Advances in the bladder cancer research using 3D culture models

Main Article Content

Yexin Gu
Ye Lu
Yunqiang Xiong
Xiangpeng Zhan
Taobin Liu
Min Tang
An Xie
Xiaoqiang Liu
Bin Fu


bladder cancer, cell culture, 3D culture models, 3D bioprinting


Bladder cancer represents the most common malignancy of the urinary system, posing a significant threat to patients' life. Animal models and two-dimensional (2D) cell cultures, among other traditional models, have been used for years to study various aspects of bladder cancer. However, these methods are subject to various limitations when mimicking the tumor microenvironment in vivo, thus hindering the further improvement of bladder cancer treatments. Recently, three-dimensional (3D) culture models have attracted extensive attention since they overcome the shortcomings of their traditional counterparts. Most importantly, 3D culture models more accurately reproduce the tumor microenvironment in the human body because they can recapitulate the cell-cell and cell-extracellular matrix interactions. 3D culture models can thereby help us gain deeper insight into the bladder cancer. The 3D culture models of tumor cells can extend the culture duration and allow for co-culturing with different cell types. Study of patient-specific bladder cancer mutations and subtypes is made possible by the ability to preserve cells isolated from particular patients in 3D culture models. It will be feasible to develop customized treatments that target relevant signaling pathways or biomarkers. This article reviews the development, application, advantages, and limitations of traditional modeling systems and 3D culture models used in the study of bladder cancer and discusses the potential application of 3D culture models.


