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Updated: Aug 7, 2025
Generation of 3D Tumor Spheroids for Drug Evaluation Studies
Published on: February 24, 2023
Microscopy-based phenotypic monitoring of MDA-MB-231 spheroids allows the evaluation of phenotype-directed therapy
Loay Mahmoud1, Antony Cougnoux1, Christina Bekiari1
1Department of Cell and Molecular Biology, Karolinska Institutet, Biomedicum, and Science for Life Laboratory, Solna, Sweden.
Abstract:
Breast cancer (BC) is the most commonly diagnosed cancer among women. Prognosis has improved over the years, to a large extent, owing to personalized therapy informed by molecular profiling of hormone receptors. However, there is a need for new therapeutic approaches for a subgroup of BCs lacking molecular markers, the Triple Negative Breast Cancer (TNBC) subgroup. TNBC is the most aggressive type of BC, lacks an effective standard of care, shows high levels of resistance and relapse is often inevitable. High resistance to therapy has been hypothesized to be associated with high intratumoral phenotypic heterogeneity. To characterize and treat this phenotypic heterogeneity, we optimized a whole-mount staining and image analysis protocol for three-dimensions (3D) spheroids. Applying this protocol to TNBC spheroids located in the outer region of the spheroid the cells with selected phenotypes: dividing, migrating, and high mitochondrial mass phenotypes. To evaluate the relevance of phenotype-based targeting these cell populations were targeted with Paclitaxel, Trametinib, and Everolimus, respectively, in a dose-dependent manner. Single agents cannot specifically target all phenotypes at the same time. Therefore, we combined drugs that should target independent phenotype. With this rationale we observed that combining Trametinib and Everolimus achieves the highest cytotoxicity at lower doses from all the tested combinations. These findings suggest a rational approach to design treatments can be evaluated in spheroids prior to pre-clinical models and potentially reduce adverse effects.
Insights
Targeting specific cell phenotypes in Triple Negative Breast Cancer (TNBC) spheroids with drug combinations like Trametinib and Everolimus shows promise for reducing resistance and improving treatment outcomes.
Area of Science:
- Oncology
- Cancer Biology
- Drug Discovery
Background:
- Triple Negative Breast Cancer (TNBC) is an aggressive subtype lacking targeted therapies.
- High intratumoral phenotypic heterogeneity contributes to treatment resistance in TNBC.
- Current treatments for TNBC have limited efficacy and high relapse rates.
Purpose of the Study:
- To optimize a 3D spheroid imaging protocol for characterizing TNBC phenotypic heterogeneity.
- To evaluate the efficacy of phenotype-targeted therapies in TNBC spheroids.
- To identify effective drug combinations for overcoming TNBC resistance.
Main Methods:
- Developed and optimized a whole-mount staining and 3D image analysis protocol for TNBC spheroids.
- Identified distinct cell phenotypes (dividing, migrating, high mitochondrial mass) within TNBC spheroids.
- Administered single agents (Paclitaxel, Trametinib, Everolimus) and drug combinations to TNBC spheroids in a dose-dependent manner.
Main Results:
- The optimized protocol successfully characterized phenotypic heterogeneity in 3D TNBC spheroids.
- The combination of Trametinib and Everolimus demonstrated the highest cytotoxicity at lower doses compared to single agents or other combinations.
- Phenotype-based drug targeting in spheroids showed potential for reducing therapeutic doses and improving efficacy.
Conclusions:
- 3D spheroid models are valuable for evaluating phenotype-targeted therapies in TNBC.
- Combined inhibition of specific cell phenotypes (e.g., migrating and high mitochondrial mass) can overcome TNBC resistance.
- This approach may lead to more effective and less toxic treatments for TNBC by enabling rational drug design prior to pre-clinical testing.
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