi.org/10.1038/s41598-019-56241-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Souza GR, Molina JR, Raphael RM, Ozawa MG, Stark DJ, Levin CS et al (2010) Three-dimensional tissue culture based on magnetic cell levitation. Nat Nanotechnol 5(4):291–296. https://doi.org/10.1038/nnano.2010.23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Verjans ET, Doijen J, Luyten W, Landuyt B, Schoofs L (2018) Three-dimensional cell culture models for anticancer drug screening: worth the effort? J Cell Physiol 233(4):2993–3003. https://doi.org/10.1002/jcp.26052

    Article  CAS  PubMed  Google Scholar 

  63. Unnikrishnan K, Thomas LV, Ram Kumar RM (2021) Advancement of scaffold-based 3D cellular models in cancer tissue engineering: an update. Front Oncol 11:733652. https://doi.org/10.3389/fonc.2021.733652

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Efraim Y, Schoen B, Zahran S, Davidov T, Vasilyev G, Baruch L et al (2019) 3D structure and processing methods direct the biological attributes of ECM-based cardiac scaffolds. Sci Rep 9(1):5578. https://doi.org/10.1038/s41598-019-41831-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Li W, Hu X, Yang S, Wang S, Zhang C, Wang H et al (2018) A novel tissue-engineered 3D tumor model for anti-cancer drug discovery. Biofabrication 11(1):015004. https://doi.org/10.1088/1758-5090/aae270

    Article  PubMed  Google Scholar 

  66. Costa EC, Moreira AF, de Melo-Diogo D, Gaspar VM, Carvalho MP, Correia IJ (2016) 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv 34(8):1427–1441. https://doi.org/10.1016/j.biotechadv.2016.11.002

    Article  PubMed  Google Scholar 

  67. Rijal G, Li W (2017) A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening. Sci Adv 3(9):e1700764. https://doi.org/10.1126/sciadv.1700764

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Xiao Y, Zhou M, Zhang M, Liu W, Zhou Y, Lang M (2019) Hepatocyte culture on 3D porous scaffolds of PCL/PMCL. Colloids Surf B Biointerfaces 173:185–193. https://doi.org/10.1016/j.colsurfb.2018.09.064

    Article  CAS  PubMed  Google Scholar 

  69. Kuriakose AE, Hu W, Nguyen KT, Menon JU (2019) Scaffold-based lung tumor culture on porous PLGA microparticle substrates. PLoS One 14(5):e0217640. https://doi.org/10.1371/journal.pone.0217640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Wang X, Dai X, Zhang X, Li X, Xu T, Lan Q (2018) Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix. Biochem Biophys Res Commun 498(4):1052–1057. https://doi.org/10.1016/j.bbrc.2018.03.114

    Article  CAS  PubMed  Google Scholar 

  71. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S et al (2013) Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol 87(8):1315–1530. https://doi.org/10.1007/s00204-013-1078-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Molina-Jimenez F, Benedicto I, Dao Thi VL, Gondar V, Lavillette D, Marin JJ et al (2012) Matrigel-embedded 3D culture of Huh-7 cells as a hepatocyte-like polarized system to study hepatitis C virus cycle. Virology 425(1):31–39. https://doi.org/10.1016/j.virol.2011.12.021

    Article  CAS  PubMed  Google Scholar 

  73. Tao F, Sayo K, Sugimoto K, Aoki S, Kojima N (2020) Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides. Sci Rep 10(1):6567. https://doi.org/10.1038/s41598-020-63621-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Meenach SA, Tsoras AN, McGarry RC, Mansour HM, Hilt JZ, Anderson KW (2016) Development of three-dimensional lung multicellular spheroids in air- and liquid-interface culture for the evaluation of anticancer therapeutics. Int J Oncol 48(4):1701–1709. https://doi.org/10.3892/ijo.2016.3376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Hughes CS, Postovit LM, Lajoie GA (2010) Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10(9):1886–1890. https://doi.org/10.1002/pmic.200900758

    Article  CAS  PubMed  Google Scholar 

  76. Book review (2012) Pharmaceutical biotechnology: drug discovery and clinical applications (2nd edition). Biotechnol J 7(9):1061–1062. https://doi.org/10.1002/biot.201200115

    Article  Google Scholar 

  77. Stock K, Estrada MF, Vidic S, Gjerde K, Rudisch A, Santo VE et al (2016) Capturing tumor complexity in vitro: comparative analysis of 2D and 3D tumor models for drug discovery. Sci Rep 6:28951. https://doi.org/10.1038/srep28951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Antoni D, Burckel H, Josset E, Noel G (2015) Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 16(3):5517–5527

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Huang L, Abdalla AME, Xiao L, Yang G (2020) Biopolymer-based microcarriers for three-dimensional cell culture and engineered tissue formation. Int J Mol Sci 21(5):1895

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Kelm JM, Fussenegger M (2004) Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol 22(4):195–202. https://doi.org/10.1016/j.tibtech.2004.02.002

    Article  CAS  PubMed  Google Scholar 

  81. Gebhardt R, Hengstler JG, Muller D, Glockner R, Buenning P, Laube B et al (2003) New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab Rev 35(2–3):145–213. https://doi.org/10.1081/dmr-120023684

