3D cell culture models have taken center stage as important pre-clinical models, bridging the gap between traditional 2D and animal testing methods. For cancer research and drug development, 3D tumor models can accurately recapitulate some of the clinical characteristics of cancer.¹

Developing more clinically relevant models is critical, as there has been a significant and growing “translation gap” in drug development. In oncology, the percentage of drugs successfully navigating the treacherous journey from pre-clinical study to market approval has been estimated to be as low as 3%, highlighting a massive loss in time and money for drug developers.^2,3

This blog discusses the utility of two types of 3D cell culture models, organoids, and spheroids, in preclinical cancer research, the challenges in extracting high-quality nucleic acids from them, and how to overcome these barriers.

The Difference Between Spheroids and Organoids

The two most common 3D tumor models are organoids and spheroids. The difference between them is subtle and still evolving as protocols for model development are being worked out.

Spheroids are spherical aggregates of one or more cell types from immortalized cancer cell lines or patient-derived cells or tissues. These 3D cell culture models are typically non-adherent. Depending on the type of tumor cells being grown, they can be cultured with or without growth factors and components of the extracellular matrix (ECM).¹ While the 3D structure of spheroids helps recapitulate some aspects of tumor physiology and treatment response, there is significant variation in establishing spheroid cultures, leading to challenges with reproducibility.⁴

By contrast, organoids form complex, heterogeneous, 3D “organ-like” structures in culture.¹ They typically include components of the ECM and are grown from pluripotent stem cells (PSCs), neonatal stem cells, adult stem cells, or patient-derived tumor cells/tissue. While organoids face similar reproducibility issues as spheroids, they can recapitulate the genetic, molecular, and cellular heterogeneity found in the tumor microenvironment (TME) incredibly well, particularly if they are derived from patient tissue or cells.

Advantages and Applications of Spheroids and Organoids in Cancer Modeling

Spheroid and organoid tumor models have several advantages and applications in pre-clinical testing, making them an attractive tool for understanding cancer and helping streamline research initiatives.

3D tumor models can:

Imitate the physiological and clinical characteristics of tumors

3D tumor models can mimic intra-tumoral heterogeneity and allow pre-clinical researchers to mimic tumor evolution and metastasis with the ease of an in vitro setting.^5,6 This helps untangle the genetic and molecular heterogeneity of tumorigenesis, metastasis, and treatment response or resistance.

Be used for rapid drug candidate screening

Because spheroids and organoids can mimic several clinical characteristics of primary and secondary tumors or healthy tissues, they can be used for drug screening and development. In addition, they are amenable to high-throughput, automated workflows for screening small molecule, biologic, and immunotherapeutic drugs. For instance, van de Wetering et al. developed a 384-well format for screening drug sensitivity in tumor organoids from colorectal carcinoma patients.⁷

Improve the genetic and molecular understanding of cancer

Genetic engineering of 3D tumor models using CRISPR-Cas systems can help researchers understand the early events that underlie tumor development.⁸ Genomics, transcriptomics, and other ‘omics techniques can further untangle the molecular underpinnings of clinically-important tumor characteristics.

Increase model accessibility through biobanking

Repositories of organoids derived from patients have become available, enabling researchers to easily access well-vetted and deeply characterized models to use them for drug screening, xenotransplantation into mice (as an in vivo model), or molecular analysis.⁹ This can help save time and money for many researchers and help provides a promising alternative to establishing a model from scratch.

As research continues, on 3D tumor models, new applications are emerging. For example, organoids-on-chips – microfluidic chips that support the culturing of organoids in physiologically-relevant conditions – have been shown to be accurate models for efficacy testing and modeling tumor traits such as development and vascularization.¹

Challenges Extracting Nucleic Acids from Spheroids and Organoids

Genomics, transcriptomics, and other ‘omics techniques are an integral part of the applications of organoids and spheroids in cancer research listed above. For these techniques to be successful, high-quality DNA and RNA extraction is essential.

Yet, 3D tumor models pose several challenges for the extraction techniques commonly used in traditional molecular biology workflows.

Challenge #1: Increased lysis times and variable lysis rates

Organoids and spheroids are small and compact structures, making extracting enough DNA or RNA for downstream applications difficult. 3D tumor models may also contain ECM components that interfere with cell lysis, and dissociation before extraction can be critical for the isolation of high-quality DNA or RNA. In addition, the cellular heterogeneity and variability in culturing protocols introduce significant variability in the quality of extracted nucleic acids. Several studies have found that RNA extraction with TRIzol™ Reagent can be variable and often requires additional lysis time.¹⁰

Challenge #2: Applications require high-throughput extraction workflows

From genome-scale CRISPR screens, drug candidate screening, biobanking, and personalized medicine applications, high-throughput workflows are essential for generating rapid, efficient, and reproducible results using organoids and spheroids. Despite this advantage, many researchers opt for time-consuming manual workflows rather than automated instrumentation to streamline sample preparation. By performing extractions manually across many different samples, isolated RNA and DNA can be vulnerable to contamination during processing, and users may require significant time and money to process samples.

Challenge #3: Significant optimization and troubleshooting

Today, many different types of DNA and RNA extraction kits and techniques exist for various applications. Because there is significant variability in organoid and spheroid culturing methods and applications, researchers may need to experiment with different kits, optimize protocols, and perform troubleshooting. Without an “out-of-the-box” solution for high-quality RNA and DNA extraction, researchers and drug developers may require significant input of upfront time, energy, and money to efficiently characterize organoid and spheroid models.

Choosing a Nucleic Acid Extraction Method for Organoids and Spheroids

Despite the challenges above, researchers are actively working to optimize protocols for developing and extracting high-quality nucleic acids from 3D tumor models, paving the way for more clinically relevant cancer models and helping improve the success rate of drug development. Thermo Fisher Scientific offers a comprehensive suite of nucleic acid extraction, isolation, and purification products, including the TaqMan™ Fast Advanced Cells-to-CT™ Kit and the MagMAX™ mirVana™ Total RNA Isolation Kit, which offer scalable, high-throughput sample processing, with KingFisher automated sample preparation instruments.

For other Interesting reads check out:

• White Paper: Isolation of RNA from organoids and spheroids

This article is for Research Use Only. Not for use in diagnostic procedures.

© 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

References

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6. Chen KY, Srinivasan T, Lin C, et al. Single-Cell Transcriptomics Reveals Heterogeneity and Drug Response of Human Colorectal Cancer Organoids. Conf Proc Annu Int Conf IEEE Eng Med Biol Soc IEEE Eng Med Biol Soc Annu Conf. 2018;2018:2378-2381. doi:10.1109/EMBC.2018.8512784
7. van de Wetering M, Francies HE, Francis JM, et al. Prospective derivation of a Living Organoid Biobank of colorectal cancer patients. Cell. 2015;161(4):933-945. doi:10.1016/j.cell.2015.03.053
8. Schutgens F, Clevers H. Human Organoids: Tools for Understanding Biology and Treating Diseases. Annu Rev Pathol Mech Dis. 2020;15(1):211-234. doi:10.1146/annurev-pathmechdis-012419-032611
9. Xie X, Li X, Song W. Tumor organoid biobank-new platform for medical research. Sci Rep. 2023;13(1):1819. doi:10.1038/s41598-023-29065-2
10. RNA Extraction from Organoids and Spheroids.; 2022. Accessed February 23, 2023. https://www.youtube.com/watch?v=bbtpiMyjcxg