Closing Keynote for Building Biology in 3D Symposium

Posters presented at the 2023 Building Biology in 3D Symposium

The need for physiologically relevant 3D, complex in vitro models of disease is steadily increasing due to the emergence of drugs targeting the immune system and their microenvironment. Further, an increasing interest in precision treatment of cancer patients has highlighted the need for microfluidic technologies capable of maximising generation and screening of 3D patient-derived models from the small cell number contained in biopsies. We have developed a versatile microfluidic platform for high quality and multiplexed screening assays on spheroid co-cultures, organoids and tissue micro-explants. When using cell suspensions, hundreds of 3D models are created within 24-48 hours within a microfluidic cell culture array. When using explants of preformed organoids, these are seeded directly into the array according to their size. The platform is designed for self-generation of multiple drug concentration gradients, offering a unique system to miniaturize drug combination studies using patient tissue and, at the same time, creating cost-effective and fast immune-oncology assays. Readouts, such as the model volume, phenotype and viability, are generated from platform-specific image analysis software, using epifluorescence or confocal microscopy images. The technology has been validated using a variety of cell sources. As examples of diverse and customisable screens: 1) human prostate biopsies were grown for the screening of clinical therapies on thousands of 3D multicellular structures1; 2) 3D co-cultures of several cell types were optimised in our platform to mechanistically study responses of the tumour microenvironment2; 3) CAR-T cells were used to assess their target specificity and cytotoxicity in 3D co-cultures3; 4) organoids from fresh and frozen tissue were cultured for precision medicine purposes, accelerating time to results. These examples show the screening capabilities of our microfluidic platform and especially its potential for extensive drug combination studies and precision medicine applications. Ultimately, the power of miniaturising combination studies on patient derived models has significant opportunities to produce faster and better preclinical data. References 1) T. Mulholland et al., Sci. Rep., 2018, 8, 14672. 2) E. Kay et l., Nature Metabolism, 2022, 4 (6), 693-710. 3) K. Paterson et al., IEEE OJEMB, 2022, 3, 86-95.

It is currently difficult to accurately predict efficacy and toxicity of emerging drugs in the clinic using conventional models of disease, particularly at the level of individual patients. This makes it challenging to nominate drug candidates to clinical studies based on accumulated preclinical data, and to match patients to these treatments. In the ideal scenario, cells from multiple patients or donors would be used early in the drug discovery pipeline to understand drug response in models that accurately reflect both disease biology and variability. Furthermore, the same readouts of efficacy and safety could follow each drug into the clinic as a companion precision diagnostic. However, there are currently few methods that enable use of primary material at a scale and cost, such that they can realistically support the full life cycle of drug discovery project at an early stage. In this talk we describe our efforts to bring highly miniaturized primary 3D models to drug discovery. We use microfluidics to generate 3D structures composed of 80-100 cells and high content imaging to characterize drug response. We have preliminary data that miniaturized liver models generated using this approach retain albumin secretion and metabolic activity characteristic of the primary cell phenotype. In addition, we touch upon similar development of primary models and imaging assays for drug profiling cells from ovarian cancer patients. We call this platform Drug Efficacy Testing in Ex Vivo Cultures (DETECt). In this setting we can discriminate between patients with a progression-free interval > 12 months and < 12 months based on the drug response score for the standard of therapy treatment, carboplatin, measured using the DETECt platform.

Stem cells have enormous potential for disease modelling and novel cell therapies. Although the two-dimensional cell culture method is routine in vitro, limitations exist for simulating the cell growth microenvironment in vivo. Therefore, the three-dimensional culture provides an alternative to achieving multicellular interactions. Furthermore, applying a three-dimensional cell culture system is exciting in drug screening.

The SLAS 2023 Building Biology in 3D Symposium will took place in Cambridge, United Kingdom 20-21 April 2023. This two-day event addresses the successes and limitations of using 3D systems in discovery and applied research while acknowledging the need for improvements to ensure widespread adoption. 

Attendees will learn about advanced 3D cellular models, advances in 3D imaging and analysis, translational models and enabling technologies.

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Purchase of the 2023 Building Biology in 3D Symposium package will gain immediate access to all talks from the event. Alternatively, talks can be purchased a la carte.