    Article  CAS  PubMed  Google Scholar 

  82. Huh D, Hamilton GA, Ingber DE (2011) From 3D cell culture to organs-on-chips. Trends Cell Biol 21(12):745–754. https://doi.org/10.1016/j.tcb.2011.09.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Sontheimer-Phelps A, Hassell BA, Ingber DE (2019) Modelling cancer in microfluidic human organs-on-chips. Nat Rev Cancer 19(2):65–81. https://doi.org/10.1038/s41568-018-0104-6

    Article  CAS  PubMed  Google Scholar 

  84. Mondadori C, Crippa M, Moretti M, Candrian C, Lopa S, Arrigoni C (2020) Advanced microfluidic models of cancer and immune cell extravasation: a systematic review of the literature. Front Bioeng Biotechnol 8:907. https://doi.org/10.3389/fbioe.2020.00907

    Article  PubMed  PubMed Central  Google Scholar 

  85. Boussommier-Calleja A, Li R, Chen MB, Wong SC, Kamm RD (2016) Microfluidics: a new tool for modeling cancer-immune interactions. Trends Cancer 2(1):6–19. https://doi.org/10.1016/j.trecan.2015.12.003

    Article  PubMed  PubMed Central  Google Scholar 

  86. Okuyama T, Yamazoe H, Mochizuki N, Khademhosseini A, Suzuki H, Fukuda J (2010) Preparation of arrays of cell spheroids and spheroid-monolayer cocultures within a microfluidic device. J Biosci Bioeng 110(5):572–576. https://doi.org/10.1016/j.jbiosc.2010.05.013

    Article  CAS  PubMed  Google Scholar 

  87. Huang CP, Lu J, Seon H, Lee AP, Flanagan LA, Kim HY et al (2009) Engineering microscale cellular niches for three-dimensional multicellular co-cultures. Lab Chip 9(12):1740–1748. https://doi.org/10.1039/b818401a

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Hsiao AY, Torisawa YS, Tung YC, Sud S, Taichman RS, Pienta KJ et al (2009) Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids. Biomaterials 30(16):3020–3027. https://doi.org/10.1016/j.biomaterials.2009.02.047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Jin HJ, Cho YH, Gu JM, Kim J, Oh YS (2011) A multicellular spheroid formation and extraction chip using removable cell trapping barriers. Lab Chip 11(1):115–119. https://doi.org/10.1039/c0lc00134a

    Article  CAS  PubMed  Google Scholar 

  90. Agastin S, Giang UB, Geng Y, Delouise LA, King MR (2011) Continuously perfused microbubble array for 3D tumor spheroid model. Biomicrofluidics 5(2):24110. https://doi.org/10.1063/1.3596530

    Article  CAS  PubMed  Google Scholar 

  91. Lee SA, No da Y, Kang E, Ju J, Kim DS, Lee SH (2013) Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects. Lab Chip 13(18):3529–3537. https://doi.org/10.1039/c3lc50197c

    Article  CAS  PubMed  Google Scholar 

  92. Leary E, Rhee C, Wilks BT, Morgan JR (2018) Quantitative live-cell confocal imaging of 3D spheroids in a high-throughput format. SLAS Technol 23(3):231–242. https://doi.org/10.1177/2472630318756058

    Article  PubMed  PubMed Central  Google Scholar 

  93. Tutty MA, Adriele P-M (2021) Pre-clinical 2D and 3D toxicity response to a panel of nanomaterials; Comparative assessment of NBM-induced liver toxicity. Drug Deliv Transl Res 12(9):2157–2217

    Article  Google Scholar 

  94. Jaros J, Petrov M, Tesarova M, Hampl A (2017) Revealing 3D ultrastructure and morphology of stem cell spheroids by electron microscopy. Methods Mol Biol 1612:417–431. https://doi.org/10.1007/978-1-4939-7021-6_30

    Article  CAS  PubMed  Google Scholar 

  95. Relucenti M, Francescangeli F, De Angelis ML, D’Andrea V, Miglietta S, Pilozzi E et al (2021) The ultrastructural analysis of human colorectal cancer stem cell-derived spheroids and their mouse xenograft shows that the same cells types have different ratios. Biology (Basel) 10(9):929. https://doi.org/10.3390/biology10090929

    Article  CAS  PubMed  Google Scholar 

  96. Kyffin JA, Sharma P, Leedale J, Colley HE, Murdoch C, Harding AL et al (2019) Characterisation of a functional rat hepatocyte spheroid model. Toxicol In Vitro 55:160–172. https://doi.org/10.1016/j.tiv.2018.12.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Correa de Sampaio P, Auslaender D, Krubasik D, Failla AV, Skepper JN, Murphy G et al (2012) A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis. PLoS One 7(2):e30753. https://doi.org/10.1371/journal.pone.0030753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Nederman T, Norling B, Glimelius B, Carlsson J, Brunk U (1984) Demonstration of an extracellular matrix in multicellular tumor spheroids. Cancer Res 44(7):3090–3097

    CAS  PubMed  Google Scholar 

  99. Granato G, Ruocco MR, Iaccarino A, Masone S, Calì G, Avagliano A et al (2017) Generation and analysis of spheroids from human primary skin myofibroblasts: an experimental system to study myofibroblasts deactivation. Cell Death Discov 3(1):17038. https://doi.org/10.1038/cddiscovery.2017.38

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Bergdorf KN, Phifer CJ, Bechard ME, Lee MA, McDonald OG, Lee E et al (2021) Immunofluorescent staining of cancer spheroids and fine-needle aspiration-derived organoids. STAR Protocols 2(2):100578. https://doi.org/10.1016/j.xpro.2021.100578

