Funding type: COFUND Marie Slodowska Curie Action
This project is under the Marie Skłodowska-Curie Actions (MSCA) program. There are no nationality conditions but the candidates must fulfill the MSCA mobility conditions, which means that she/he must not have stayed more than 1 year in France during the last 3 years immediately before the call deadline (31/05/2019)

Expected start date: 01/10/2019

Contacts: 

Ass. Prof. Emmanuelle Laurenceau
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+33 4 72 18 62 40

Ass. Prof Anne Laure Deman
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+33 4 72 43 14 37

Dr. Cesar Sanchez Huertas
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websites:

• https://www.rmit.edu.au/about/our-locations-and-facilities/facilities/research-facilities/micronano-research-facility
• http://inl.cnrs.fr/en/

Collaborations/External partners: Lyon-Sud Hospital

Scientific context:

Circulating tumor cells (CTCs) play an important role in metastasis dissemination. CTCs are tumor cells that separate from the tumor and join the bloodstream. They are characteristic of the tumor from which they shed. In these last decades, the count of CTCs from total blood has been clearly associated with bad prognosis in many cancer types. In particular, CTC-clusters demonstrate increased metastatic potential compared to single CTCs and their presence is strongly correlated with a dramatically shorter overall survival time. Thus, baseline CTC counts and monitoring of CTCs numbers under treatment is a prognostic factor of survival. However, since CTCs are rare cells (some CTCs for millions of blood cells) their capture remains challenging. Moreover, in order to study the metastatic power of these CTCs and to test their chemosensitivity towards new molecules, it is necessary to capture them alive, to cultivate them in dedicated micro-environments and to analyze their secretions in various conditions. Microfluidic devices are a proven technology for cellular handling as they can offer precise spatial and temporal control in a miniaturized environment compatible with cell or cell cluster size. This approach is particularly suitable for rare cell manipulation and multiplex biosensor technology that will allow the real-time detection of cell secretions.

Key-words: microfluidic platform; multiplex biosensor; circulating tumor cell; monitoring growth cell; chemotherapy prognosis.

Objectives of PhD thesis:

The subject of the PhD thesis aims at developing microfluidic platforms for the specific isolation and the numeration of living sub-populations of CTCs from pre-purified blood sample. These platforms will be associated with cell culture micro-reactors to assess the chemosensitivity level of the sub-populations of CTCs, and with biosensors to detect cell secretions. Magnetic forces will be used to operate cell trapping and separation.

Scientific hurdles:

Owing to the extremely low abundance and heterogeneity of CTCs populations (one CTC in millions of blood cells), their isolation and numeration is still technically challenging. Moreover, the CTCs integrity needs to be preserved in order to perform functional analysis on CTC-cluster.
Concerning the isolation of all CTCs from other blood cells and especially white blood cells, we chose magnetic immuno-capture separation of the white blood cells (WBC) in order to preserve the integrity of the CTCs. The WBC will therefore be specifically labeled with magnetic nanoparticles (NP) functionalized with the anti-CD45 antibody. The capture of these labeled WBC will be in a microsystem having a magnetic function provided by the integration of heterogeneous material metals (iron particles) / polymer (polydimethylsiloxane). Thus, one key point is the design and optimization of the magnetic trapping function.

The numeration of CTCs could be achieved by the integration of an optical sensor at the end of the magnetic trapping zone. The optical biosensor will consist of an array of nano-plasmonic biosensors covering a glass substrate. The biosensors will consist of gold nanostructures manufactured uniformly with low-cost and wafer-scale photolithography. The working principle of this biosensor would be based on the generation of localized surface plasmon resonance by in-plane excitation of guided modes in total internal reflection, creating an effective RI that is sufficiently large to support a guided electromagnetic mode inside this thin and sparse monolayer of nanostructures. For optical detection, we will employ a camera which will converts each imaged point to an effective sensing element. For single cell analyses, we will design a microfluidic system equipped with pneumatic valves that enables both the in-flow introduction of cells into the device and isolation of a micro-reactor on the nanostructured sensor chip to confine and analyze individual cells, as demonstrated in a previous work (Xiaokang Li, Maria Soler, Crispin Szydzik, Khashayar Khoshmanesh, Julien Schmidt, George Coukos, Arnan Mitchell, 2018). A valve-gated microchannel will allow us to control the flow when introducing the cells that are then isolated within a low volume cell chamber for the efficient monitoring of single cell secretion.

