Scale up of organoid culture for their widespread use as a polarity model


Supervising PIs

Trevor Dale & Marianne Ellis

ESR15 Nuria Abajo Lima

Project Description

Recent studies showing cancer organoids recapitulate the biology of primary cancers have driven tremendous excitement in the potential for cancer organoids to revolutionise drug discovery and personalized medicine. At present, freshly-prepared organoids from colorectal cancers are composed of genetically and phenotypically diverse populations. Tumour heterogeneity at the genetic and phenotypic level drives differential responses to therapeutic agents. Differences in cell polarity / differentiation within multicellular cancer organoids underlies their differential drug responses. In this project, chemical engineering / bioreactor technologies will be used to separate distinct organoid subpopulations based on their size, shape and density. Purified populations of organoids will be used to study differences in organoid subtypes, function and polarity, and to relate genetic and phenotypic differences back to drug response and primary tumour heterogeneity. The outputs of this cross-disciplinary project will be 1. The development of a novel biophysical technique for the isolation of multicellular organoid subtypes. 2. The identification of genetic and phenotypic / cell polarity differences predict intra-tumour heterogeneity in response to therapeutics.

Objectives

  • Analysis of cancer-induced changes in cell polarity using biophysically-purified organoid subpopulations.
  • Design and analysis of a device for biophysical purification of organoids.

Summary of Results

Organoids have the potential to be used for testing efficacy and toxicity of drug compounds and this system enables the discard of ineffective drugs before animal trials. Using organoids as an alternative to 2D cultures is growing in popularity but there is a bottleneck to their widespread utilization. Organoids need to be produced on a large enough scale to adequately supply endusers, from university researchers to pharmaceutical companies; importantly batch-to-batch variation needs to be minimized. Currently, manual processing results in organoids of varied size and while the majority are suitably functional, the smallest, comprising only a few cells may be too small to polarise and differentiate, while the bigger ones may have necrotic cores.

Cellesce Ltd has developed a new bioprocessing technology by semi-automating the process of organoid culture in a bioreactor, thus improving the control of the process conditions, which yields a narrower and thus more desirable range of organoid sizes. A quality control step after the expansion process is proposed in this work and consists of fractionating organoids based on their size in order to get a more consistent product and reduce batch-to-batch variability.

This work showed that fractionated colorectal cancer organoids respond to known colorectal cancer targeting compounds (LGK974 and 5FU) and drug response variability is reduced by including a fractionation step.

The effect of organoid size on its biology has been studied by RNA-seq and a time course experiment and size fractionation have been performed to generate different size organoids. Hypoxia has been identified as the main contributor of differences in gene expression between smaller and larger organoids. Adaptation to hypoxia requires reprogramming of essential elements of cellular metabolism as for example energy and lipid metabolism. Oxygen limitation has shifted energy production in larger organoids by increasing glycolysis and decreasing mitochondrial function. Also, an increase in cholesterol biosynthesis in larger organoids showed the dysregulation of lipid metabolism under hypoxic conditions.

These findings show that aggregate size is a key parameter in multicellular structures due to diffusional limitations and needs to be taken into account in the planning phase of specific drug testing, accordingly to the mechanism of action of the drug.

References

  1. Young, M. & Reed, K. R. Organoids as a Model for Colorectal Cancer. Current Colorectal Cancer Reports1–7 (2016). doi:10.1007/s11888-016-0335-4
  2. Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nature Publishing Group18,246–254 (2016).
  3. Rohwer, N. & Cramer, T. Hypoxia-mediated drug resistance: Novel insights on the functional interaction of HIFs and cell death pathways. Drug Resistance Updates14,191–201 (2011).
  4. Daster, S. Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment. 1–12 (2017).
  5. Wu, J., Rostami, M. R., Cadavid Olaya, D. P. & Tzanakakis, E. S. Oxygen Transport and Stem Cell Aggregation in Stirred-Suspension Bioreactor Cultures. PLoS ONE9,e102486–12 (2014).
  6. Ilias Mylonis, George Simos and Efrosyni Paraskeva. Hypoxia-Inducible Factors and the Regulation of Lipid Metabolism. (2019)
  7. Guomin Shen and Xiaobo Li. The Multifaceted Role of Hypoxia-Inducible Factor 1 (HIF1) in Lipid Metabolism. (2017)