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Piccinini F, Tesei A, Arienti C, Bevilacqua A (2015) Cancer multicellular spheroids: volume assessment from a single 2D projection. Comput Methods Prog Biomed 118(2):95–106. https://doi.org/10.1016/j.cmpb.2014.12.003

    Article  Google Scholar 

  102. Gebhard C, Gabriel C, Walter I (2016) Morphological and Immunohistochemical characterization of canine osteosarcoma spheroid cell cultures. Anat Histol Embryol 45(3):219–230. https://doi.org/10.1111/ahe.12190

    Article  CAS  PubMed  Google Scholar 

  103. 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. https://doi.org/10.1186/1471-2407-13-73

    Article  PubMed  PubMed Central  Google Scholar 

  104. Huang C-H, Lei KF, Tsang N-M (2019) Apoptosis and cell cycle arrest of hepatocellular carcinoma spheroids treated by an alternating electric field. Biotechnol Prog 35(3):e2787. https://doi.org/10.1002/btpr.2787

    Article  CAS  PubMed  Google Scholar 

  105. Chelobanov B, Poletaeva J, Epanchintseva A, Tupitsyna A, Pyshnaya I, Ryabchikova E (2020) Ultrastructural features of gold nanoparticles interaction with HepG2 and HEK293 cells in monolayer and spheroids. Nanomaterials (Basel) 10(10):2040. https://doi.org/10.3390/nano10102040

    Article  CAS  PubMed  Google Scholar 

  106. Ma H-l, Jiang Q, Han S, Wu Y, Tomshine JC, Wang D et al (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):7290.2012.00012. https://doi.org/10.2310/7290.2012.00012

    Article  CAS  Google Scholar 

  107. England CG, Gobin AM, Frieboes HB (2015) Evaluation of uptake and distribution of gold nanoparticles in solid tumors. Eur Phys J Plus 130(11). https://doi.org/10.1140/epjp/i2015-15231-1

  108. Wen Z, Liao Q, Hu Y, You L, Zhou L, Zhao Y (2013) A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay. Braz J Med Biol Res 46(7):634–642. https://doi.org/10.1590/1414-431x20132647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Bresciani G, Hofland LJ, Dogan F, Giamas G, Gagliano T, Zatelli MC (2019) Evaluation of spheroid 3D culture methods to study a pancreatic neuroendocrine neoplasm cell line. Front Endocrinol 10:682. https://doi.org/10.3389/fendo.2019.00682

    Article  Google Scholar 

  110. Eilenberger C, Kratz SRA, Rothbauer M, Ehmoser EK, Ertl P, Küpcü S (2018) Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids. MethodsX 5:781–787. https://doi.org/10.1016/j.mex.2018.07.011

    Article  PubMed  PubMed Central  Google Scholar 

  111. Zanoni M, Piccinini F, Arienti C, Zamagni A, Santi S, Polico R et al (2016) 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6(1):19103. https://doi.org/10.1038/srep19103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Murali VS, Chang B-J, Fiolka R, Danuser G, Cobanoglu MC, Welf ES (2019) An image-based assay to quantify changes in proliferation and viability upon drug treatment in 3D microenvironments. BMC Cancer 19(1):502. https://doi.org/10.1186/s12885-019-5694-1

    Article  PubMed  PubMed Central  Google Scholar 

  113. Tutty MA, Prina-Mello A, Vella G (2022) Pre-clinical 2D and 3D toxicity response to a panel of nanomaterials; comparative assessment of NBM-induced liver toxicity. Res Square. https://doi.org/10.21203/rs.3.rs-1277778/v1

  114. Patra B, Peng C-C, Liao W-H, Lee C-H, Tung Y-C (2016) Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device. Sci Rep 6(1):21061. https://doi.org/10.1038/srep21061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Beaumont KA, Anfosso A, Ahmed F, Weninger W, Haass NK (2015) Imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3D melanoma spheroids. J Vis Exp 106:e53486. https://doi.org/10.3791/53486

    Article  CAS  Google Scholar 

  116. Grässer U, Bubel M, Sossong D, Oberringer M, Pohlemann T, Metzger W (2018) Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis. Ann Anat – Anat Anz 216:1–8. https://doi.org/10.1016/j.aanat.2017.10.002

    Article  Google Scholar 

  117. Pulak R (2006) Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system. Methods Mol Biol 351:275–286. https://doi.org/10.1385/1-59745-151-7:275

    Article  PubMed  Google Scholar 

  118. K. McBain*, M. Oliver, L. Kelsey, C. Szybut and T. Dale (2021) Quantifying T cell response in 3D tumor spheroids using advanced flow cytometry workflows. https://www.sartorius.com/en/products/flow-cytometry/flow-cytometryresources/quantifying-t-cell-response-in-3d-tumor-spheroids-using-advanced-flow-cytometry-workflows-applicationnote

  119. Senthebane DA, Rowe A, Thomford NE, Shipanga H, Munro D, Mazeedi M et al (2017) The role of tumor microenvironment in Chemoresistance: to survive, keep your enemies closer. Int J Mol Sci 18(7). https://doi.org/10.3390/ijms18071586

  120. Zheng HC (2017) The molecular mechanisms of chemoresistance in cancers. Oncotarget 8(35):59950–59964. https://doi.org/10.18632/oncotarget.19048

