New Approaches for Testing Anti-Cancer Drugs on Tumoroids: Flow Chips, Imaging, and Metabolite Analysis
As we saw in the first article in this series, a transition from two-dimensional (2D) to three-dimensional (3D) tumor models, or tumoroids, provides a powerful way to recapitulate the cellular composition of tumors. As a result, tumoroids are a much better predictive tool for drug discovery compared to 2D models, and show great promise in guiding personalized medicine, including immunotherapy.
This article gives an overview of methods for handling and analyzing tumoroids and how challenges can be overcome, for example by using a combination of miniaturization and magnetic immobilization in a flow cell.
Tumoroid methodology - meeting the challenge of a new dimension
Transitioning from a relatively simple 2D monolayer cell culture to 3D makes the situation much more complex, and many methods have been developed to create, maintain, and analyze tumoroids [1,2], including:
- Static suspension
- Hanging drop methods
- Spinner or rotational bioreactors
- Scaffold-based methods, e.g., hydrogels
- Magnetic 3D bioprinting & levitation
- Microfluidic systems
The desire to mimic extracellular matrix (ECM) has led to several gel-based approaches that are widely used, especially hydrogels based on tumor extracts containing basement membrane ECM proteins. These are, however, prone to lot-to-lot variability that can greatly impact data reproducibility [3]. Synthetic scaffolds have been developed as alternatives, but these do not provide a one-size-fits-all solution and require laborious and costly tuning of parameters that direct cellular behavior. Added to that, matching the complexity of native tissues using synthetic scaffolds is very challenging.
Exploiting the power of magnetic bioprinting and levitation
One particularly innovative approach to creating 3D cell models involves using magnetic forces to promote cell aggregation while inducing ECM synthesis and promoting cell-cell interactions. This is achieved by tagging the cells with magnetic nanoparticles, then inducing them to form spheroids by exposing them to a magnetic field [4]. Magnetic levitation or bioprinting using, for example, NanoShuttle-PL (Greiner Bio-One) has several advantages [2]:
- There is no need for a specific medium
- The method works with normal 2D cell culture techniques
- A wide range of cell types can be used
- It only takes approximately 16h to form spheroids
- A 3D culture can be formed without the need for an artificial protein substrate
- The ECM can be synthesized while the spheroid is forming
One example of this approach focused on replacing patient-derived xenograft (PDX) models. These are effective preclinical models, but their cost and laborious preparation limit their value as platforms for high-throughput drug screens [5]. An in vitro 3D model was generated by isolating cells harvested from a mouse PDX tumor, magnetizing them with NanoShuttle-PL, and then using magnetic bioprinting to promote cell-cell interaction, which is essential for the reproducibility of such models. This is also a cost- and time-efficient in vitro first-pass drug screening platform.
As we will see below, magnetizing a spheroid is also a very good way of simplifying handling, and immobilizing the structure for analysis, such as in high content imaging.
The benefits of miniaturization
Another development that is becoming established in 3D cell culture is miniaturization, including the use of microfluidics. This combination has become a well-established approach in the life sciences to reduce the consumption of precious materials (e.g., reagents and patient samples), enable automation, and increase sensitivity and throughput of data generation from multiple complex assays.
Microfluidic devices are now being used to enable the formation, maintenance, and testing of tumoroids within a single device. The spatial control over fluids that can be achieved in micrometer sized channels increases the physiological relevance of 3D tumor models, and with the miniaturization sensitive analysis of, for example, metabolites is possible. As a result, “organoids-on-a-chip” have a promising application in drug discovery [6].
Magnetic immobilization improves flow cell performance
One example of a miniaturized platform for organoid handling and analysis is Pu·MA System® (Protein Fluidics, Inc.), which has 3D flow chips designed for streamlined automated measurements on organoids, including high-content imaging and fast fluorescence kinetic assays [7].
The system can be combined with magnetic immobilization to hold the tumoroids in a protected sample chamber during the assay and subsequent imaging. This simplifies the handling of the small fragile tumoroids, which are sensitive to manipulations such as media exchange, supernatant sampling, and immunofluorescence staining.
In the next article in this series, we will look at one example of how this combination has been used for multi-functional assay profiling on tumoroids treated with anti-cancer drugs.
Magnetic immobilization improves flow cell performance
One example of a miniaturized platform for organoid handling and analysis is Pu·MA System® (Protein Fluidics, Inc.), which has 3D flow chips designed for streamlined automated measurements on organoids, including high-content imaging and fast fluorescence kinetic assays [7].
The system can be combined with magnetic immobilization to hold the tumoroids in a protected sample chamber during the assay and subsequent imaging. This simplifies the handling of the small fragile tumoroids, which are sensitive to manipulations such as media exchange, supernatant sampling, and immunofluorescence staining.
In the next article in this series, we will look at one example of how this combination has been used for multi-functional assay profiling on tumoroids treated with anti-cancer drugs.
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References
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[3] Aisenbrey EA, Murphy WL. Synthetic alternatives to Matrigel. Nat Rev Mater. 2020 Jul;5(7):539-551. doi: 10.1038/s41578-020-0199-8. Epub 2020 May 27. PMID: 32953138; PMCID: PMC7500703.
[4] Souza, G., Molina, J., Raphael, R. et al. Three-dimensional tissue culture based on magnetic cell levitation. Nature Nanotech 5, 291–296 (2010). https://doi.org/10.1038/nnano.2010.23.
[5] Eckhardt BL, Gagliardi M, Iles L, Evans K, Ivan C, Liu X, Liu CG, Souza G, Rao A, Meric-Bernstam F, Ueno NT, Bartholomeusz GA. Clinically relevant inflammatory breast cancer patient-derived xenograft-derived ex vivo model for evaluation of tumor-specific therapies. PLoS One. 2018 May 16;13(5):e0195932. doi: 10.1371/journal.pone.0195932. PMID: 29768500; PMCID: PMC5955489.
[6] Park SE, Georgescu A, Huh D. Organoids-on-a-chip. Science. 2019 Jun 7;364(6444):960-965. doi: 10.1126/science.aaw7894. PMID: 31171693; PMCID: PMC7764943.
[7] Cromwell EF, Leung M, Hammer M, et al. Disease modeling with 3D cell-based assays using a novel flowchip system and high-content imaging. SLAS Technol 2021;26:237–48.