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Systems microscopy – a key enabling methodology for next-generation systems biology

Periodic Report Summary 4 - SYSTEMS MICROSCOPY (Systems microscopy – a key enabling methodology for next-generation systems biology)

Project Context and Objectives:
The mission: Single cells in 4D
The Systems Microscopy Network of Excellence (NoE) is developing a methodological platform that will enable the studies of single cells in the three dimensional of space and time at the systems level. This is an important analysis capacity in cases where spatio-temporal events in cellular subpopulations play a critical role, such as in cancer development, progression and metastasis. Advanced light microscopy forms the technological basis for the project as it is the only technology that can deliver sufficient sensitivity, as well as spatial and temporal resolution in single living cells. When employed quantitatively and with high throughput, microscopy allows for the acquisition of complex and rich data sets at the systems level. Efforts to increase the speed and the confidence with which high content data from microscopic images can be produced, will strengthen the emerging methodology referred to as “systems microscopy” and will establish it as a corner stone in the systems biology analysis of the living cell.
As a paradigm to enable systems biology at the cellular scale of biological organization, the network addresses, as its core biological theme, two major mechanisms underlying cancer progression and metastasis: cell division and cell migration. The network joins world authorities in the fields of microscopy, systems biology, bioinformatics, mathematical statistics and modeling, as well as in cell migration and cell division and provides a unique opportunity for Europe to acquire global lead in systems microscopy.

Project objectives
The overall objective of the Systems Microscopy NoE is to develop systems microscopy into a powerful enabling platform for next-generation systems biology.

The overall objective will be achieved through the following sub-objectives:
• To develop a novel pan-European systems microscopy infrastructure for systems biology, including new Imaging platforms and Image extraction software; new tailored methods for Statistics, Bioinformatics, and Modeling; and Standards as well as a Database for Systems Microscopy.
• By using cell division and migration as our core subjects; To gain a systems-level understanding of two basic mechanisms underlying cancer, and identify novel pathways for translational research.
• To perform translational research using results and methodology from our systems microscopy research.
• To disseminate our results to the research community, industry and other interested parties.
• To train the next generation of scientists in systems microscopy.
• To make our network as well as its results and structures durable beyond the EC financing period.

Project Results:
Improving throughput
The work on high-throughput screening platform development continued with further improvement of the automated fluid delivery system FCS/FCCS that utilizes microfluidic imaging chambers for high-throughput correlated and multimodal microscopy on both the Leica SP5 SMD and Zeiss LSM780 ConFoCor3 imaging system using Micropilot or Micronaut software plugins. We have also focused on further development and optimization of 384 well micro-plate based screening approach for drug sensitivity and resistance testing (DSRT) as well as siRNA screening.

Maximizing content, data processing, modeling and query
Available high-throughput image analysis software offer efficient algorithms for analysis of single time-point assays while the existing tools for the analysis of cellular dynamics in multi-dimensional large-scale imaging are very limited. During our fourth year, we developed the software Micronaut, which provides an extended and improved interface between the software controlling a confocal microscope and the computer vision / machine learning methods. The EBImage software package for R was further enriched with tools to transform images, segment cells and extract quantitative cellular descriptors. The open source standard format for high-content screening data, CellH5 has been extended to further include common analysis tasks such as dimension reduction techniques and unsupervised learning methods on extracted morphometric cell features. We continued to improve software (WP5) that performs primary statistical analysis of complex data along with quality assessment and significance analysis developing the RBioFormats software package that provides an interface between the powerful statistical and visualization environment R and the Bio-Formats microscopy image formats reader.

Development and application of modelling methods for systems microscopy
Data derived from a number of different experimental set-ups have been used to commence bold modeling projects that will ultimately serve to describe the dynamic processes of cell migration and division. During P4, we have been testing and further exploring an approach to measure the heterogeneity of single cell descriptors in a population of cells from single-cell RNA-Seq data to also make it applicable to cellular descriptors derived from microscopy imaging. We made tremendous advances in the area of mechanistic modelling on protein localization on steady-state structures with the development of a third original method to calculate the localization of molecules transported by filaments.

Standardization measures
Development of compatibility and exchangeability of data from the packages developed by the NoE members (original multidimensional images and segmentation masks, analyzed features and statistical results) continued by including import/export modules based on HDF5 database structures and OME image file standards and the agreed common nomenclature This allows application of complementary software (e.g. screens analysis by scores based on multidimensional statistics, by supervised or by unsupervised machine learning classification) .
Database Development
The Cellular Phenotype Database (CPD) running on a production server since 2013 it now contains 10 datasets associated with scientific publications. Phenotypes associated with these 10 studies have been mapped to Cellular Microscopy Phenotype Ontology (CMPO) terms and ontology-enabled query has been implemented through the CPD user interface for phenotype search and browsing.

The biological systems
The characteristics of cancer biology and its clinical consequences render a strong case for studying cell division and migration at the systems level. During the fourth project period we continued to work on a tertiary RNAi screens and validated the earlier identified candidate regulator of chromosome-chromosome adhesion. Further, we complemented the RNAi screening method by recently developed technologies based on CRISPR/Cas9 gene editing. Screening activities searching for genes whose knockdown affects individual or collective cell migration led to the validation of highly promising hits. Super-resolution studies using FRET and dual color super-resolution of adhesion sites were performed which shed new light on the molecular architecture of the adhesion sites.

Potential Impact:
The area of systems microscopy is just emerging, but has already proven its impact in being able to powerfully combine systems biology with detailed visual analysis of cellular processes. This NoE aims to enable systems biology of complex cellular processes. This aim will be fulfilled by the development and provision of common technical and computational tools for systems microscopy, as well as experimental and theoretical resources to carry out systems experiments and model their results. A generic approach for next-generation systems biology will be offered to the research community.
The joint research programme will generate data and models that are expected to further our systems-level understanding of cell division and migration, both of which are crucially involved in the development and progression of cancer, thereby providing new avenues for the translation of basic research into applied research. Novel testing systems for drug sensitivities and gene dependencies in primary cultures of cancer patients will be developed, tests that are expected to help guide future cancer diagnosis and therapy choices thereby paving the way for personalized medicine for the benefit of European citizens.
New technologies are developed in an area with significant future potential for innovation in biology and medicine, including translation to industry. We believe that at the end of this program, we will have developed significant tools, including software, screening systems and more, for translational applications that will provide opportunity for industrial collaborations to promote the NoE science and technology, thus strengthening European competitiveness. The results obtained in this project on cell division and migration, particularly in relation to cancer, may highlight and illuminate numerous opportunities for translational research breakthroughs, and consequent industrial benefits such as novel drug targets, as well as mechanisms of drug action. Furthermore, it is likely that systems microscopy will become an indispensable tool to monitor drug effects in cellular systems in vitro, thereby playing a crucial role towards the development of future diagnostic applications.
We will execute a multidisciplinary training programme, providing European research institutions with well-trained scientists to further develop this emerging area. The training programs will create a large user base, as well as contribute to the creation of future experts in this field across Europe. The power of the technology, the dissemination of knowledge and infrastructure will together serve as guarantor for the durability of the systems microscopy strategy.

List of Websites:
www.systemsmicroscopy.eu