    Article  PubMed  PubMed Central  Google Scholar 

  121. Barbone D, Yang TM, Morgan JR, Gaudino G, Broaddus VC (2008) Mammalian target of rapamycin contributes to the acquired apoptotic resistance of human mesothelioma multicellular spheroids. J Biol Chem 283(19):13021–13030. https://doi.org/10.1074/jbc.M709698200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Huanwen W, Zhiyong L, Xiaohua S, Xinyu R, Kai W, Tonghua L (2009) Intrinsic chemoresistance to gemcitabine is associated with constitutive and laminin-induced phosphorylation of FAK in pancreatic cancer cell lines. Mol Cancer 8:125. https://doi.org/10.1186/1476-4598-8-125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Longati P, Jia X, Eimer J, Wagman A, Witt M-R, Rehnmark S et al (2013) 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 13(1):95. https://doi.org/10.1186/1471-2407-13-95

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Thomas F, Holly JMP, Persad R, Bahl A, Perks CM (2010) Fibronectin confers survival against chemotherapeutic agents but not against radiotherapy in DU145 prostate cancer cells: involvement of the insulin like growth factor-1 receptor. The Prostate 70(8):856–865. https://doi.org/10.1002/pros.21119

    Article  CAS  PubMed  Google Scholar 

  125. Weigelt B, Lo AT, Park CC, Gray JW, Bissell MJ (2010) HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment. Breast Cancer Res Treat 122(1):35–43. https://doi.org/10.1007/s10549-009-0502-2

    Article  CAS  PubMed  Google Scholar 

  126. Sethi T, Rintoul RC, Moore SM, MacKinnon AC, Salter D, Choo C et al (1999) Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat Med 5(6):662–668. https://doi.org/10.1038/9511

    Article  CAS  PubMed  Google Scholar 

  127. Aoudjit F, Vuori K (2001) Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells. Oncogene 20(36):4995–5004. https://doi.org/10.1038/sj.onc.1204554

    Article  CAS  PubMed  Google Scholar 

  128. Nunes AS, Barros AS, Costa EC, Moreira AF, Correia IJ (2019) 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnol Bioeng 116(1):206–226. https://doi.org/10.1002/bit.26845

    Article  CAS  PubMed  Google Scholar 

  129. Frankel A, Man S, Elliott P, Adams J, Kerbel RS (2000) Lack of multicellular drug resistance observed in human ovarian and prostate carcinoma treated with the proteasome inhibitor PS-341. Clin Cancer Res 6(9):3719–3728

    CAS  PubMed  Google Scholar 

  130. Ferrante A, Rainaldi G, Indovina P, Indovina PL, Santini MT (2006) Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation. Int J Oncol 28(1):111–118

    CAS  PubMed  Google Scholar 

  131. Salata OV (2004) Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2(1):3. https://doi.org/10.1186/1477-3155-2-3

    Article  Google Scholar 

  132. Shi Y, van der Meel R, Chen X, Lammers T (2020) The EPR effect and beyond: strategies to improve tumor targeting and cancer nanomedicine treatment efficacy. Theranostics 10(17):7921–7924. https://doi.org/10.7150/thno.49577

    Article  PubMed  PubMed Central  Google Scholar 

  133. Wick P, Grafmueller S, Petri-Fink A, Rothen-Rutishauser B (2014) Advanced human in vitro models to assess metal oxide nanoparticle-cell interactions. MRS Bull 39(11):984–989. https://doi.org/10.1557/mrs.2014.219

    Article  CAS  Google Scholar 

  134. Alepee N, Bahinski A, Daneshian M, De Wever B, Fritsche E, Goldberg A et al (2014) State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. Altex 31(4):441–477. https://doi.org/10.14573/altex.1406111

    Article  PubMed  PubMed Central  Google Scholar 

  135. Mó I, Sabino IJ, Melo-Diogo D, Lima-Sousa R, Alves CG, Correia IJ (2020) The importance of spheroids in analyzing nanomedicine efficacy. Nanomedicine 15(15):1513–1525. https://doi.org/10.2217/nnm-2020-0054

    Article  CAS  PubMed  Google Scholar 

  136. Zhang X, Jiang T, Chen D, Wang Q, Zhang LW (2020) Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation. Crit Rev Toxicol 50(4):279–309. https://doi.org/10.1080/10408444.2020.1756219

    Article  CAS  PubMed  Google Scholar 

  137. Capek I (2006) Chapter 1: Nanotechnology and nanomaterials. In: Capek I (ed) Studies in interface science. Elsevier, Amsterdam, pp 1–69

    Google Scholar 

  138. Millard M, Yakavets I, Zorin V, Kulmukhamedova A, Marchal S, Bezdetnaya L (2017) Drug delivery to solid tumors: the predictive value of the multicellular tumor spheroid model for nanomedicine screening. Int J Nanomedicine 12:7993–8007. https://doi.org/10.2147/ijn.s146927

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Huang K, Boerhan R, Liu C, Jiang G (2017) Nanoparticles penetrate into the multicellular spheroid-on-chip: effect of surface charge, protein Corona, and exterior flow. Mol Pharm 14(12):4618–4627. https://doi.org/10.1021/acs.molpharmaceut.7b00726

    Article  CAS  PubMed  Google Scholar 

  140. Zhao J, Lu H, Wong S, Lu M, Xiao P, Stenzel MH (2017) Influence of nanoparticle shapes on cellular uptake of paclitaxel loaded nanoparticles in 2D and 3D cancer models. Polym Chem 8(21):3317–3326. https://doi.org/10.1039/C7PY00385D

