AccScience Publishing / IJB / Volume 9 / Issue 6 / DOI: 10.36922/ijb.0985

3D bioprinting-based single liver tumor spheroid analysis for aflatoxin B1-induced drug-resistant cancer cell

Viet Phuong Cao1 Sera Hong1 Joon Myong Song1 *
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1 College of Pharmacy, Seoul National University, Seoul 08826, South Korea
Submitted: 24 May 2023 | Accepted: 1 July 2023 | Published: 18 August 2023
© 2023 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( )

Aflatoxin B1, found in a variety of foods, is a mycotoxin known to cause cancer. Therefore, humans may be exposed to it through their daily diet. In this study, a three-dimensional (3D) tumor spheroid model was developed via 3D bioprinting to examine whether exposure of HepG2 liver tumor spheroids to aflatoxin B1 can increase the population of drug-resistant liver cancer cells in a single tumor spheroid. Two biomarkers, CD133 (prominin-1) and aldehyde dehydrogenase 1 (ALDH1), were used to identify drug-resistant cancer cells formed in the single liver tumor spheroids. The induction of drug-resistant cancer cells in the single tumor spheroids was examined through single spheroid imaging and fluorescence-activated cell sorting (FACS). The increase of drug-resistant cancer cells, which was caused by aflatoxin B1 in a dose-dependent manner, was quantitatively monitored at the single tumor spheroid level using both methods. 3D bioprinting-fabricated single liver tumor spheroid model successfully determined drug-resistant liver cancer cells caused by aflatoxin B1

3D bioprinting
Single tumor spheroid imaging
Aflatoxin B1
Drug-resistant cancer cell
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (MEST) (No. 2018M3A7B4071235, 2023R1A2C3006991, and RS-2023-00218543).
  1. Dzobo K, Senthebane DA, Ganz C, et al., 2020, Advances in therapeutic targeting of cancer stem cells within the tumor microenvironment: An updated review. Cells, 9(8): 1896.



  1. Dzobo K, Senthebane DA, Rowe A, et al., 2016, Cancer stem cell hypothesis for therapeutic innovation in clinical oncology? Taking the root out, not chopping the leaf. Omics J Integr Biol, 20: 681–691.



  1. Batlle E, Clevers H, 2017, Cancer stem cells revisited. Nat Med 23: 1124–1134.



  1. Ahn J, Choi HJ, Bang J, et al., 2022, Ink-lithographic fabrication of silver-nanocrystal-based multiaxial strain gauge sensors through the coffee-ring effect for voice recognition applications. Nano Converg, 9: 46.



  1. Walcher L, Kistenmacher AK, Suo H, et al., 2020, Cancer stem cells-origins and biomarkers: perspectives for targeted personalized therapies. Front Immunol, 11: 1280.



  1. Huang T, Song X, Xu D, et al., 2020, Stem cell programs in cancer initiation, progression, and therapy resistance. Theranostics, 10(19): 8721–843.



  1. Najafi M, Mortezaee K, Majidpoor J, 2019, Cancer stem cell (CSC) resistance drivers. Life Sci, 234: 116781.



  1. Li Y, Wang Z, Ajani JA, et al., 2021, Drug resistance and cancer stem cells. Cell Commun Signal, 19(1): 19.



  1. Dzobo K, Sinkala M, 2021, Cancer stem cell marker CD44 plays multiple key roles in human cancers: Immune suppression/evasion, drug resistance, epithelial-mesenchymal transition, and metastasis. Omics Journal of Integr Biol, 25(5): 313–332



  1. Fu JJ, Zhou Y, Shi XX, et al., 2019, Spontaneous formation of tumor spheroid on a hydrophilic filter paper for cancer stem cell enrichment Colloids Surf B: Biointerfaces, 174: 426–434



  1. Lapidot T, Sirard C, Vormoor J, et al., 1994, A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature, 367(6464): 645–648.



  1. Masciale V, Grisendi G, Banchelli F, et al., 2019, Isolation and identification of cancer stem-like cells in adenocarcinoma and squamous cell carcinoma of the lung: A pilot study. Front Oncol, 9: 1394.



  1. Suetsugu A, Nagaki M, Aoki H, et al., 2006, Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/ progenitor cells Biochem Biophys Res Commun, 351(4): 820–824.



  1. Verma RJ, 2004, Aflatoxin cause DNA damage. Int J Hum Genet, 4: 231–236.



  1. Jayaratne WMSC, Abeyratne AHMAK, De Zoysa HKS, et al., 2020, Detection and quantification of Aflatoxin B1 in corn and corn-grown soils in the district of Anuradhapura, Sri Lanka. Heliyon, 6(10): e05319.



  1. Asl GB, Arvand M, Habibi MF, 2022, High affinity aptamers for ultra-sensitive detection of aflatoxin B1 in milk and groundnut samples with label-free photo-electrochemical aptasensor. Food Chem, 397: 133829.



  1. Romero-Sánchez I, Ramírez-García L, Gracia-Lor E, et al., 2022, Simultaneous determination of aflatoxins B1, B2, G1 and G2 in commercial rices using immunoaffinity column clean-up and HPLC-MS/MS. Food Chem, 395: 133611.



