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Three-dimensional culture systems in cancer research: Focus on tumor spheroid model
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Cancer cells propagated in three-dimensional (3D) culture systems exhibit physiologically relevant cell-cell and cell-matrix interactions, gene expression and signaling pathway profiles, heterogeneity and structural complexity that reflect in vivo tumors. In recent years, development of various 3D models has improved the study of host-tumor interaction and use of high-throughput screening platforms for anti-cancer drug discovery and development. This review attempts to summarize the various 3D culture systems, with an emphasis on the most well characterized and widely applied model - multicellular tumor spheroids. This review also highlights the various techniques to generate tumor spheroids, methods to characterize them, and its applicability in cancer research.
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“This physiochemical gradient generally induces the formation of three concentric zones on a spheroid -an anoxic core in the center containing necrotic cells, a middle hypoxic zone that has a low concentration of oxygen and nutrients, and an outer zone containing highly proliferative cells [53].”
From 3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions (2021) · cites this paper as [53]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Cancer spheroids are arguably the simplest in vitro 3D tumor models [114,115].”
From The Tumor-on-Chip: Recent Advances in the Development of Microfluidic Systems to Recapitulate the Physiology of Solid Tumors (2019) · cites this paper as [114]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“Currently, various tumor spheroids have been fabricated by embedding cancer cells into protein‐based hydrogels such as Matrigel,[ 64 ] fibrin,[ 65 ] and collagen[ 66 ] as they incorporate specific cell‐adhesive motifs and enzymatic‐mediated matrix degradation which are beneficial to cell proliferation.”
From 3D Biomimetic Models to Reconstitute Tumor Microenvironment In Vitro: Spheroids, Organoids, and Tumor‐on‐a‐Chip (2023) · cites this paper as [66]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“An important aspect to consider is uniformity in size and homogeneity to ensure that the results are not in fl uenced by these factors [53].”
From 3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions (2021) · cites this paper as [53]
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In the citing paper
“Tumor cell spheroids are especially useful for studying cancers that form tumor embolus, or a closely packed tumor cell cluster, as happens in in fl ammatory breast cancer [53].”
From 3D In Vitro Model (R)evolution: Unveiling Tumor–Stroma Interactions (2021) · cites this paper as [53]
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In the citing paper
“Three-dimensional multicellular tumor spheroids have emerged as more representative in vitro models that capture essential features of tumor architecture, stromal interactions, and microenvironmental resistance mechanisms.”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [17]
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In the citing paper
“Spheroids exhibit spatially organized regions of proliferation, quiescence, and hypoxia, and can incorporate non-tumor cells to mimic tumor–stroma crosstalk.”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [17]
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“By mimicking the hierarchical structure, proliferation gradients, and stromal interactions found in native tumor tissues, spheroids provide a more physiologically relevant context for evaluating anticancer agents and delivery systems [16,17].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [17]
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“These gradients typically emerge when spheroids exceed ~500 μm in diameter, with proliferative cells localized to the outer rim, quiescent cells residing in intermediate zones, and hypoxic or necrotic cores forming at the center due to limited diffusion [17,184,185].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [17]
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In the citing paper
“Furthermore, 2D cultured cells are artificially polarized when their cytoskeleton rearranges as they are stretched on the culture plate, which in turn causes these cultured cells to express genes and proteins which are atypical [56].”
From Microfluidic modelling of the tumor microenvironment for anti-cancer drug development (2019) · cites this paper as [56]
In the citing paper
“Spheroids [98, 99] are, in general, considered similar to an early vascular stage of tumors”
In the citing paper
“Moreover, this gradient leads to the formation of concentric zones in the spheroids with a center containing an anoxic core with necrotic cells, a middle zone containing hypoxic cells, and an outer zone of highly proliferative cells, recapitulating the properties of an in vitro tumor [379].”
From Tumor microenvironment signaling and therapeutics in cancer progression (2023) · cites this paper as [379]
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Metadata
- Authors
- Nath, S., & Devi, G. R.
- Affiliations
- Duke University (US)
- Corresponding author
- Devi, Gayathri R.
- ORCID
- Devi, Gayathri R.
- Venue
- Pharmacology & Therapeutics
- Publisher
- Elsevier BV
- Published
- 2016-07
- Volume
- 163
- Pages
- 94-108
- ISSN
- 0163-7258 (ISSN-L)
- PMC
- PMC4961208
- OpenAlex
- W2332155194
- Cited by
- 919
- References
- 175
- Citations by year
- 2026: 67 · 2025: 95 · 2024: 100 · 2023: 92 · 2022: 110 · 2021: 125 · 2020: 137 · 2019: 81 · 2018: 63 · 2017: 38 · 2016: 11
- FWCI
- 6.47
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Pharmacology & Therapeutics (acceptedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- NIH (CA014236)
- Department of Defense (W81XWH-13-1-0047)
- 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, tumor cell culture, cell-matrix interactions, tumor heterogeneity, tumor-host interaction, high-throughput drug screening, anticancer drug discovery
- MeSH terms
- Antineoplastic Agents, Cell Communication, Cell Movement, Flow Cytometry, Humans, Microscopy, Electron, Scanning, Neoplasm Invasiveness, Neoplasms, Tumor Cells, Cultured, Microscopy, Confocal, Spheroids, Cellular, Cell Culture Techniques, Hydrogels, Tumor Microenvironment
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