Three-dimensional (3D) bioprinting offers a suitable in vitro preclinical model system to reduce or replace animal experiments; however, published studies are difficult to compare. In this study, we characterized growth dynamics, tissue architecture, and mammalian target of rapamycin (mTOR) pathway activity in a 3D bioprinted breast carcinoma model of T47D cell line and compared these features with conventional two-dimensional (2D) monolayer cultures. Tissue-mimetic structures (TMSs) were generated by 3D bioprinting and analyzed for cell viability, proliferation, autophagy, and apoptosis, as well as the expression of cell–cell and cell–extracellular matrix (ECM) adhesion proteins. In addition, mTOR pathway activity and responsiveness to mTOR inhibitors (rapamycin and ipatasertib) and chemotherapeutic agents (cisplatin) were assessed. The bioprinted TMSs remained viable for up to 3 weeks and developed a tissue-like architecture characterized by heterogeneous marker expression (β-catenin, E-cadherin, N-cadherin, fibronectin, and syndecan) and complex cellular organization. Compared with 2D monolayer cultures, 3D TMSs exhibited reduced mTOR signaling activity, which led to significantly decreased sensitivity to mTOR inhibition. These findings indicate that 3D bioprinted breast cancer models recapitulate key structural and signaling features of in situ tumors more accurately than 2D systems, highlighting their potential value for preclinical drug testing and mechanistic studies.
| [1] |
C. H. Wong, K. W. Siah, and A. W. Lo, “Corrigendum: Estimation of Clinical Trial Success Rates and Related Parameters,” Biostatistics 20, no. 2 (2019): 366.
|
| [2] |
J. Lexchin, “How Safe Are New Drugs? Market Withdrawal of Drugs Approved in Canada Between 1990 and 2009,” Open Medicine 8, no. 1 (2014): e14.
|
| [3] |
N. S. Craveiro, B. S. Lopes, L. Tomás, and S. F. Almeida, “Drug Withdrawal due to Safety: A Review of the Data Supporting Withdrawal Decision,” Current Drug Safety 15, no. 1 (2020): 4–12.
|
| [4] |
E. Kim, J. Yang, S. Park, and K. Shin, “Factors Affecting Success of New Drug Clinical Trials,” Therapeutic Innovation & Regulatory Science 57, no. 4 (2023): 737–750.
|
| [5] |
M. Kapałczyńska, T. Kolenda, W. Przybyła, et al., “2D and 3D Cell Cultures—A Comparison of Different Types of Cancer Cell Cultures,” Archives of Medical Science 14, no. 4 (2018): 910–919.
|
| [6] |
A. Akhtar, “The Flaws and Human Harms of Animal Experimentation,” Cambridge Quarterly of Healthcare Ethics 24, no. 4 (2015): 407–419.
|
| [7] |
G. A. Van Norman, “Limitations of Animal Studies for Predicting Toxicity in Clinical Trials: Is It Time to Rethink Our Current Approach?,” JACC: Basic to Translational Science 4, no. 7 (2019): 845–854.
|
| [8] |
A. Knight, “Animal Experiments Scrutinised: Systematic Reviews Demonstrate Poor human Clinical and Toxicological Utility,” ALTEX: Alternativen zu Tierexperimenten 24, no. 4 (2007): 320–325.
|
| [9] |
I. Hutchinson, C. Owen, and J. Bailey, “Modernizing Medical Research to Benefit People and Animals,” Animals 12, no. 9 (2022): 1173.
|
| [10] |
“Regulation (EC) No. 1223/2009 of the European Parliament and of the Council of 30 November 2009 on Cosmetic Products (Recast) (Text with EEA Relevance),” European Parliament CotEU, accessed December 22, 2009.
|
| [11] |
M. Wadman, “FDA no Longer Has to Require Animal Testing for New Drugs,” Science 379, no. 6628 (2023): 127–128.
|
| [12] |
S. K. Niazi, “End Animal Testing for Biosimilar Approval,” Science 377, no. 6602 (2022): 162–163.
|
| [13] |
J. J. Han, “FDA Modernization Act 2.0 Allows for Alternatives to Animal Testing,” Artificial Organs 47, no. 3 (2023): 449–450.
