See the big picture

The phenotype is making a strong comeback when it comes to screening cancer drugs for efficacy and toxicology – and this could be the key to a personalised approach. The trick, says Dr Magnus Jansson, is to make the 3D culture environment so close to the in vivo situation that grand metabolic changes can be truly predictive

Modern drug discovery is based predominantly on parameter research. Individual factors like gene expression levels, protein levels, protein activity and localisation are measured and extrapolated into in vivo function for treatment in humans.

The measurement of these factors and levels is limited in time and space and to a large extent performed in synthetic cell models based on the two-dimensional growth of immortalized cell lines – far away from the tissue/organ/whole organism that will eventually be treated, usually for an extended period.

The extrapolation of the effect in a two-dimensional (2D) cell culture to the in vivo human situation has time and again displayed less favorable outcomes. There are numerous examples where clinical trials have been unsuccessful due to limited efficacy or toxicological side effects. One simple example is the screening of anti-cancer drugs performed in oxygen rich 2D cell culture in which very high specific metabolism gives very different chemical hits compared to an oxygen-starved situation that would be a closer match to the situation in solid tumors in vivo.

There are numerous examples where clinical trials have been unsuccessful due to limited efficacy or toxicological side effects

Another example is in leukemia research, where drug development is known to have faced serious setbacks due to the limitations of 2D culturing. Despite the knowledge that the leukemic cells’ interaction with the bone marrow’s stromal microenvironment can provide them protection, the usual methods are unable to investigate this “relationship”. Thus, a multitude of promising compounds that would otherwise be successful in combating the leukemic cells, fail due to the lack of an in vitro testing method that can mimic the in vivo system adequately.

Accounting for life Cases like these are one key reason why only 1 in 50 anti-cancer drug candidates are making it to the market. 2D culturing and parameter testing limitations are partly responsible for this problematic number. A proposal to counteract the issue, is to try and increase the knowledge of cell-response upon treatment and to reduce the gap between synthetic assay conditions and real live whole human response. The way to do this is to introduce the phenotypic assay where the total response of all the mechanisms of life is accounted for.

The use of a calorimetric approach enables direct measurement of the cell activity without any labels or additions of any kind. The calorimetry-based assay directly measures the energy release from the total metabolic response. This is an optimal phenotypic screening solution for improving drug discovery and development processes.

The unique upside is that it is not parameter testing and presents a novel label-free, real-time and non-destructive technology that can be used to study the net effect of all cellular parameters at once and in the correct context, regardless of sample composition and morphology. The isothermal microcalorimetry assay is unique in this capacity.

An example of such a technology is the calScreener, an accurate phenotype (functional) assay, as compared to most other technologies that use molecular target-based screening. Most assays are also predominantly end-point assays, where samples are gathered at one or a limited number of time points, in contrast to the continuous kinetic measurement of the calScreener.

Direct Metabolic Readout (DMR), provided by this kind of technology, gives the scientist access to an unbiased assay where the effects of treatment are studied in a correct biological context.

The approach can bridge the gap between screening on isolated components to 2D cell cultures to 3D tissue to the in vivo human situation. It is this which can result in greater predictive value for cost-effective, rapid and fruitful drug development. The possibility to direct continuous measurement of effects in hard or previously impossible to monitor environments like 3D cell cultures or bacterial infections in complex matrices also increases the predictive power of the scientific hypothesis in drug development, as well as the physiological relevance of the produced data.

One size doesn't fit all The extension of 3D based drug discovery would be to apply the same assay principle on actual patient tissue samples to evaluate the best possible treatment based on the real patient response. The screening of these tissues for a prolonged period, through non-destructive and real-time monitoring which mimics the in vivo condition, can provide invaluable information about the disease as well as the specific patients.

So, with the usage of patient samples, the microcalorimetry assay can also act as a novel approach to developing personalised medicine by shifting the focus from genotypic to phenotypic screening. While standard approaches, such as whole genome sequencing, are of great value, in fields like oncology where there is significant differentiation of tumor cells between patients or even within the same patient, utilizing phenotypic screening can provide better solutions. They can also avoid relapse in patients that did not “fit the mold” of a genotypic-derived therapy that serves specific patient subgroups with similar characteristics.

Even outside oncology, the development of cell-based disease models and the use of phenotypic screening in general is becoming highly relevant in drug discovery, as molecular target-based screening alone is not able to provide the desired quota of approved drugs or effectiveness on distinct patient populations.

Of course, there are cases where phenotypic screening would not be enough on its own. The importance of parameter testing in many aspects of drug development is not in question, but what is undoubtedly clear is that adding phenotypic screening to the various approaches that are already in place would enhance the drug discovery process and complement genotypic methods, when these are unable to provide the necessary data. Calorimetry, specifically, measures one of the two fundamentals of life - metabolism. Using it along with the methods related to the other fundamental – the self-replication of the genetic information ­– there is every chance that a well-researched drug compound will make it rather than fail, if the new and improved assays are utilized to their maximum potential.

We predict that the recent developments in calorimetric equipment – enabling small volume samples and multichannel equipment adapted to microbiological laboratory settings – will lead the way to new 3D assay tools in the development of novel oncology treatments. Beyond that, this technology will also serve as a tool for in vitro diagnostics and personalised medicine, by revolutionising the processes used for research, drug development and treatment of multiple diseases.

Author Dr Magnus Jansson is Chief Scientific Officer at Symcel Sverige AB. He joined Symcel in 2009 and is responsible for technology and applications development. He has 25 years of experience in the biotechnology and pharmaceutical development industry.

magnus.jansson@symcel.se