Advanced 3D Cellular Models

Patient-derived scaffolds and analyses of adapting cancer cell lines can monitor malignant properties of a cell-free cancer microenvironment highlighting distinct links between scaffold influences and clinical aggressiveness in cancer. The protein composition of the cell-free cancer microenvironments influencing adapting cancer cells have been defined by quantitative mass spectrometry and results indicate clear clustering of PDS differing in extracellular matrix related proteins as well as immunoregulatory and metabolic regulators. Interestingly both the defined clusters as well as individual imprinted proteins in the cell-free scaffolds are linked to clinical behaviours of the cancer and data from breast cancer and colorectal cancer will be presented. The multitude of proteins imprinted in the cell-free cancer scaffolds representing various tumour biological activities and cell types, supports the importance of the cancer microenvironment in influencing varying disease behaviours. The in vivo identified proteins can be used for improved disease subtyping, cancer drug targeting and importantly to construct optimal synthetic 3D-models that can be used for human-like drug discovery and validation of novel cancer treatments. Data from the first prototypes of human-mimicking synthetic scaffolds will be presented and discussed in relation to other 3D-based growth models available for cancer discovery.

Traditional two-dimensional (2D) cancer cell culture has been used extensively to evaluate the efficacy of drug delivery systems, but it has limitations that can deviate results from a real tumour mass in vivo. The lack of accurate cellular interactions and extracellular matrix (ECM) components are the primary differences between 2D cancer models and an actual tumour in vivo. 2D cell culture also lose tumour heterogeneity. The results obtained from 2D models do not represent the complexity of drug delivery and diffusion that occurs in vivo. In recent years, three-dimensional (3D) tumour models have gained popularity as they can overcome the limitations of 2D models and are physiologically comparable to a real tumour mass. 3D models possess ECM components which resembles in vivo tumours. In 3D culture, drug penetration and distribution are more representative of the tumour microenvironment, allowing us to investigate the efficacy and toxicity of a drug in a more clinically relevant model. Together, this makes 3D culture models suitable for drug delivery research. Phage Cancer Therapy group has employed 3D tumour models to evaluate the efficacy of a gene delivery platform derived from a harmless filamentous bacteriophage (Phage); Transmorphic Phage/AAV (TPA). The TPA particle has many advantages over other gene delivery vectors including low production cost, very low toxicity to normal cells, can be targetable to specific cell type, and does not require low temperature storage. Phage Cancer Therapy group has developed TPA as a multifunctional cancer cell targeted nanocarrier to guide the delivery of therapeutic genes (or drugs) to cancer cells. Capsids of TPA particles were engineered to equip cancer targeting ligand (RGD4C) on the capsids with a therapeutic transgene cassette inside the capsids. The RGD4C ligand specifically binds to ?v?3 and ?v?5, which are specifically overexpressed on tumour cell surface. It is subsequently up taken by, and delivers the therapeutic gene to, cancer cells. We adopted 3D tumour models of various cancer cell types as models to evaluate the efficacy of the novel TPA nanocarrier. We found that cancer stem cell population in our 3D tumour sphere models resemble tumours in vivo. Our novel TPA particle demonstrates promising gene delivery efficiency across many types of 3D cancer models. TPA efficiently penetrated through the ECM and delivered a transgene to targeted cells, ultimately resulting in tumour regression. Furthermore, toxicity tests, in vitro and in vivo, showed that the TPA particle itself does not harm normal cells and organs. We confirmed the efficacy of TPA in an animal model by systemic administration of the particle through the teil vein. TPA can selectively deliver a transgene to tumour cells but spare other organs unharmed. Altogether, the TPA particle has the potential to be a powerful cargo for tumour-targeted gene delivery.

There is an urgent need for scalable microphysiological systems (MPS) that can better predict drug efficacy and toxicity at the preclinical drug screening stage. An ideal preclinical model that can accurately predict clinical response does not exist however the use of MPS can help bridge the translational gap by providing more accurate representation of human susceptibility to drug response. Mera is an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has microfluidic flow control and multiple possible fluidics modes, the primary mode allowing cells to be matured into a desired microtissue type and the secondary mode where the fluid flow can be re-orientated to create a body-on-a-plate-format with recirculating circuits composed of inter-connected channels to allow microtissue communication. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM (CalAM) and Ethidium Homodimer (EtHD1) viability stains. The prototype microtissue culture plate wells are laid out in a 3 x 3 or 4 x 10 grid format with viability (multi-organ models) and toxicity assays demonstrated in both formats. We present the groundwork for the Mera system to be used as a viable option for scalable microtissue culture and assay development for preclinical drug development.