|
| [14] |
A. C. Duarte, E. C. Costa, H. A. Filipe, et al., “Animal-Derived Products in Science and Current Alternatives,” Biomaterials Advances 151 (2023): 213428.
|
| [15] |
A. Honkala, S. V. Malhotra, S. Kummar, and M. R. Junttila, “Harnessing the Predictive Power of Preclinical Models for Oncology Drug Development,” Nature Reviews Drug Discovery 21, no. 2 (2022): 99–114.
|
| [16] |
H. Sajjad, S. Imtiaz, T. Noor, Y. H. Siddiqui, A. Sajjad, and M. Zia, “Cancer Models in Preclinical Research: A Chronicle Review of Advancement in Effective Cancer Research,” Animal Models and Experimental Medicine 4, no. 2 (2021): 87–103.
|
| [17] |
S. Pozzi, A. Scomparin, S. Israeli Dangoor, et al., “Meet Me Halfway: Are In Vitro 3D Cancer Models on the Way to Replace In Vivo Models for Nanomedicine Development?,” Advanced Drug Delivery Reviews 175 (2021): 113760.
|
| [18] |
L. Neufeld, E. Yeini, S. Pozzi, and R. Satchi-Fainaro, “3D Bioprinted Cancer Models: From Basic Biology to Drug Development,” Nature Reviews Cancer 22, no. 12 (2022): 679–692.
|
| [19] |
A. Li, R. Liang, W. Sheng, et al., “Global Research Trends in Bone/Cartilage Organoids From 2010 to 2024: A Bibliometric and Visualization Study,” Organoid Research 1, no. 3 (2025): 8295.
|
| [20] |
D. Hanahan, “Hallmarks of Cancer: New Dimensions,” Cancer Discovery 12, no. 1 (2022): 31–46.
|
| [21] |
G. Bassi, S. Panseri, S. M. Dozio, et al., “Scaffold-Based 3D Cellular Models Mimicking the Heterogeneity of Osteosarcoma Stem Cell Niche,” Scientific Reports 10, no. 1 (2020): 22294.
|
| [22] |
T. Dankó, G. Petővári, R. Raffay, et al., “Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting,” International Journal of Molecular Sciences 23, no. 13 (2022): 7444.
|
| [23] |
W. Ma, H. Lu, Y. Xiao, and C. Wu, “Advancing Organoid Development With 3D Bioprinting,” Organoid Research 1, no. 1 (2025): 025040004.
|
| [24] |
D. Sztankovics, D. Moldvai, G. Petővári, et al., “3D Bioprinting and the Revolution in Experimental Cancer Model Systems—A Review of Developing New Models and Experiences With In Vitro 3D Bioprinted Breast Cancer Tissue-Mimetic Structures,” Pathology Oncology Research 29 (2023): 1610996.
|
| [25] |
G. Petővári, D. Moldvai, R. Raffay, et al., “Mimicking Breast Cancer Tissue—3D Bioprinted Models in Accurate Drug Sensitivity Tests,” View 6, no. 5 (2025): 20250092.
|
| [26] |
M. Pickl and C. H. Ries, “Comparison of 3D and 2D Tumor Models Reveals Enhanced HER2 Activation in 3D Associated With an Increased Response to Trastuzumab,” Oncogene 28, no. 3 (2009): 461–468.
|
| [27] |
M. Watanabe, T. Yano, T. Sato, et al., “mTOR Inhibitors Modulate the Physical Properties of 3D Spheroids Derived From H9c2 Cells,” International Journal of Molecular Sciences 24, no. 14 (2023): 11459.
|
| [28] |
A. Filipiak-Duliban, K. Brodaczewska, A. Kajdasz, and C. Kieda, “Spheroid Culture Differentially Affects Cancer Cell Sensitivity to Drugs in Melanoma and RCC Models,” International Journal of Molecular Sciences 23, no. 3 (2022): 1166.
|
| [29] |
J. Heid, A. Affolter, Y. Jakob, et al., “3D Cell Culture Alters Signal Transduction and Drug Response in Head and Neck Squamous Cell Carcinoma,” Oncology Letters 23, no. 6 (2022): 177.