    Article  CAS  Google Scholar 

  141. Cho W, Kim MS, Lee KH, Park SJ, Shin HJ, Lee YJ et al (2020) Ionizing radiation attracts tumor targeting and apoptosis by radiotropic lysyl oxidase traceable nanoparticles. Nanomedicine 24:102141. https://doi.org/10.1016/j.nano.2019.102141

    Article  CAS  PubMed  Google Scholar 

  142. Sadri A, Changizi V, Eivazadeh N (2015) Evaluation of glioblastoma (U87) treatment with ZnO nanoparticle and X-ray in spheroid culture model using MTT assay. Radiat Phys Chem 115:17–21. https://doi.org/10.1016/j.radphyschem.2015.05.035

    Article  CAS  Google Scholar 

  143. Gaspar VM, Baril P, Costa EC, de Melo-Diogo D, Foucher F, Queiroz JA et al (2015) Bioreducible poly(2-ethyl-2-oxazoline)-PLA-PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin. J Control Release 213:175–191. https://doi.org/10.1016/j.jconrel.2015.07.011

    Article  CAS  PubMed  Google Scholar 

  144. Gaspar VM, Costa EC, Queiroz JA, Pichon C, Sousa F, Correia IJ (2015) Folate-targeted multifunctional amino acid-chitosan nanoparticles for improved cancer therapy. Pharm Res 32(2):562–577. https://doi.org/10.1007/s11095-014-1486-0

    Article  CAS  PubMed  Google Scholar 

  145. Wu J, Feng S, Liu W, Gao F, Chen Y (2017) Targeting integrin-rich tumors with temoporfin-loaded vitamin-E-succinate-grafted chitosan oligosaccharide/d-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles to enhance photodynamic therapy efficiency. Int J Pharm 528(1–2):287–298. https://doi.org/10.1016/j.ijpharm.2017.06.021

    Article  CAS  PubMed  Google Scholar 

  146. Jin G, He R, Liu Q, Dong Y, Lin M, Li W et al (2018) Theranostics of triple-negative breast cancer based on conjugated polymer nanoparticles. ACS Appl Mater Interfaces 10(13):10634–10646. https://doi.org/10.1021/acsami.7b14603

    Article  CAS  PubMed  Google Scholar 

  147. Kumari P, Jain S, Ghosh B, Zorin V, Biswas S (2017) Polylactide-based block Copolymeric micelles loaded with Chlorin e6 for photodynamic therapy: in vitro evaluation in monolayer and 3D spheroid models. Mol Pharm 14(11):3789–3800. https://doi.org/10.1021/acs.molpharmaceut.7b00548

    Article  CAS  PubMed  Google Scholar 

  148. Madsen SJ, Christie C, Hong SJ, Trinidad A, Peng Q, Uzal FA et al (2015) Nanoparticle-loaded macrophage-mediated photothermal therapy: potential for glioma treatment. Lasers Med Sci 30(4):1357–1365. https://doi.org/10.1007/s10103-015-1742-5

    Article  PubMed  PubMed Central  Google Scholar 

  149. Cheng X, Li D, Sun M, He L, Zheng Y, Wang X et al (2019) Co-delivery of DOX and PDTC by pH-sensitive nanoparticles to overcome multidrug resistance in breast cancer. Colloids Surf B Biointerfaces 181:185–197. https://doi.org/10.1016/j.colsurfb.2019.05.042

    Article  CAS  PubMed  Google Scholar 

  150. Du AW, Lu H, Stenzel MH (2015) Core-cross-linking accelerates antitumor activities of paclitaxel-conjugate micelles to prostate multicellular tumor spheroids: a comparison of 2D and 3D models. Biomacromolecules 16(5):1470–1479. https://doi.org/10.1021/acs.biomac.5b00282

    Article  CAS  PubMed  Google Scholar 

  151. Lu H, Utama RH, Kitiyotsawat U, Babiuch K, Jiang Y, Stenzel MH (2015) Enhanced transcellular penetration and drug delivery by crosslinked polymeric micelles into pancreatic multicellular tumor spheroids. Biomater Sci 3(7):1085–1095. https://doi.org/10.1039/C4BM00323C

    Article  CAS  PubMed  Google Scholar 

  152. Malarvizhi GL, Retnakumari AP, Nair S, Koyakutty M (2014) Transferrin targeted core-shell nanomedicine for combinatorial delivery of doxorubicin and sorafenib against hepatocellular carcinoma. Nanomedicine 10(8):1649–1659. https://doi.org/10.1016/j.nano.2014.05.011

    Article  CAS  PubMed  Google Scholar 

  153. Sarisozen C, Dhokai S, Tsikudo EG, Luther E, Rachman IM, Torchilin VP (2016) Nanomedicine based curcumin and doxorubicin combination treatment of glioblastoma with scFv-targeted micelles: in vitro evaluation on 2D and 3D tumor models. Eur J Pharm Biopharm 108:54–67. https://doi.org/10.1016/j.ejpb.2016.08.013