  1. Ju H, Shim Y, Arumugam P, et al., 2016, Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1. Toxicol Lett, 243: 31–39.



  1. Birgersdotter A, Sandberg R, Ernberg I, 2005, Gene expression perturbation in vitro—A growing case for three-dimensional (3D) culture systems. Semin Cancer Biol, 15(5): 405–412.



  1. Yip D, Cho CH, 2013, A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing. Biochem Biophys Res Commun, 433(3): 327–332.



  1. Antoni D, Burckel H, Josset E, et al., 2015, Three-dimensional cell culture: A breakthrough in vivo. Int J Mol Sci, 16(3): 5517–5527.



  1. Khalaf K, Hana D, Chou JT, et al., 2021, Aspects of the tumor microenvironment involved in immune resistance and drug resistance. Front Immunol, 12: 656364.



  1. Senthebane DA, Rowe A, Thomford NE, et al., 2017, The role of tumor microenvironment in chemoresistance: to survive, keep your enemies closer. Int J Mol Sci, 18(7): 1586.



  1. Senthebane DA, Jonker T, Rowe A, et al., 2018, The role of tumor microenvironment in chemoresistance: 3D extracellular matrices as accomplices. Int J Mol Sci, 19(10): 2861.



  1. Dzobo K, Senthebane DA, Dandara C, 2023, The tumor microenvironment in tumorigenesis and therapy resistance revisited. Cancers (Basel), 15(2): 376.



  1. Dzobo K, Rowe A, Senthebane DA, et al. 2018, Three-dimensional organoids in cancer research: The search for the holy grail of preclinical cancer modeling. Omics Journal Integr Biol, 22(26): 733–748.



  1. Dzobo K, Dandara C, 2023, The extracellular matrix: Its composition, function, remodeling, and role in tumorigenesis. Biomimetics (Basel, Switzerland), 8(2): 146.



  1. Najafi M, Farhood B, Mortezaee K, 2019, Extracellular matrix (ECM) stiffness and degradation as cancer drivers. J Cell Biochem, 120(3): 2782–2790.



  1. Ravi M, Paramesh V, Kaviya SR, et al., 2015, 3D cell culture systems: Advantages and applications. J Cell Physiol 230(1): 16–26.



  1. Jensen C, Teng Y, 2020, Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci, 7: 33.



  1. Bokhari M, Carnachan RJ, Cameron NR, et al., 2007, Culture of HepG2 liver cells on three dimensional polystyrene scaffolds enhances cell structure and function during toxicological challenge. J Anatom, 211(4): 567–576.



  1. Rodriguez-Salvador M, Fox-Miranda I, Perez-Benitez BE, et al., 2022, Research dynamics of tissue spheroids as building blocks: A scientometric analysis. Int J Bioprint, 8(3): 585.



  1. Kim JY, Rhim W-K, Cha S-G, et al., 2022, Bolstering the secretion and bioactivities of umbilical cord MSC-derived extracellular vesicles with 3D culture and priming in chemically defined media. Nano Converg, 9(1): 57.



  1. Schyschka L, Sánchez JJM, Wang Z, et al., 2013, Hepatic 3D cultures but not 2D cultures preserve specific transporter activity for acetaminophen-induced hepatotoxicity. Arch Toxicol, 87(8): 1581–1593.



  1. Huang YJ, Hsu SH, 2014, Acquisition of epithelial-mesenchymal transition and cancer stem-like phenotypes within chitosan-hyaluronan membrane-derived 3D tumor spheroids. Biomaterials, 35(38): 10070–10079.



  1. Kawashima D, Yuki T, Li S, et al., 2022, Non-invasive imaging of ion concentration distribution around cell spheroids by electrical impedance tomographic sensor printed on circuit board under temporal compensation by ion transport impedance model Biosens Bioelectron, 212: 114432.



  1. Langhans SA, 2018, Three-dimensional in vitro cell culture models in drug discovery and drug repositioning, Front Pharmacol, 9: 6.



  1. Ahmed A, Azam A, Wang Y, et al., 2021, Additively manufactured nano-mechanical energy harvesting systems: Advancements, potential applications, challenges and future perspectives. Nano Converg, 8(1): 37.



  1. Ranjan P, Gaur S, Yadav H, et al., 2022, 2D materials: Increscent quantum flatland with immense potential for applications. Nano Converg, 9(1): 26.



  1. Kim J, Lee J-K, Chae B, et al., 2022, Near-field infrared nanoscopic study of EUV- and e-beam-exposed hydrogen silsesquioxane photoresist. Nano Converg, 9(1): 53.



  1. Pérez B, Nykvist H, Brøgger AF, et al., 2019, Impact of macronutrients printability and 3D-printer parameters on 3D-food printing: A review. Food Chem, 287: 249–257.



  1. Samson AAS, Balwe SG, Hong S, et al., 2023, Verification of nanomaterial-induced size-dependent human ether-à-go-go-related gene potassium channel blockage using three-dimensional bioengineered functional cardiac tissue constructs. Chem Mater, 35(2): 658–671.