|
| [30] |
K. Baghy, A. Ladányi, A. Reszegi, and I. Kovalszky, “Insights Into the Tumor Microenvironment—Components, Functions and Therapeutics,” International Journal of Molecular Sciences 24, no. 24 (2023): 17536.
|
| [31] |
J. Liu, Q. Xiao, and J. Xiao, “Wnt/β-Catenin Signalling: Function, Biological Mechanisms, and Therapeutic Opportunities,” Signal Transduction and Targeted Therapy 7, no. 1 (2022): 3.
|
| [32] |
A. Barzegar Behrooz, Z. Talaie, F. Jusheghani, M. J. Łos, T. Klonisch, and S. Ghavami, “Wnt and PI3K/Akt/mTOR Survival Pathways as Therapeutic Targets in Glioblastoma,” International Journal of Molecular Sciences 23, no. 3 (2022): 1353.
|
| [33] |
T. C. Lin, C. H. Yang, L. H. Cheng, W. T. Chang, Y. R. Lin, and H. C. Cheng, “Fibronectin in Cancer: Friend or Foe,” Cells 9, no. 1 (2019): 27.
|
| [34] |
B. D. Manning and L. C. Cantley, “AKT/PKB Signaling: Navigating Downstream,” Cell 129, no. 7 (2007): 1261–1274.
|
| [35] |
A. Glaviano, A. S. C. Foo, H. Y. Lam, et al., “PI3K/AKT/mTOR Signaling Transduction Pathway and Targeted Therapies in Cancer,” Molecular Cancer 22, no. 1 (2023): 138.
|
| [36] |
J. A. Engelman, J. Luo, and L. C. Cantley, “The Evolution of Phosphatidylinositol 3-Kinases as Regulators of Growth and Metabolism,” Nature Reviews Genetics 7, no. 8 (2006): 606–619.
|
| [37] |
R. A. Saxton and D. M. Sabatini, “mTOR Signaling in Growth, Metabolism, and Disease,” Cell 169, no. 2 (2017): 361–371.
|
| [38] |
R. Watanabe, L. Wei, and J. Huang, “mTOR Signaling, Function, Novel Inhibitors, and Therapeutic Targets,” Journal of Nuclear Medicine 52, no. 4 (2011): 497–500.
|
| [39] |
S. Menon and B. D. Manning, “Common Corruption of the mTOR Signaling Network in Human Tumors,” Oncogene 27, no. S2 (2008): S43–S51.
|
| [40] |
L. Ciuffreda, C. Di Sanza, U. C. Incani, and M. Milella, “The mTOR Pathway: A New Target in Cancer Therapy,” Current Cancer Drug Targets 10, no. 5 (2010): 484–495.
|
| [41] |
K. S. Abdelrahman, H. A. Hassan, S. A. Abdel-Aziz, et al., “JNK Signaling as a Target for Anticancer Therapy,” Pharmacological Reports 73, no. 2 (2021): 405–434.
|
| [42] |
B. C. Mak and R. S. Yeung, “The Tuberous Sclerosis Complex Genes in Tumor Development,” Cancer Investigation 22, no. 4 (2004): 588–603.
|
| [43] |
A. Riedl, M. Schlederer, K. Pudelko, et al., “Comparison of Cancer Cells in 2D vs 3D Culture Reveals Differences in AKT-mTOR-S6K Signaling and Drug Responses,” Journal of Cell Science 130, no. 1 (2017): 203–218.
|
| [44] |
A. C. Luca, S. Mersch, R. Deenen, et al., “Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines,” PLoS One 8, no. 3 (2013): e59689.
|
| [45] |
T. Desigaux, L. Comperat, N. Dusserre, et al., “3D Bioprinted Breast Cancer Model Reveals Stroma-Mediated Modulation of Extracellular Matrix and Radiosensitivity,” Bioactive Materials 42 (2024): 316–327.
|
| [46] |
E. H. Y. Lam, F. Yu, S. Zhu, and Z. Wang, “3D Bioprinting for Next-Generation Personalized Medicine,” International Journal of Molecular Sciences 24, no. 7 (2023): 6357.
