Challenges of applying multicellular tumor spheroids in preclinical phase
Open accessAbstract
The three-dimensional (3D) multicellular tumor spheroids (MCTs) model is becoming an essential tool in cancer research as it expresses an intermediate complexity between 2D monolayer models and in vivo solid tumors. MCTs closely resemble in vivo solid tumors in many aspects, such as the heterogeneous architecture, internal gradients of signaling factors, nutrients, and oxygenation. MCTs have growth kinetics similar to those of in vivo tumors, and the cells in spheroid mimic the physical interaction of the tumors, such as cell-to-cell and cell-to-extracellular matrix interactions. These similarities provide great potential for studying the biological properties of tumors and a promising platform for drug screening and therapeutic efficacy evaluation. However, MCTs are not well adopted as preclinical tools for studying tumor behavior and therapeutic efficacy up to now. In this review, we addressed the challenges with MCTs application and discussed various efforts to overcome the challenges.
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Conventional viability assays such as MTT and MTS are commonly used in monolayer systems but require optimization for three-dimensional cultures due to diffusion limitations.
“Conventional viability assays, such as MTT and MTS, which rely on the enzymatic reduction of tetrazolium salts to formazan by metabolically active cells, are commonly used in monolayer systems but require optimization for three-dimensional cultures due to diffusion limitations [306].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [306]
The ‘Issues of cell viability assay using MCTs’ section says viability assays in spheroids may need optimization because reagents penetrate differently depending on spheroid size and compactness; its Table 3 lists MTT as a 2D assay. Our copy does not identify MTS or describe MTT and MTS reducing tetrazolium salts to formazan, so those assay-specific parts of the citing sentence are not supported. (LLM, conf=0.84)
Experimental — accuracy not yet measuredReagent penetration can be insufficient in compact spheroids, necessitating extended incubation times or modifications to assay protocols.
“Reagent penetration can be insufficient in compact spheroids, necessitating extended incubation times or modifications to assay protocols [306].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [306]
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Flagged first, then by citations
Research works printed but not linked: 2
- [117]Gang C Janet T Rakesh KJ Lance LM Micro-environmental mechanical stress controls tumor spheroid size and morphology by suppressing proliferation and inducing apoptosis in cancer cells PLoS ONE 2009 4 e4632 10.1371/journal.pone.0004632 19247489 PMC2645686 · DOI
- [119]Koike C McKee TD Pluen A Ramanujan A Burton K Munn LL Solid stress facilitates spheroid formation: potential involvement of hyaluronan Br J Cancer 2002 86 947 953 10.1038/sj.bjc.6600158 11953828 PMC2364140 · DOI
Cited by
Sorted by citations
Shah, S., & D’Souza, G. G. M. (2025) · Cells · doi:10.3390/cells14100732 · Cited by 66 · see citing works
“Conventional viability assays, such as MTT and MTS, which rely on the enzymatic reduction of tetrazolium salts to formazan by metabolically active cells, are commonly used in monolayer systems but require optimization for three-dimensional cultures due to diffusion limitations [306].”
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- Authors
- Han, S. J., Kwon, S., & Kim, K. S.
- Affiliations
- Kyung Hee University (KR)
- Corresponding author
- Kim, Kyung Sook
- Venue
- Cancer Cell International
- Published
- 2021-03-04
- Volume
- 21
- Issue
- 1
- Pages
- 152-152
- ISSN
- 1475-2867 (ISSN-L)
- PMC
- PMC7934264
- OpenAlex
- W3134164720
- Cited by
- 428
- References
- 171
- Citations by year
- 2026: 66 · 2025: 95 · 2024: 106 · 2023: 80 · 2022: 63 · 2021: 18
- FWCI
- 8.21
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Cancer Cell International (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Research Foundation of Korea (2020R1F1A1057505)
- National Assembly (2020R1F1A1057505)
- Topics
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Physical Sciences › Mathematics › Modeling and Simulation › Mathematical Biology Tumor Growth
- Keywords
- tumor spheroids, 3D tumor models, preclinical drug screening, treatment efficacy assessment, tumor heterogeneity, tumor microenvironment, growth kinetics
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