    Article  CAS  PubMed  Google Scholar 

  154. Cheheltani R, Ezzibdeh RM, Chhour P, Pulaparthi K, Kim J, Jurcova M et al (2016) Tunable, biodegradable gold nanoparticles as contrast agents for computed tomography and photoacoustic imaging. Biomaterials 102:87–97. https://doi.org/10.1016/j.biomaterials.2016.06.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Xiao Y-D, Paudel R, Liu J, Ma C, Zhang Z-S, Zhou S-K (2016) MRI contrast agents: classification and application (review). Int J Mol Med 38(5):1319–1326. https://doi.org/10.3892/ijmm.2016.2744

    Article  CAS  PubMed  Google Scholar 

  156. Cole LE, Ross RD, Tilley JM, Vargo-Gogola T, Roeder RK (2015) Gold nanoparticles as contrast agents in x-ray imaging and computed tomography. Nanomedicine (Lond) 10(2):321–341. https://doi.org/10.2217/nnm.14.171

    Article  CAS  PubMed  Google Scholar 

  157. Miyamoto Y, Koshidaka Y, Noguchi H, Oishi K, Saito H, Yukawa H et al (2013) Observation of positively charged magnetic nanoparticles inside HepG2 spheroids using electron microscopy. Cell Med 5(2–3):89–96. https://doi.org/10.3727/215517913x666530

    Article  PubMed  PubMed Central  Google Scholar 

  158. Peng X, Wang B, Yang Y, Zhang Y, Liu Y, He Y et al (2019) Liver tumor spheroid reconstitution for testing mitochondrial targeted magnetic hyperthermia treatment. ACS Biomater Sci Eng 5(3):1635–1644. https://doi.org/10.1021/acsbiomaterials.8b01630

    Article  CAS  PubMed  Google Scholar 

  159. Yohan D, Cruje C, Lu X, Chithrani D (2015) Elucidating the uptake and distribution of nanoparticles in solid tumors via a multilayered cell culture model. Nanomicro Lett 7(2):127–137. https://doi.org/10.1007/s40820-014-0025-1

    Article  CAS  PubMed  Google Scholar 

  160. Tutty MA, Movia D, Prina-Mello A (2022) Three-dimensional (3D) liver cell models – a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials. Drug Deliv Transl Res. https://doi.org/10.1007/s13346-022-01147-0

  161. Park J-K, Utsumi T, Seo Y-E, Deng Y, Satoh A, Saltzman WM et al (2016) Cellular distribution of injected PLGA-nanoparticles in the liver. Nanomedicine 12(5):1365–1374. https://doi.org/10.1016/j.nano.2016.01.013

    Article  CAS  PubMed  Google Scholar 

  162. Erler JT, Weaver VM (2009) Three-dimensional context regulation of metastasis. Clin Exp Metastasis 26(1):35–49. https://doi.org/10.1007/s10585-008-9209-8

    Article  PubMed  Google Scholar 

  163. Lefranc F, Brotchi J, Kiss R (2005) Possible future issues in the treatment of glioblastomas: special emphasis on cell migration and the resistance of migrating glioblastoma cells to apoptosis. J Clin Oncol 23(10):2411–2422. https://doi.org/10.1200/jco.2005.03.089

    Article  CAS  PubMed  Google Scholar 

  164. Vinci M, Box C, Zimmermann M, Eccles SA (2013) Tumor spheroid-based migration assays for evaluation of therapeutic agents. Methods Mol Biol 986:253–266. https://doi.org/10.1007/978-1-62703-311-4_16

    Article  CAS  PubMed  Google Scholar 

  165. Weiswald LB, Bellet D, Dangles-Marie V (2015) Spherical cancer models in tumor biology. Neoplasia 17(1):1–15. https://doi.org/10.1016/j.neo.2014.12.004

    Article  PubMed  PubMed Central  Google Scholar 

  166. Cattin S, Ramont L, Rüegg C (2018) Characterization and in vivo validation of a three-dimensional multi-cellular culture model to study heterotypic interactions in colorectal cancer cell growth, invasion and metastasis. Front Bioeng Biotechnol 6:97. https://doi.org/10.3389/fbioe.2018.00097

    Article  PubMed  PubMed Central  Google Scholar 

  167. Gao Q, Yang Z, Xu S, Li X, Yang X, Jin P et al (2019) Heterotypic CAF-tumor spheroids promote early peritoneal metastasis of ovarian cancer. J Exp Med 216(3):688–703. https://doi.org/10.1084/jem.20180765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Almahmoudi R, Salem A, Murshid S, Dourado MR, Apu EH, Salo T et al (2019) Interleukin-17F has anti-tumor effects in Oral tongue cancer. Cancers 11(5):650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. Liu H, Lu T, Kremers G-J, Seynhaeve ALB, ten Hagen TLM (2020) A microcarrier-based spheroid 3D invasion assay to monitor dynamic cell movement in extracellular matrix. Biol Proced Online 22(1):3. https://doi.org/10.1186/s12575-019-0114-0

    Article  PubMed  PubMed Central  Google Scholar 

  170. Conti S, Kato T, Park D, Sahai E, Trepat X, Labernadie A (2021) CAFs and cancer cells co-migration in 3D spheroid invasion assay. Methods Mol Biol 2179:243–256. https://doi.org/10.1007/978-1-0716-0779-4_19