  1. Yu J, Lee S, Song J, et al., 2022, Perfusable micro-vascularized 3D tissue array for high-throughput vascular phenotypic screening. Nano Converg, 9(1): 16.



  1. Dong Z, Gong J, Zhang H, et al., 2022, Preparation and characterization of 3D printed porous 45S5 bioglass bioceramic for bone tissue engineering application. Int J Bioprint, 8(4): 613.



  1. Abdelrahim AA, Hong S, Song JM, 2022, Integrative in situ photodynamic therapy-induced cell death measurement of 3D-bioprinted MCF-7 tumor spheroids. Anal Chem, 94: 13936–13943.



  1. Hong S, Song JM, 2021, A 3D cell printing-fabricated HepG2 liver spheroid model for high-content in situ quantification of drug-induced liver toxicity. Biomater Sci, 9(17): 5939–5950.



  1. Hong S, Song JM, 2022, 3D bioprinted drug-resistant breast cancer spheroids for quantitative in situ evaluation of drug resistance. Acta Biomater, 138: 228–239.



  1. Hong S, Song JM, 2023, High-resolution in situ high-content imaging of 3D-bioprinted single breast cancer spheroids for advanced quantification of benzo(a)pyrene carcinogen-induced breast cancer stem cells. ACS Appl Mater Interfaces, 15(9): 11416–11430.



  1. Shao L, Hou R, Zhu Y, et al., 2021, Pre-shear bioprinting of highly oriented porous hydrogel microfibers to construct anisotropic tissues. Biomater Sci, 9(20): 6763–6771.



  1. Tetsuka H, Shin SR, 2020, Materials and technical innovations in 3D printing in biomedical applications. J Mater Chem B, 8(15): 2930–2950.



  1. Wang T-Y, Li X-F, Liu S-M, et al., 2022, Self-assembled wide bandgap nanocoatings enabled outstanding dielectric characteristics in the sandwich-like structure polymer composites. Nano Converg, 9(1): 55.



  1. Clark DW, Palle KJA, 2016, Aldehyde dehydrogenases in cancer stem cells: Potential as therapeutic targets. Ann Transl Med, 4(24): 518.



  1. Sung H, Ferlay J, Siegel RL, et al., 2021, Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J Clin, 71(3): 209–249.



  1. Miao Y, Yang T, Yang S, et al., 2022, Protein nanoparticles directed cancer imaging and therapy. Nano Converg, 9(1): 2.



  1. Zhou H-M, Zhang J-G, Zhang X, et al., 2021, Targeting cancer stem cells for reversing therapy resistance: Mechanism, signaling, and prospective agents. Signal Transduct Target Ther, 6(1): 62.



  1. Yu Z, Pestell TG, Lisanti MP, et al., 2012, Cancer stem cells. Int J Biochem Cell Biol, 44(12): 2144–2151.



  1. Nguyen ST, Nguyen LS, Nguyen THP, et al., 2020, Isolation of cancer stem-like cells from hepatocellular carcinoma cell line HepG2 by methods of magnetic-activated cell sorting, spheroid culture, and anti-tumor drug-resistant selection: A primary evaluation. Prog Stem Cell, 7(1-2): 279–289.



  1. Dzobo K, Ganz C, Thomford NE, et al., 2021, Cancer stem cell markers in relation to patient survival outcomes: Lessons for integrative diagnostics and next-generation anticancer drug development. Omics J Integr Biol, 25(2): 81–92.



  1. Lu R, Zhao G, Yang Y, et al., 2019, Inhibition of CD133 overcomes cisplatin resistance through inhibiting PI3K/ AKT/mTOR signaling pathway and autophagy in CD133- positive gastric cancer cells. Technol Cancer Res Treatment, 18: 1533033819864311.



  1. Zhou L, Sun Y, Ye G, et al., 2022, Effects of CD133 expression on chemotherapy and drug sensitivity of adenoid cystic carcinoma. Mol Med Rep, 25(1): 18.



  1. Hiroyuki T, Tomohiro K, Ayumi N, et al., 2017, Updates in Liver Cancer, Mohamed AH (ed.), IntechOpen, Rijeka.



  1. Wei Y, Li Y, Chen Y, et al., 2022, ALDH1: A potential therapeutic target for cancer stem cells in solid tumors. Front Oncol, 12: 1026278.



  1. Link T, Iwakuma T, 2017, Roles of p53 in extrinsic factor-induced liver carcinogenesis Hepatoma Res, 3(6): 95–104.



  1. Kawasaki Y, Adachi N, Yamazaki T, et al., 2007, Cancer stem cells in aflatoxin B1-induced rat hepatocellular carcinoma K2 cells. JSM Mycotoxins, 57: 87–93.



  1. Feng W-H, Xue KS, Tang L, et al., 2017, Aflatoxin B1- induced developmental and DNA damage in caenorhabditis elegans. Toxins, 9(1): 9.

Conflict of interest
The authors declare no conflicts of interests.
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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing