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Chromatin organization PROfiling with high-throughput super-resolution microscopy on a CHIP

Periodic Reporting for period 1 - PROCHIP (Chromatin organization PROfiling with high-throughput super-resolution microscopy on a CHIP)

Reporting period: 2018-09-01 to 2019-08-31

Metastasis is the leading cause of cancer death among cancer patients. Cancer genomics showed that tumor progression to metastasis formation is poorly supported by further genetic alterations, implying that the adaptation capacity of disseminating tumor cells to foreign microenvironments rely on epigenetic alterations. Epigenomic reprogramming plays a central role in cancer progression and metastasis formation, supporting tumor heterogeneity, which represents a challenge for precise diagnosis and targeted therapy.
The aim of the project is to study cancer heterogeneity at single cell level by classifying freshly isolated cancer samples based on their chromatin architecture and to use chromatin alteration as a marker for cancer. To achieve this result we will develop a superresolution microscope with high throughput capabilities. Extracting and imaging large number of samples will allow performing statistically relevant biological studies that take into account the variability of cellular behavior and creating highly specific phenotyping procedures required for personalized medicine.
To fulfill this goal we will exploit the Femtosecond Laser Irradiation followed by Chemical Etching (FLICE) technology to develop a lattice light sheet microscope on a chip. FLICE allows fabricating with accurate precision optofluidic components as waveguides, microchannels and lenses in a glass substrate. The illumination path and the sample delivery systems will be fully integrated in a mm-scaled lab on chip, permitting easy operation, since no optical or fluidic alignment of the components is needed.
The long term vision of PROCHIP project is to develop a new enabling technology, able to extract cancer cell from a patient, assess alterations in chromatin architecture at single cell level, and define a targeted personalized therapy or drive the development of new drugs. In details the project objectives are:
1) the development of a miniaturized 3D fluidic network integrated on a glass chip and its relative pumping system for automatic sample scanning under a microscope
2) the development of a superresolution microscope, whose illumination and sample scanning components are integrated in a lab on chip
3) the identification of the protocols for isolation of tumor cells suitable for high-throughput imaging and for data analysis at super-resolution based on the image simulator
4) the assessment of cancer cell heterogeneity by querying chromatin domains as functional biomarkers.
The project is structured in four work packages for research and development activities and two for project management and dissemination:
WP1 is dedicated to identify the best protocols both for sample handling and data processing. In the first year the procedures for the isolation of cancer cells from primary and metastatic organs and their labelling have been identified by UNITN. Optimization of handling protocols is in progress by CNR and ELVESYS. The image simulator that generates artificial 3D image stacks to help the image processing have been implemented by UA and INSA Lyon and it is now available online for download.
WP2 aims to set the basis for advanced high-throughput microscopy in a fluidic chip. In Year1 a milestone concerning the development of a closed-loop fluidic system for single cell movement by ELVESYS and CNR has been achieved. The cell flow is controlled within a prototypical light sheet microscope on chip with standard optical resolution that has been fabricated to test and improve the throughput of the microscope.
WP3 is devoted to realize on chip lattice light sheet illumination. In Year1 extensive numerical and experimental analyses have been conducted. The first allowed designing aspherical profiles for the lenses used for dual color light sheet generation. On the other hand interesting results have been obtained in the selective etching of brosilicate glass and in thermal annealing processes for the smoothening of the etched surfaces. Preliminary experiments on the generation of interference patterns in a microchannel have been conducted. These results will lead to the final design of the on-chip superresolution microscope.
The activity of WP4 will begin at Year2, anyway IMPERIAL started to image samples produced by UNITN with a standard Structured Illumination Microscope already available. These results will be used as a reference for the images achieved with the on chip microscope.
WP5 and WP6 concern respectively the dissemination and management of the project. The main results achieved in Year1 are:
- for WP5 the launch of the Project website the preparation of the Data Management Plan and the Dissemination and Exploitation Plan;
- for WP6 the setting up of the governance of the project, e.g. the Steering and the Advisory Committees, the organization of the kick off meeting (Milan, 13th September 2018). The organization of the First Year Annual meeting and the Review meeting, which will take place at the beginning of November 2019, is in progress.
As discussed in the DoA, the project proposes to go well beyond the state of the art in different field as: (1) photonics, (2) computer sciences and (3) cancer research.
1) A notable development in the field of microscopy is Lattice Light Sheet Microscopy, which uses a periodic 2D interference pattern to illuminate the sample. The project aims at providing high-throughput imaging of single cells with superresolution resolution in a glass chip by integrating the sample delivery system and the illumination path. At the state of the art an optofluidic microscope on chip able to optically section a single cell have been produced and preliminary results in the generation of 1D interference pattern in a microfluidic channels have been obtained. These results might impact the fabrication of glass components by femtosecond laser writing, an industrial sector that is experiencing an investment increase.
2) Quantitative analysis on large datasets is a challenging task in computer vision. So far, phenotypic profiling has been addressed from classical image analysis methods either in a targeted approach to quantify a single process or involving a less targeted approach, allowing for the identification of many properties of the sample. Such complex situations require advanced techniques relying on machine learning. The project proposes to develop an image simulator to automatically simulate annotated synthetic ground truth images and associated simulated acquired images. At the state of the art a first version of MATLAB simulator application aiming to generate synthetic superresolution chromatin microscopy image stack is available and freely downloadable.
3) Chromatin topology plays a key role in genome functions such as controlling gene expression and maintenance of genome stability. Distinct epigenetic states are demarked by specific chromatin modifications. Defining the 3D-organization of cancer-associated chromatin domains represents a new frontier to decipher tumor heterogeneity. The project proposes to image the chromatin structure to gain insights in its alterations during tumor progression. At the state of the art the procedures to prepare the samples have been identified, thus ensuring the feasibility of microfluidic imaging analysis. We therefore aim to impact on clinical procedures to dissect cell heterogeneity and to support clinician in choosing the most successful therapeutic regimes.
PROCHIP roadmap; steps towards the goal: chromatin profiling with superresolution microscopy on chip