    Article  CAS  PubMed  Google Scholar 

  171. Stejskalová A, Fincke V, Nowak M, Schmidt Y, Borrmann K, von Wahlde M-K et al (2021) Collagen I triggers directional migration, invasion and matrix remodeling of stroma cells in a 3D spheroid model of endometriosis. Sci Rep 11(1):4115. https://doi.org/10.1038/s41598-021-83645-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Nazari SS (2020) Generation of 3D tumor spheroids with encapsulating basement membranes for invasion studies. Curr Protoc Cell Biol 87(1):e105. https://doi.org/10.1002/cpcb.105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Bell HS, Wharton SB, Leaver HA, Whittle IR (1999) Effects of N-6 essential fatty acids on glioma invasion and growth: experimental studies with glioma spheroids in collagen gels. J Neurosurg 91(6):989–996. https://doi.org/10.3171/jns.1999.91.6.0989

    Article  CAS  PubMed  Google Scholar 

  174. Berens EB, Holy JM, Riegel AT, Wellstein A (2015) A cancer cell spheroid assay to assess invasion in a 3D setting. Vis Exp 105:e53409. https://doi.org/10.3791/53409

    Article  Google Scholar 

  175. Verma M (2012) Personalized medicine and cancer. J Perinat Med 2(1):1–14. https://doi.org/10.3390/jpm2010001

    Article  CAS  Google Scholar 

  176. Pauli C, Hopkins BD, Prandi D, Shaw R, Fedrizzi T, Sboner A et al (2017) Personalized in vitro and in vivo cancer models to guide precision medicine. Cancer Discov 7(5):462–477. https://doi.org/10.1158/2159-8290.Cd-16-1154

    Article  PubMed  PubMed Central  Google Scholar 

  177. Offit K (2011) Personalized medicine: new genomics, old lessons. Hum Genet 130(1):3–14. https://doi.org/10.1007/s00439-011-1028-3

    Article  PubMed  PubMed Central  Google Scholar 

  178. Gilazieva Z, Ponomarev A, Rutland C, Rizvanov A, Solovyeva V (2020) Promising applications of tumor spheroids and organoids for personalized medicine. Cancers (Basel) 12(10). https://doi.org/10.3390/cancers12102727

  179. Ong SM, Zhao Z, Arooz T, Zhao D, Zhang S, Du T et al (2010) Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies. Biomaterials 31(6):1180–1190. https://doi.org/10.1016/j.biomaterials.2009.10.049

    Article  CAS  PubMed  Google Scholar 

  180. Kobayashi H, Man S, Graham CH, Kapitain SJ, Teicher BA, Kerbel RS (1993) Acquired multicellular-mediated resistance to alkylating agents in cancer. Proc Natl Acad Sci 90(8):3294–3298. https://doi.org/10.1073/pnas.90.8.3294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. Lemmo S, Atefi E, Luker GD, Tavana H (2014) Optimization of aqueous biphasic tumor spheroid microtechnology for anti-cancer drug testing in 3D culture. Cell Mol Bioeng 7(3):344–354. https://doi.org/10.1007/s12195-014-0349-4

    Article  CAS  PubMed  Google Scholar 

  182. Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E et al (2012) HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer 12(1):4. https://doi.org/10.1186/1471-2407-12-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. Pattni BS, Nagelli SG, Aryasomayajula B, Deshpande PP, Kulkarni A, Hartner WC et al (2016) Targeting of micelles and liposomes loaded with the pro-apoptotic drug, NCL-240, into NCI/ADR-RES cells in a 3D spheroid model. Pharm Res 33(10):2540–2551. https://doi.org/10.1007/s11095-016-1978-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  184. Sachs N, de Ligt J, Kopper O, Gogola E, Bounova G, Weeber F et al (2018) A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172(1–2):373–86.e10. https://doi.org/10.1016/j.cell.2017.11.010

    Article  CAS  PubMed  Google Scholar 

  185. Nanki K, Toshimitsu K, Takano A, Fujii M, Shimokawa M, Ohta Y et al (2018) Divergent routes toward Wnt and R-spondin niche independency during human gastric carcinogenesis. Cell 174(4):856–69.e17. https://doi.org/10.1016/j.cell.2018.07.027

    Article  CAS  PubMed  Google Scholar 

  186. Vlachogiannis G, Hedayat S, Vatsiou A, Jamin Y, Fernández-Mateos J, Khan K et al (2018) Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359(6378):920–926. https://doi.org/10.1126/science.aao2774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  187. Antunes N, Kundu B, Kundu SC, Reis RL, Correlo V (2022) In vitro cancer models: a closer look at limitations on translation. Bioengineering (Basel) 9(4). https://doi.org/10.3390/bioengineering9040166

  188. Ekert JE, Deakyne J, Pribul-Allen P, Terry R, Schofield C, Jeong CG et al (2020) Recommended guidelines for developing, qualifying, and implementing complex in vitro models (CIVMs) for drug discovery. SLAS Discov 25(10):1174–1190. https://doi.org/10.1177/2472555220923332

    Article  CAS  PubMed  Google Scholar 

  189. Zanoni M, Cortesi M, Zamagni A, Arienti C, Pignatta S, Tesei A (2020) Modeling neoplastic disease with spheroids and organoids. J Hematol Oncol 13(1):97. https://doi.org/10.1186/s13045-020-00931-0

    Article  PubMed  PubMed Central  Google Scholar 

  190. Foster AJ, Chouhan B, Regan SL, Rollison H, Amberntsson S, Andersson LC et al (2019) Integrated in vitro models for hepatic safety and metabolism: evaluation of a human Liver-Chip and liver spheroid. Arch Toxicol 93(4):1021–1037. https://doi.org/10.1007/s00204-019-02427-4