|
| [47] |
Z. Li, Y. Luo, M. Lu, et al., “Design, Characterisation, and Clinical Evaluation of a Novel Porous Ti-6Al-4V Hemipelvic Prosthesis Based on Voronoi Diagram,” Biomaterials Translational 5, no. 3 (2024): 314–324.
|
| [48] |
G. Huang, Y. Zhao, D. Chen, et al., “Applications, Advancements, and Challenges of 3D Bioprinting in Organ,” Biomaterials Science 12, no. 6 (2024): 1425–1448.
|
| [49] |
C. M. Ketcham and J. M. Crawford, “The Impact of Review Articles,” Laboratory Investigation 87, no. 12 (2007): 1174–1185.
|
| [50] |
S. Đokić, B. Gazić, B. Grčar Kuzmanov, et al., “Clinical and Analytical Validation of Two Methods for Ki-67 Scoring in Formalin Fixed and Paraffin Embedded Tissue Sections of Early Breast Cancer,” Cancers 16, no. 7 (2024): 1405.
|
| [51] |
C. Follo, D. Barbone, W. G. Richards, R. Bueno, and V. C. Broaddus, “Autophagy Initiation Correlates With the Autophagic Flux in 3D Models of Mesothelioma and With Patient Outcome,” Autophagy 12, no. 7 (2016): 1180–1194.
|
| [52] |
J. Li and B. P. Zhou, “Activation of β-Catenin and Akt Pathways by Twist Are Critical for the Maintenance of EMT Associated Cancer Stem Cell-Like Characters,” BMC Cancer 11 (2011): 49.
|
| [53] |
N. Borcherding, K. Cole, P. Kluz, et al., “Re-Evaluating E-Cadherin and β-Catenin: A Pan-Cancer Proteomic Approach With an Emphasis on Breast Cancer,” American Journal of Pathology 188, no. 8 (2018): 1910–1920.
|
| [54] |
C. Nagi, M. Guttman, S. Jaffer, et al., “N-Cadherin Expression in Breast Cancer: Correlation With an Aggressive Histologic Variant–Invasive Micropapillary Carcinoma,” Breast Cancer Research and Treatment 94, no. 3 (2005): 225–235.
|
| [55] |
Y. L. Park, H. P. Kim, Y. W. Cho, et al., “Activation of WNT/β-Catenin Signaling Results in Resistance to a Dual PI3K/mTOR Inhibitor in Colorectal Cancer Cells Harboring PIK3CA Mutations,” International Journal of Cancer 144, no. 2 (2019): 389–401.
|
| [56] |
P. Debnath, R. S. Huirem, A. Bhowmick, et al., “Epithelial Mesenchymal Transition Induced Nuclear Localization of the Extracellular Matrix Protein Fibronectin,” Biochimie 219 (2024): 142–145.
|
| [57] |
F. Adkins, A. Akcakanat, H. Chen, et al., “Epithelial-Mesenchymal Transition Confers Resistance to Rapamycin,” Journal of the American College of Surgeons 213, no. S3 (2011): S144.
|
| [58] |
J. Dudás, G. Ramadori, T. Knittel, et al., “Effect of Heparin and Liver Heparan Sulphate on Interaction of HepG2-Derived Transcription Factors and Their Cis-Acting Elements: Altered Potential of Hepatocellular Carcinoma Heparan Sulphate,” Biochemical Journal 350 (2000): 245–251.
|
| [59] |
T. Szatmári, R. Ötvös, A. Hjerpe, and K. Dobra, “Syndecan-1 in Cancer: Implications for Cell Signaling, Differentiation, and Prognostication,” Disease Markers 2015 (2015): 796052.
|
| [60] |
T. L. Nguyen, W. E. Grizzle, K. Zhang, O. Hameed, G. P. Siegal, and S. Wei, “Syndecan-1 Overexpression Is Associated With Nonluminal Subtypes and Poor Prognosis in Advanced Breast Cancer,” American Journal of Clinical Pathology 140, no. 4 (2013): 468–474.