Concerning the functional analysis of CTC-clusters (growth, chemosensitivity, characterization of cell secretions), the separation of the different sub-populations of CTCs is of major concern. One possibility is to use the differential expression of cell markers according to the level of aggressiveness of these CTCs. Indeed, the phenotype of CTCs (determined by the expression of cell surface markers: EpCAM, PD-L1, N-Cadherin) evolves during the course of the disease. But the main obstacle is that the expression of these cell surface markers can vary concomitantly and in small proportions relative to each other. It is therefore necessary to develop a discriminating method allowing the specific adhesion (through chemical and biological functionalization of the micro-reactor surface) of CTC-clusters in culture micro-reactors.

Then, the influence of various environments or treatments on the growth and secretion of CTCs will be evaluated in real time in the micro-reactors. Various culture conditions or treatment could be applied through microfluidic channels directly into the micro-reactor. Cell growth will be achieved by integration of a cell counting sensor and analysis of cell secretion through the integration of specific biosensors that will contain specific bioreceptors able to interact with the secreted molecules of interest and to provide a quantitative sensor response.

Innovative contributions:

One major challenge consists in integrating all the functions described above into the same fluidic microsystem, namely: the WBC magnetic separation function, the efficient separation of the CTC sub-populations in micro-reactors, the cell culture and counting function of CTCs, and the biosensing analysis of CTC secretions. Each of these functions will be optimized first separately, and then associated and integrated into the designed lab-on-chip device.

Working plan:

1. Functionalization of magnetic NP with antibodies: Different magnetic NP sizes (200 nm, 300 nm, 500 nm, 20 μm) will be tested. The immobilization rate of antibodies (anti-CD45) by type of NP will be studied and the efficiency of the separation will be evaluated according to the targeted cells (WBC).
2. Study of the structuration of the particles according to the magnetic fields applied and their concentration in the polymer matrix. Study of interaction mechanisms between particles and magnetic properties obtained according to their self-organization (SQUID magnetometer, MFM (magnetic force microscopy).
3. Chemical and biological functionalization of surface of cell culture micro-reactor with SAMs carrying different chemical functions (COOH, NH2, CH3, OH). Characterization of functionalized surfaces (XPS, Tof-SIMs, contact angle, AFM).
4. Culture of CTCs on functionalized surfaces: Evaluation of the adhesion and the proliferation of CTCs on the different functionalized surfaces. Integration and performance evaluation of the cell counting sensor.
5. Design and optimization of the biosensor and lab-on-a-chip platforms for the analysis of cell secretion.
6. Design of the different functions and integration in microsystem: evaluation and optimization of the efficiency of each function with model samples from cell lines, then with samples from patients (in collaboration with Hospital). Development of the lab-on-chip with all the functions integrated.

PhD funding : Co-Fund ECL/RMIT (ECLAUsion program)

Profile of the PhD student:

The candidate must have a Master's degree or an engineering degree with a specialization in Micro-Nanotechnologies, Physics or Chemistry. In addition, he (she) will have to demonstrate his particular interest in microfluidics, magnetism and optics. Finally, the candidate is expected to be motivated in the field of health. He (she) will not have studied in France for more than 2 years over the last 3 years.

Benefits of the project:

Publications in peer-review international journals and international conferences.

Patent proposal depending on the results.

Skills developed during the PhD:

The skills that will be developed during the thesis will be very multidisciplinary and concern the chemical and biological surface functionalization, the physico-chemical characterization of surfaces, micro-nanofabrication, the design and characterization of microfluidic systems and optical sensors, magnetophoresis, numerical simulations, immuno-labeling and cell culture.

He (she) will participate to the writing of publications and eventually patents, and will present his (her) research work to international conferences.

References:

1. Xiaokang Li, Maria Soler, Crispin Szydzik, Khashayar Khoshmanesh, Julien Schmidt, George Coukos, Arnan Mitchell,  and H. A. (2018). Label‐Free Optofluidic Nanobiosensor Enables Real‐Time Analysis of Single‐Cell Cytokine Secretion. Small. https://doi.org/10.1002/smll.201800698
2. S. Mekkaoui, D. Le Roy, M.-C. Audry, J. Lachambre, V. Dupuis, J. Desgouttes, A.-L. Deman. (2018). Arrays of high aspect ratio magnetic microstructures for large trapping throughput in lab-on-chip systems. Microfluid. Nanofluid., 22: 119, https://doi.org/10.1007/s10404-018-2141-6
3. A.-L. Deman, S. Mekkaoui, D. Dhungana, J.-F. Chateaux, A. Tamion, J. Degouttes, V. Dupuis and D. Le Roy. (2017). Anisotropic composite polymer for high magnetic force in microfluidic systems. Microfluid. Nanofluid., 21: 170.  https://doi.org/10.1007/s10404-017-2008-2