Replacement of animals in cancer drug development by using 3D in vitro functional assays for increased predictive power

Project background

Cancer therapeutics have a higher attrition rate comparatively to many other therapeutic areas. Preclinical testing of potential candidates typically involves applying the compound to 2D tumour cell culture systems and assessing the impact of the compound on cell proliferation. The best candidates are then assessed in a relevant animal model. The inability of 2D in vitro assays to accurately predict the in vivo response is one of the contributing factors to the high attrition rate of cancer therapeutics. This is likely due to the relative simplicity of the 2D cell culture, which does not accurately replicate the complexity of the tumour in the body.

Why we funded it

This Project Grant aims to develop 3D in vitro functional assays as a replacement for animals required in the preclinical testing stage of cancer drug development.

The Cancer Therapeutics department of the Institute of Cancer Research requires approximately 2000 mice per year for in vivo testing of molecularly targeted drug discovery projects. Dr Chesler estimates using the 3D assays in preclinical testing could reduce the number of compounds progressing to in vivo testing, reducing the numbers of animals required by 400 a year. To exemplify the assays, this project grant focusses on malignant brain tumours, which also use transgenic murine models to better replicate the disease. Transgenic models require breeding colonies to sustain the necessary population needed for studies. Using the in vitro assay as a replacement for one such study requiring a transgenic model could further replace up to 750 to 1000 animals per year.

Research methods

3D culture systems, compared to 2D cultures systems, more faithfully reproduce the in vivo tumour microenvironment in terms of gene expression profiles, signalling pathway activation, cell-cell interactions, matrix deposition, and nutrient and oxygen gradients. In initial experiments, Dr Chesler and colleagues have demonstrated 40 different cancer cell lines were able to form 3D spheroids spontaneously in their multicellular tumour culture system. In this project grant, functional assays, which are both reproducible and quantifiable, will be developed for tumour growth, invasion and angiogenic potential for a panel of 3D cancer spheroids. Once optimised, the response of the cells to inhibitors will be compared to results previously obtained in both 2D and in vivo systems. The developed assays will also be modifiable for a high-throughput format, allowing screening of novel cancer drugs in a more in vivo like environment. 

Animal testing is mandatory for any drugs entering clinical trials. However, in cancer in particular there is a high attrition rate prior to clinical deployment. Simple 2-dimensional monolayer cultures of tumour cells are not sufficiently predictive of in vivo responses and thus many animals are used for subsequent tests without legitimate in vitro validation of drug candidates. Our objective is to bridge the gap between standard in vitro cell cultures and in vivo efficacy studies. We focus on malignant brain tumours because they are almost universally fatal in both adults and children. Using a well-characterised panel of human adult and paediatric gliomas and medulloblastomas, we will optimise 96-well plate 3D spheroid assays of growth, invasion and angiogenesis. We have already shown proof of principle of the assays and that they are amenable to high throughput drug evaluation. Spheroids are established in suspension culture and their growth kinetics measured over time by quantitative image analysis. Endpoint cell viability assays enable GI50 values to be determined for direct comparison with 2D cultures. By addition of matrigel, we convert the assay into an invasion assay which can be similarly quantified. Finally spheroids co-cultured with endothelial cell monolayers provide a model of perivascular invasion and confrontation cultures between embryoid bodies and tumour spheroids mimic angiogenesis. We will test in these models inhibitors of PI3Kinase and the HSP90 chaperone (validated targets in these diseases and where we have extensive preclinical and clinical experience). We will then compare results with those already obtained in 2D assays and in vivo as a benchmark for qualification of our proposed alternative in vitro 3D systems. Parallel mechanistic studies will evaluate the effects of hypoxia and stem cell populations (prevalent in 3D cultures but lacking in 2D systems) for their impact on drug responses. In addition, we will undertake detailed pharmacodynamic biomarker assays (e.g. phosphoproteins levels for PI3K inhibitors; client protein expression for HSP90 inhibitors) to understand the basis for differences in response in the three systems. By developing a more sophisticated triage system for experimental compounds we will be able to replace a significant proportion of the animals currently required to bring new drugs to the clinic. Although we will exemplify the assays in brain tumours, we have already shown that they are applicable to many other tumour types and hence will have wide applicability in academic and pharmaceutical drug discovery.

• Research Review 2013: 3D micro-cancers reduce animal use in drug development
• Engagement Activities: SET for Britain, March 2013
• Blog: Getting the 3Rs message out there