|
| [61] |
S. Kind, A. Jaretzke, F. Büscheck, et al., “A Shift From Membranous and Stromal Syndecan-1 (CD138) Expression to Cytoplasmic CD138 Expression Is Associated With Poor Prognosis in Breast Cancer,” Molecular Carcinogenesis 58, no. 12 (2019): 2306–2315.
|
| [62] |
B. J. Burbach, A. Friedl, C. Mundhenke, and A. C. Rapraeger, “Syndecan-1 Accumulates in Lysosomes of Poorly Differentiated Breast Carcinoma Cells,” Matrix Biology 22, no. 2 (2003): 163–177.
|
| [63] |
Y. Deng, V. Adam, E. Nepovimova, et al., “c-Jun N-Terminal Kinase Signaling in Cellular Senescence,” Archives of Toxicology 97, no. 8 (2023): 2089–2109.
|
| [64] |
P. Phukhum, J. Phetcharaburanin, K. Chaleekarn, et al., “The Impact of Hypoxia and Oxidative Stress on Proteo-Metabolomic Alterations of 3D Cholangiocarcinoma Models,” Scientific Reports 13, no. 1 (2023): 3072.
|
| [65] |
B. E. Grottkau, Z. Hui, and Y. Pang, “Bioprinting Native-Like 3D Micro Breast Cancer Tissues Utilizing Existing Cancer Cell Lines,” International Journal of Bioprinting 10, no. 3 (2024): 2911.
|
| [66] |
Y. Fujimoto, T. Y. Morita, A. Ohashi, et al., “Combination Treatment With a PI3K/Akt/mTOR Pathway Inhibitor Overcomes Resistance to Anti-HER2 Therapy in PIK3CA-Mutant HER2-Positive Breast Cancer Cells,” Scientific Reports 10, no. 1 (2020): 21762.
|
| [67] |
N. Rhodes, D. A. Heerding, D. R. Duckett, et al., “Characterization of an Akt Kinase Inhibitor With Potent Pharmacodynamic and Antitumor Activity,” Cancer Research 68, no. 7 (2008): 2366–2374.
|
| [68] |
T. Okuzumi, D. Fiedler, C. Zhang, et al., “Inhibitor Hijacking of Akt Activation,” Nature Chemical Biology 5, no. 7 (2009): 484–493.
|
| [69] |
K. Lin, J. Lin, W. I. Wu, et al., “An ATP-Site On-Off Switch That Restricts Phosphatase Accessibility of Akt,” Science Signaling 5, no. 223 (2012): ra37.
|
| [70] |
B. Weigelt, A. T. Lo, C. C. Park, J. W. Gray, and M. J. Bissell, “HER2 Signaling Pathway Activation and Response of Breast Cancer Cells to HER2-Targeting Agents Is Dependent Strongly on the 3D Microenvironment,” Breast Cancer Research and Treatment 122, no. 1 (2010): 35–43.
|
| [71] |
A. Frtús, B. Smolková, M. Uzhytchak, et al., “Hepatic Tumor Cell Morphology Plasticity Under Physical Constraints in 3D Cultures Driven by YAP-mTOR Axis,” Pharmaceuticals 13, no. 12 (2020): 430.
|
| [72] |
B. Xue, J. Schüler, C. M. Harrod, K. Lashuk, Z. Bomya, and K. C. Hribar, “A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies,” Cells 12, no. 8 (2023): 1145.
|
| [73] |
R. Laczkó-Rigó, É. Bakos, R. Jójárt, C. Tömböly, E. Mernyák, and C. Özvegy-Laczka, “Selective Antiproliferative Effect of C-2 Halogenated 13α-Estrones on Cells Expressing Organic Anion-Transporting Polypeptide 2B1 (OATP2B1),” Toxicology and Applied Pharmacology 429 (2021): 115704.
|
| [74] |
G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, “Interstitial pH and pO2 Gradients in Solid Tumors In Vivo: High-Resolution Measurements Reveal a Lack of Correlation,” Nature Medicine 3, no. 2 (1997): 177–182.
|
RIGHTS & PERMISSIONS
2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.
PDF
(5842KB)
