Development of an in vitro tumor spheroid culture model amenable to high-throughput testing of potential anticancer nanotherapeutics
Abstract
CONTEXT: Three-dimensional tumor spheroid cultures are a better representative of in vivo solid tumors than monolayer cultures and should be used for testing potential nanotherapeutics in vitro. OBJECTIVE: To develop techniques to test the disposition and efficacy of nanocarrier formulations in spheroids in a cost-effective manner amenable to high-throughput testing. METHODS: Spheroids were obtained using a modified liquid overlay technique in a 96-well plate. Several nanocarrier formulations were prepared and tested in the spheroid model. The disposition of the formulations in the spheroids was determined by confocal microscopy while the effect of the drug-loaded formulations was assessed in terms of the cell viability, loss of membrane integrity, induction of caspases and inhibition of growth of the spheroids. RESULTS: The surface charge of the formulations influenced the accumulation of the nanocarrier and drug in the spheroid, with the cationic formulation accumulating to the greatest extent. Also, the smallest particle size formulation, micelles, penetrated to the greatest extent in the spheroid. The iRGD tumor-penetrating peptide co-administered with unmodified liposomes exhibited both high accumulation and penetration. The effect studies revealed that the formulations that penetrated or accumulated to the highest extent in the spheroid exhibited better antitumor activity compared to the other formulations. CONCLUSION: The 96-well plate format spheroid model developed in the study can be used toward the rational selection of nanocarrier therapeutics prior to their testing in in vivo models.
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Spheroids can incorporate non-tumor cells to mimic tumor-stroma crosstalk.
“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 [186]
Cells in the outermost layer of a spheroid are typically highly proliferative due to proximity to nutrients and oxygen.
“Cells in the outermost layer of the spheroid are typically highly proliferative due to their proximity to nutrients and oxygen.”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
These interactions are absent in monolayers but are necessary for recapitulating drug responses observed in vivo.
“These interactions are absent in monolayers but are necessary for recapitulating drug responses observed in vivo [101,186].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
Cells in the intermediate zone of a spheroid are often senescent.
“cells located in the intermediate zone are often senescent”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
Spheroid models can be co-cultured with stromal elements such as fibroblasts, endothelial cells, or immune cells, permitting reconstitution of cell-cell and cell-ECM interactions essential for modeling tumor-stroma crosstalk, which governs survival signaling, drug resistance, and cell migration.
“Importantly, spheroid models can be co-cultured with stromal elements such as fibroblasts, endothelial cells, or immune cells. This permits the reconstitution of cell–cell and cell–ECM interactions that are essential for modeling tumor–stroma crosstalk, which governs processes such as survival signaling, drug resistance, and cell migration (Figure 2).”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
Cells in the central core of a spheroid exist in a hypoxic state.
“those in the central core exist in a hypoxic state [101,184,186,187]”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
Spheroids allow for the evaluation of drug penetration, metabolic adaptation, and regional sensitivity within a tumor-like context.
“As a result, spheroids allow for the evaluation of drug penetration, metabolic adaptation, and regional sensitivity within a tumor-like context [101,186].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
Additional markers of cellular integrity, including lactate dehydrogenase release, caspase activation, and changes in mitochondrial membrane potential, have been adapted to evaluate apoptotic and necrotic responses within 3D tumor models.
“Additional markers of cellular integrity, including lactate dehydrogenase release, caspase activation, and changes in mitochondrial membrane potential, have also been adapted to evaluate apoptotic and necrotic responses within 3D tumor models [186,308,309,310].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [186]
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“For example, extended reagent exposure or parallel quantification using monolayer standards have been implemented to ensure more accurate estimation of viable cell numbers in 3D systems [186,305].”
InfluentialSelf-citation
- Authors
- Solomon, M. A., Lemera, J., & D’Souza, G. G. M.
- Affiliations
- Massachusetts College of Pharmacy and Health Sciences (US)
- Corresponding author
- D’Souza, Gerard G. M.
- ORCID
- Solomon, Melani A.
- Venue
- Journal of Liposome Research
- Publisher
- Informa UK Limited
- Published
- 2016-01-18
- Volume
- 26
- Issue
- 3
- Pages
- 246-260
- ISSN
- 0898-2104 (ISSN-L)
- 1532-2394
- OpenAlex
- W2335836427
- Cited by
- 30
- References
- 49
- Citations by year
- 2026: 1 · 2025: 3 · 2024: 1 · 2023: 2 · 2022: 1 · 2021: 3 · 2020: 7 · 2019: 4 · 2018: 4 · 2017: 4
- FWCI
- 1.63
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Liposome Research (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Harvard NeuroDiscovery Center
- Topics
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Keywords
- tumor spheroids, 3D tumor models, nanocarrier formulation, high-throughput screening, surface charge, particle size, micelles, liposomes, iRGD peptide, tumor-penetrating peptide, drug accumulation, anticancer activity, membrane integrity
- MeSH terms
- Antineoplastic Agents, Cell Survival, Flow Cytometry, Humans, Models, Biological, Spheroids, Cellular, Cell Culture Techniques, Cell Line, Tumor, Cell Proliferation, Nanomedicine, High-Throughput Screening Assays
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