    Article  CAS  PubMed  Google Scholar 

  191. Walker PA, Ryder S, Lavado A, Dilworth C, Riley RJ (2020) The evolution of strategies to minimise the risk of human drug-induced liver injury (DILI) in drug discovery and development. Arch Toxicol 94(8):2559–2585. https://doi.org/10.1007/s00204-020-02763-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Williams DP, Lazic SE, Foster AJ, Semenova E, Morgan P (2020) Predicting drug-induced liver injury with Bayesian machine learning. Chem Res Toxicol 33(1):239–248. https://doi.org/10.1021/acs.chemrestox.9b00264

    Article  CAS  PubMed  Google Scholar 

  193. Wax PM (1995) Elixirs, diluents, and the passage of the 1938 Federal Food, drug and cosmetic act. Ann Intern Med 122(6):456–461. https://doi.org/10.7326/0003-4819-122-6-199503150-00009

    Article  CAS  PubMed  Google Scholar 

  194. Administration USFaD (2017) Investigational new drug (IND) application. U.S. Food and Drug Administration

    Google Scholar 

  195. Tutty MA (2021) Three-dimensional (3D) hepatic cell culture models to improve the clinical translation of nanobiomaterials (NBMs). Trinity College Dublin

    Google Scholar 

  196. Courau T, Bonnereau J, Chicoteau J, Bottois H, Remark R, Assante Miranda L et al (2019) Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment. J Immunother Cancer 7(1):74. https://doi.org/10.1186/s40425-019-0553-9

    Article  PubMed  PubMed Central  Google Scholar 

  197. Aref AR, Campisi M, Ivanova E, Portell A, Larios D, Piel BP et al (2018) 3D microfluidic ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade. Lab Chip 18(20):3129–3143. https://doi.org/10.1039/C8LC00322J

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  198. Jeong SY, Lee JH, Shin Y, Chung S, Kuh HJ (2016) Co-culture of tumor spheroids and fibroblasts in a collagen matrix-incorporated microfluidic Chip mimics reciprocal activation in solid tumor microenvironment. PLoS One 11(7):e0159013. https://doi.org/10.1371/journal.pone.0159013

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  199. Shao H, Moller M, Wang D, Ting A, Boulina M, Liu ZJ (2020) A novel stromal fibroblast-modulated 3D tumor spheroid model for studying tumor-stroma interaction and drug discovery. J Vis Exp 156. https://doi.org/10.3791/60660

  200. Yakavets I, Francois A, Benoit A, Merlin J-L, Bezdetnaya L, Vogin G (2020) Advanced co-culture 3D breast cancer model for investigation of fibrosis induced by external stimuli: optimization study. Sci Rep 10(1):21273. https://doi.org/10.1038/s41598-020-78087-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  201. Korff T, Augustin HG (1998) Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol 143(5):1341–1352. https://doi.org/10.1083/jcb.143.5.1341

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  202. Upreti M, Jamshidi-Parsian A, Koonce NA, Webber JS, Sharma SK, Asea AA et al (2011) Tumor-endothelial cell three-dimensional spheroids: new aspects to enhance radiation and drug therapeutics. Transl Oncol 4(6):365–376. https://doi.org/10.1593/tlo.11187

    Article  PubMed  PubMed Central  Google Scholar 

  203. Heiss M, Hellström M, Kalén M, May T, Weber H, Hecker M et al (2015) Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro. FASEB J 29(7):3076–3084. https://doi.org/10.1096/fj.14-267633

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We acknowledged the partial financial support from the European Union under H2020 REFINE (ref 761104), and EXPERT (ref 825828).

The authors would also like to thank Dr. G. McManus, Microscopy and Imaging facility manager; Professor C. O’Farrelly, Chair in Comparative Immunology, Trinity College Dublin; Dr. Dania Movia, Trinity College Dublin; and Professor M. Santin, from University of Brighton, for the technical discussions and insight on 3D spheroids imaging and biology.

Author information

Authors and Affiliations

  1. Laboratory for Biological Characterization of Advanced Materials (LBCAM), Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Trinity College Dublin, Dublin, Ireland

    Melissa Anne Tutty & Adriele Prina-Mello

  2. Nanomedicine and Molecular Imaging Group, Trinity Translational Medicine Institute, (TTMI), School of Medicine, Trinity College Dublin, Dublin, Ireland

    Adriele Prina-Mello

  3. Trinity St. James’s Cancer Institute, St. James’s Hospital, Trinity College Dublin, Dublin, Ireland

    Adriele Prina-Mello

  4. Advanced Materials and Bioengineering Research (AMBER) Centre, CRANN Institute, Trinity College Dublin, Dublin, Ireland

    Adriele Prina-Mello

Authors
  1. Melissa Anne Tutty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adriele Prina-Mello
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Melissa Anne Tutty .

Editor information

Editors and Affiliations

  1. Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Dublin, Ireland

    Dania Movia

  2. Trinity Translational Medicine Institute, Trinity Centre for Health Sciences, Dublin, Ireland

    Adriele Prina-Mello

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Tutty, M.A., Prina-Mello, A. (2023). Three-Dimensional Spheroids for Cancer Research. In: Movia, D., Prina-Mello, A. (eds) Cancer Cell Culture. Methods in Molecular Biology, vol 2645. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3056-3_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3056-3_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3055-6

  • Online ISBN: 978-1-0716-3056-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation