Cellular therapy: from bench to bedside

Cell-based therapies show increasing promise in the treatment of many life-threatening conditions but the development of these depends on a strong foundation of cellular knowledge

 Technological developments have the capacity to advance scientific knowledge and drive research - one area currently benefitting is cellular research. Cell structure and function have long been investigated on a component basis to better characterise diseases, to design better drugs and, more recently, to find novel, more targeted cell-based therapies. Recent innovations in the field of cellular imaging, along with improved data analysis systems, have enabled scientists to look at the cell as a whole entity, to a greater level of detail than before, and in an environment closer to natural conditions.

The development of sophisticated cellular imaging tools for high-throughput screening (HTS) microscopy and super-resolution microscopy is increasing our understanding of basic cellular mechanisms and functions. This greater insight will uncover new opportunities in drug discovery through identification of novel drug targets and therapeutic strategies leading to safer drugs with less potential for adverse side effects and late-stage clinical failure due to toxicity. In addition, more targeted treatments with well-defined mechanisms of action will usher in an era of more precise, cell-based personalised therapies.

 There are a number of ways imaging tools can unravel the complexity of the cell.  Using live-cell assays for high content screening is one method being increasingly applied to study intricate signalling networks and integrated biochemical pathways. In this way, researchers can better appreciate cellular behaviour and the role a cell plays within a tissue or organism depending on its type, location, and environmental cues. When used alongside other advanced technologies for growing, processing and analysing cells this method gives valuable knowledge which can then be used to not only develop cell-based models for toxicity testing but also to further the industrialisation and standardisation of cell production for therapeutic applications.

 The ultimate value of all of this information will only be attained through its integration and interpretation in the context of human cells. By studying and developing accurate models of human heart cells, for example, grown and maintained in surroundings that approximate their natural environments, it becomes possible to screen drug candidates in preclinical testing and achieve truly predictive toxicology. The development of cardiomyocytes that can be propagated in large-scale cell culture will foster advances in regenerative therapies and improved cell-based assays to assess cardiotoxicity. Research aimed at characterising stem cells and identifying the signals and stimuli that trigger and direct their growth, differentiation, and death, will accelerate progress in developing stem-cell derived cell lines for research, preclinical studies, and HTS, and for advancing cell-based strategies to repair and replace damaged or diseased tissues and organs.

Current research by the Cell Technologies team at GE Healthcare Life Sciences is investigating one aspect of cardiotoxicity – that caused by anti-cancer drugs targeting cellular kinases. It is known that many anti-cancer drugs target key parts of the cell signalling pathway but some treatments have been found to be cardiotoxic due to off-target effects. Our team is using high content analysis screening to characterise this mechanism with the aim of developing cardiomyocyte-based models for drug toxicity testing.

[caption id="attachment_32344" align="alignleft" width="147" caption="3D-SIM images of FtsZ-GFP localisation in live cells of B. subtilis. (A) Conventional wide-field fluorescence microscopy image of B. subtilis strain SU570 (ftsZ-gfp) stained with the membrane dye FM4-64 shows how FtsZ-GFP assembles into Z rings. Scale bar, 5 µm. (B) When the same strain is imaged using 3D-SIM (OMX V3), regions of interest from the image can be selected (dashed box) to zoom in and rotate the image around the z-axis to view 3D FtsZ structures in the axial plane1"][/caption]

The team has grown cardiomyocytes under different culture conditions which mimic the metabolic changes that occur in cells in the heart under normal and stress conditions. We used mitochondrial, calcium and membrane probes to profile the actions of anti-cancer drugs and gain insight into the mechanisms of toxicity using high content analysis.  Our results show that cardiomyocytes grown under conditions where their mitochondria are fully active are between 100 and 1000 times more sensitive to some anti-cancer compounds than those grown under standard culture conditions. This suggests that the conditions in which cells are grown is key to designing optimum cardiomyocyte-based assays for drug toxicity testing.

Crucial to the success of this approach is the depth and breadth of information generated from HCA compound screening using the latest technologies. It is now possible to create a multiparametric profile of each compound, which allows comparison and grouping of different types of compound. The team are looking at drugs already in the clinic and some still in development. The aim is to develop a bank of ‘signatures’ for compounds, especially those with known clinical toxicity. In the future, the results of the same assay on new drugs in development will allow comparison of signatures and flag up which drugs need further investigation.

The data generated by HCA also provide a good indication of mechanism of action (i.e. how the drugs are interacting with cells and what they are doing). This information could be used to prioritise compounds and guide investigations into engineering the toxicity out of a compound whilst maintaining efficacy. In this way, HCA is becoming a powerful, efficient and informative way of carrying out investigative biology.

 In addition to tools that allow the capture of multi-parameter data, innovative high-speed, high-resolution imaging technologies have dramatically increased the speed and resolution at which imaging data can be captured. Super-resolution microscopy technologies, which can achieve 80-100 nm spatial resolution, are now enabling live cell imaging and opening a window into the mechanistic workings of intracellular processes.  This in turn is helping enable discoveries and insights not previously possible. The combination of nanometer-scale resolution, high speed signal detection (so that "real-time" imaging means capturing cellular and subcellular processes as they are occurring), and the capability to record, store, and analyse the large amounts of data generated is yielding a wealth of new knowledge.

The ability to conduct live cell imaging at the super-resolution level will help cell biologists overcome a major challenge: fluorescence microscopy is largely performed in fixed tissue samples, meaning scientists can only infer what is driving cell behaviour based on relatively limited findings. Advanced cellular imaging technologies such as super-resolution microscopy can give researchers the ability to follow a sequence of events over time under changing conditions. Innovative label-free technologies can be used to observe cell activity and nanoparticle-based approaches can probe the intracellular space, both without disturbing normal cellular function.

A super-resolution imaging system enables this type of research through the use of 3D-SIM (Structured Illumination Microscopy), a super resolution technique that approximately doubles the resolution in all three dimensions compared to conventional fluorescence-based optical microscopy methods. The result is an eight times improvement in volume resolution. Professor Liz Harry of the ithree institute, University of Technology Sydney, and colleagues, have used this groundbreaking imaging system to gain new insights into the conserved spatial organisation of proteins in the "divisome" of bacteria and how that relates to the cytokinetic processes essential for cell division.

In a recent paper published in PLoS Biology[1], Professor Harry and her team described the application of 3D-SIM to study the dynamic localisation of the FtsZ protein in two types of bacteria undergoing cell division: the rod-shaped Bacillus subtilis and the spherical Staphylococcus aureus. FtsZ is a tubulin-like cytoskeletal protein that polymerises to form the Z ring, which acts as a scaffold during bacterial cell division, recruits other proteins needed for cell division, and generates a contractile force through constriction of the Z ring that is required for cytokinesis.

Previously, conventional fluorescence microscopy had indicated that the Z ring is a continuous dynamic structure of uniform density, assembled from FtsZ precursors, that undergoes continuous subunit turnover. In contrast, 3D-SIM revealed a heterogeneous distribution of fluorescently labeled FtsZ throughout the Z ring. Furthermore, it suggested a discontinuous structure with gaps of approximately 118-200 nm, which could not be resolved using conventional fluorescence microscopy.

The findings indicated that FtsZ molecules maintain a dynamic bead-like distribution across the Z ring that can change before and during constriction of the ring, challenging the existing concept of a homogeneous, continuous structure that wraps around the cell. These new insights were made possible by the ability to view the entire 3D architecture of the Z ring in live bacteria at high resolution over time, and have led to novel theories of how the Z ring constricts and what triggers constriction and cytokinesis. New insights into the process of bacterial cell division may ultimately help in the quest to find novel antibiotics which target this mechanism.

So what does the future hold for cellular research? Cell therapy, the use of cells to treat disease, is already showing enormous promise for the treatment of many life- threatening and life-limiting diseases.   The concept is not new - for over forty years, clinicians have used the transplantation of bone marrow, a rich source of haematopoietic stem cells, as a way of treating patients with serious disorders of the blood.  What is new is the pace and rapid acceleration of research efforts worldwide to find novel, more targeted cell-based therapies for a wide range of diseases such as cancer, heart failure, macular degeneration and Parkinson’s.

Globally, there are more than two thousand clinical trials underway in some of the world’s most prestigious institutes and hospitals.  Researchers are exploring a wide range of different strategies and approaches – two examples being direct transplantation of stem cells into the body to repair or replace tissue damaged by disease or trauma, and immunotherapy where the patient’s own white blood cells are collected, expanded outside the body and then reintroduced to the patient.

As with any new therapeutic approach, the use of cell therapy in the routine clinical setting will require the introduction of new technological, manufacturing and supply chain approaches, and there are significant obstacles that will have to be overcome. Already, there are investments in the development of: sophisticated cellular imaging tools to increase our understanding of the basic cellular mechanisms involved in cell therapy; new technologies for the isolation, purification and expansion of millions of cells for thousands of patients on an individual, yet safe and routine, basis: and quality control protocols to support this new approach for routine and reliable manufacturing.

The development of cell therapies depends on a strong foundation of knowledge about the cells targeted for use in a clinical application. Well-defined metrics and methods are needed to quantify ranges for what represents normal and optimal cell function and growth in a laboratory and commercial setting. Current efforts will not only support the development of robust, reproducible, and standardised protocols for early-stage development, testing of therapeutic strategies and later-stage scale-up and manufacturing, but will ultimately be critical to meeting regulatory requirements and ensuring patient safety.

Reference: Strauss M, Liew A T F, Turnball L, Whitchurch C B, Monahan L G, Harry, EJ. 2012. 3D-SIM Super Resolution Microscopy Reveals a Bead-Like Arrangement for FtsZ and the Division Machinery: Implications for Triggering Cytokinesis. PLoS Biology 10 (9):e10011389

Author: Nick Thomas, Principal Scientist, Cell Technologies, GE Healthcare Life Sciences