Metrics Loading ...
Abstract 291 | HTML Downloads 42 PDF Downloads 91


1. Antoni S, Ferlay J, Soerjomataram I, Znaor A, Jemal A, Bray F. Bladder Cancer Incidence and Mortality: A Global Overview and Recent Trends. Eur Urol. 2017 Jan;71(1):96–108. PMID:27370177
2. Chen W, Xia C, Zheng R, Zhou M, Lin C, Zeng H, et al. Disparities by province, age, and sex in site-specific cancer burden attributable to 23 potentially modifiable risk factors in China: a comparative risk assessment. Lancet Glob Health. 2019 Feb;7(2):e257–69. PMID:30683243
3. Puzio-Kuter AM, Castillo-Martin M, Kinkade CW, Wang X, Shen TH, Matos T, et al. Inactivation of p53 and Pten promotes invasive bladder cancer. Genes Dev. 2009 Mar;23(6):675–80. PMID:19261747
4. Langhans SA. Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front Pharmacol. 2018 Jan;9:6. PMID:29410625
5. Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FD. 3D cell culture systems: advantages and applications. J Cell Physiol. 2015 Jan;230(1):16–26. PMID:24912145
6. Imamura Y, Mukohara T, Shimono Y, Funakoshi Y, Chayahara N, Toyoda M, et al. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol Rep. 2015 Apr;33(4):1837–43. PMID:25634491
7. Bodgi L, Bahmad HF, Araji T, Al Choboq J, Bou-Gharios J, Cheaito K, et al. Assessing Radiosensitivity of Bladder Cancer in vitro: A 2D vs. 3D Approach. Front Oncol. 2019 Mar;9:153. PMID:30941305
8. Ikari R, Mukaisho KI, Kageyama S, Nagasawa M, Kubota S, Nakayama T, et al. Differences in the Central Energy Metabolism of Cancer Cells between Conventional 2D and Novel 3D Culture Systems. Int J Mol Sci. 2021 Feb;22(4):1805. PMID:33670390
9. Jiang X, Lei T, Zhang M. Expression and Functions of Formyl Peptide Receptor 1 in Drug-Resistant Bladder Cancer. Technol Cancer Res Treat. 2018 Jan;17:1533034618769413. PMID:29665744
10. Yu S, Meng Q, Hu H, Zhang M. Correlation of ANXA1 expression with drug resistance and relapse in bladder cancer. Int J Clin Exp Pathol. 2014 Aug;7(9):5538–48. PMID:25337195
11. Zuiverloon TC, de Jong FC, Costello JC, Theodorescu D. Systematic Review: Characteristics and Preclinical Uses of Bladder Cancer Cell Lines. Bladder Cancer. 2018 Apr;4(2):169–83. PMID:29732388
12. Jamieson LE, Harrison DJ, Campbell CJ. Chemical analysis of multicellular tumour spheroids. Analyst (Lond). 2015 Jun;140(12):3910–20. PMID:25923379
13. Reis LO, Pereira TC, Favaro WJ, Cagnon VH, Lopes-Cendes I, Ferreira U. Experimental animal model and RNA interference: a promising association for bladder cancer research. World J Urol. 2009 Jun;27(3):353–61. PMID:19214530
14. Oliveira PA, Arantes-Rodrigues R, Vasconcelos-Nóbrega C. Animal models of urinary bladder cancer and their application to novel drug discovery. Expert Opin Drug Discov. 2014 May;9(5):485–503. PMID:24670247
15. Asanuma H, Arai T, Seguchi K, Kawauchi S, Satoh H, Kikuchi M, et al. Successful diagnosis of orthotopic rat superficial bladder tumor model by ultrathin cystoscopy. J Urol. 2003 Feb;169(2):718–20. PMID:12544350
16. Holmäng S, Cano M, Grenabo L, Hedelin H, Johansson SL. Effect of indomethacin on N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced urinary tract carcinogenesis. Carcinogenesis. 1995 Jul;16(7):1493–8. PMID:7614682
17. Montie JE. Profiling the evolution of human metastatic bladder cancer. J Urol. 2005 Aug;174(2):485–6. PMID:16006872
18. Watanabe T, Shinohara N, Sazawa A, Harabayashi T, Ogiso Y, Koyanagi T, et al. An improved intravesical model using human bladder cancer cell lines to optimize gene and other therapies. Cancer Gene Ther. 2000 Dec;7(12):1575–80. PMID:11228536
19. Wilmanns C, Fan D, Obrian C, Radinsky R, Bucana C, Tsan R, et al. Modulation of Doxorubicin sensitivity and level of p-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice. Int J Oncol. 1993 Sep;3(3):413–22. PMID:21573380
20. Overdevest JB, Thomas S, Kristiansen G, Hansel DE, Smith SC, Theodorescu D. CD24 offers a therapeutic target for control of bladder cancer metastasis based on a requirement for lung colonization. Cancer Res. 2011 Jun;71(11):3802–11. PMID:21482678
21. Costa EC, Moreira AF, de Melo-Diogo D, Gaspar VM, Carvalho MP, Correia IJ. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv. 2016 Dec;34(8):1427–41. PMID:27845258
22. Patel VG, Oh WK, Galsky MD. Treatment of muscle-invasive and advanced bladder cancer in 2020. CA Cancer J Clin. 2020 Sep;70(5):404–23. PMID:32767764
23. Zhang ZT, Pak J, Shapiro E, Sun TT, Wu XR. Urothelium-specific expression of an oncogene in transgenic mice induced the formation of carcinoma in situ and invasive transitional cell carcinoma. Cancer Res. 1999 Jul;59(14):3512–7. PMID:10416618
24. Kompier LC, Lurkin I, van der Aa MN, van Rhijn BW, van der Kwast TH, Zwarthoff EC. FGFR3, HRAS, KRAS, NRAS and PIK3CA mutations in bladder cancer and their potential as biomarkers for surveillance and therapy. PLoS One. 2010 Nov;5(11):e13821. PMID:21072204
25. Ayala de la Peña F, Kanasaki K, Kanasaki M, Tangirala N, Maeda G, Kalluri R. Loss of p53 and acquisition of angiogenic microRNA profile are insufficient to facilitate progression of bladder urothelial carcinoma in situ to invasive carcinoma. J Biol Chem. 2011 Jun;286(23):20778–87. PMID:21388952
26. Badr-Eldin SM, Aldawsari HM, Kotta S, Deb PK, Venugopala KN. Three-Dimensional In Vitro Cell Culture Models for Efficient Drug Discovery: Progress So Far and Future Prospects. Pharmaceuticals (Basel). 2022 Jul;15(8):926. PMID:36015074
27. Knight E, Przyborski S. Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat. 2015 Dec;227(6):746–56. PMID:25411113
28. Burgués JP, Gómez L, Pontones JL, Vera CD, Jiménez-Cruz JF, Ozonas M. A chemosensitivity test for superficial bladder cancer based on three-dimensional culture of tumour spheroids. Eur Urol. 2007 Apr;51(4):962–9. PMID:17084017
29. Medle B, Sjödahl G, Eriksson P, Liedberg F, Höglund M, Bernardo C. Patient-Derived Bladder Cancer Organoid Models in Tumor Biology and Drug Testing: A Systematic Review. Cancers (Basel). 2022 Apr;14(9):2062. PMID:35565191
30. Lee SH, Hu W, Matulay JT, Silva MV, Owczarek TB, Kim K, et al. Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer. Cell. 2018 Apr;173(2):515–528.e17. PMID:29625057
31. Elbadawy M, Usui T, Mori T, Tsunedomi R, Hazama S, Nabeta R, et al. Establishment of a novel experimental model for muscle-invasive bladder cancer using a dog bladder cancer organoid culture. Cancer Sci. 2019 Sep;110(9):2806–21. PMID:31254429
32. Mullenders J, de Jongh E, Brousali A, Roosen M, Blom JP, Begthel H, et al. Mouse and human urothelial cancer organoids: A tool for bladder cancer research. Proc Natl Acad Sci USA. 2019 Mar;116(10):4567–74. PMID:30787188
33. Amaral R, Zimmermann M, Ma AH, Zhang H, Swiech K, Pan CX. A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models. Cancers (Basel). 2020 May;12(5):1304. PMID:32455634
34. Lamy P, Nordentoft I, Birkenkamp-Demtröder K, Thomsen MB, Villesen P, Vang S, et al. Paired Exome Analysis Reveals Clonal Evolution and Potential Therapeutic Targets in Urothelial Carcinoma. Cancer Res. 2016 Oct;76(19):5894–906. PMID:27488526
35. Breslin S, O’Driscoll L. Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today. 2013 Mar;18(5-6):240–9. PMID:23073387
36. Buchanan CF, Verbridge SS, Vlachos PP, Rylander MN. Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model. Cell Adhes Migr. 2014;8(5):517–24. PMID:25482628
37. Sackett SD, Tremmel DM, Ma F, Feeney AK, Maguire RM, Brown ME, et al. Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas. Sci Rep. 2018 Jul;8(1):10452. PMID:29993013
38. Odawara A, Gotoh M, Suzuki I. Control of neural network patterning using collagen gel photothermal etching. Lab Chip. 2013 Jun;13(11):2040–6. PMID:23615759
39. Wang L, Smith BA, Balanis NG, Tsai BL, Nguyen K, Cheng MW, et al. A genetically defined disease model reveals that urothelial cells can initiate divergent bladder cancer phenotypes. Proc Natl Acad Sci USA. 2020 Jan;117(1):563–72. PMID:31871155
40. Yu L, Li Z, Mei H, Li W, Chen D, Liu L, et al. Patient-derived organoids of bladder cancer recapitulate antigen expression profiles and serve as a personal evaluation model for CAR-T cells in vitro. Clin Transl Immunology. 2021 Jan;10(2):e1248. PMID:33552510
41. Kim S, Kim Y, Kong J, Kim E, Choi JH, Yuk HD, et al. Epigenetic regulation of mammalian Hedgehog signaling to the stroma determines the molecular subtype of bladder cancer. eLife. 2019 Apr;8:e43024. PMID:31036156
42. Kloskowski T, Uzarska M, Gurtowska N, Olkowska J, Joachimiak R, Bajek A, et al. How to isolate urothelial cells? Comparison of four different methods and literature review. Hum Cell. 2014 Apr;27(2):85–93. PMID:24368576
43. Ringuette Goulet C, Bernard G, Chabaud S, Couture A, Langlois A, Neveu B, et al. Tissue-engineered human 3D model of bladder cancer for invasion study and drug discovery. Biomaterials. 2017 Nov;145:233–41. PMID:28888113
44. Varley CL, Southgate J. Organotypic and 3D reconstructed cultures of the human bladder and urinary tract. Methods Mol Biol. 2011;695:197–211. PMID:21042974
45. Gu Q, Hao J, Lu Y, Wang L, Wallace GG, Zhou Q. Three-dimensional bio-printing. Sci China Life Sci. 2015 May;58(5):411–9. PMID:25921944
46. Hansen CJ, Saksena R, Kolesky DB, Vericella JJ, Kranz SJ, Muldowney GP, et al. High-throughput printing via microvascular multinozzle arrays. Adv Mater. 2013 Jan;25(1):96–102. PMID:23109104
47. Abdella S, Youssef SH, Afinjuomo F, Song Y, Fouladian P, Upton R, et al. 3D Printing of Thermo-Sensitive Drugs. Pharmaceutics. 2021 Sep;13(9):1524. PMID:34575600
48. Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. 2014 Aug;32(8):773–85. PMID:25093879
49. Ozbolat IT, Yu Y. Bioprinting toward organ fabrication: challenges and future trends. IEEE Trans Biomed Eng. 2013 Mar;60(3):691–9. PMID:23372076
50. Wang Z, Abdulla R, Parker B, Samanipour R, Ghosh S, Kim K. A simple and high-resolution stereolithography-based 3D bioprinting system using visible light crosslinkable bioinks. Biofabrication. 2015 Dec;7(4):045009. PMID:26696527
51. Nahmias Y, Schwartz RE, Verfaillie CM, Odde DJ. Laser-guided direct writing for three-dimensional tissue engineering. Biotechnol Bioeng. 2005 Oct;92(2):129–36. PMID:16025535
52. Guillotin B, Souquet A, Catros S, Duocastella M, Pippenger B, Bellance S, et al. Laser assisted bioprinting of engineered tissue with high cell density and microscale organization. Biomaterials. 2010 Oct;31(28):7250–6. PMID:20580082
53. Bejrananda T, Liawrungrueang W. Successful transitional cell carcinoma of bladder underwent laparoscopic radical cystectomy with orthotopic intracorporeal Y pouch neobladder using a 3D digital printing model for surgical post op pouch evaluation. Urol Case Rep. 2020 Apr;31:101190. PMID:32292705
54. Kim MJ, Chi BH, Yoo JJ, Ju YM, Whang YM, Chang IH. Structure establishment of three-dimensional (3D) cell culture printing model for bladder cancer. PLoS One. 2019 Oct;14(10):e0223689. PMID:31639124
55. Sontheimer-Phelps A, Hassell BA, Ingber DE. Modelling cancer in microfluidic human organs-on-chips. Nat Rev Cancer. 2019 Feb;19(2):65–81. PMID:30647431
56. Kim JH, Lee S, Kang SJ, Choi YW, Choi SY, Park JY, et al. Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guérin. Int J Mol Sci. 2021 Aug;22(16):8887. PMID:34445591