Corinne Laplace-Builhé

The Origin of Phenotypic Heterogeneity in a Clonal Cell Population In Vitro

Background The spontaneous emergence of phenotypic heterogeneity in clonal populations of mammalian cells in vitro is a rule rather than an exception. We consider two simple, mutually non-exclusive models that explain the generation of diverse cell types in a homogeneous population. In the first model, the phenotypic switch is the consequence of extrinsic factors. Initially identical cells may become different because they encounter different local environments that induce adaptive responses. According to the second model, the phenotypic switch is intrinsic to the cells that may occur even in homogeneous environments. Principal Findings We have investigated the “extrinsic” and the “intrinsic” mechanisms using computer simulations and experimentation. First, we simulated in silico the emergence of two cell types in a clonal cell population using a multiagent model. Both mechanisms produced stable phenotypic heterogeneity, but the distribution of the cell types was different. The “intrinsic” model predicted an even distribution of the rare phenotype cells, while in the “extrinsic” model these cells formed small clusters. The key predictions of the two models were confronted with the results obtained experimentally using a myogenic cell line. Conclusions The observations emphasize the importance of the “ecological” context and suggest that, consistently with the “extrinsic” model, local stochastic interactions between phenotypically identical cells play a key role in the initiation of phenotypic switch. Nevertheless, the “intrinsic” model also shows some other aspects of reality: The phenotypic switch is not triggered exclusively by the local environmental variations, but also depends to some extent on the phenotypic intrinsic robustness of the cells.

Confocal laser endomicroscopy for non-invasive head and neck cancer imaging: A comprehensive review

Histological assessment is an essential tool in the diagnosis and guidance of the treatment of various diseases, in particular cancer, of the head and neck. Recent major advances in optical imaging techniques have made it possible to acquire high-resolution in vivo images at the cellular scale. Confocal endomicroscopy is a non-invasive technique, which can be highly useful whenever meaningful in situ histological information is required. The technical aspects of confocal endomicroscopy are introduced, followed by an overview of major clinical studies in the field of head and neck cancer. Ongoing technical developments, contributing to improvements in imaging of the upper aero-digestive tract, are also discussed. Finally, the potential complementarities of functional and molecular imaging, as compared to morphological endomicroscopy, are highlighted.

Combination of quantification and observation methods for study of “Side Population” cells in their “in vitro” microenvironment

Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide‐based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines.

Red and far-red fluorescent dyes for the characterization of head and neck cancer at the cellular level.

BACKGROUND Primary upper aerodigestive tract malignancy remains a cancer having a poor prognosis, despite current progress in treatment, due to a generally late diagnosis. OBJECTIVES We conducted a preliminary assessment of five dyes approved for human use for the imaging of head and neck tissues at the cellular level, which could be considered for clinical examination. METHODS We investigated fluorescence endomicroscopic images on fresh samples obtained from head and neck surgeries after staining with hypericin, methylene blue, toluidine blue, patent blue or indocyanine green to provide a preliminary consideration as to whether these images contain enough information for identification of non-pathologic and pathologic tissues. The distribution pattern of dye has been examined using probe-based confocal laser endomicroscopy (pCLE) in ex vivo specimens and compared with corresponding histology. RESULTS In most samples, the image quality provided by pCLE with both dyes allowed pathologists to recognize histological characteristics to identify the tissues. CONCLUSION The combination of pCLE imaging with these dyes provides interpretable images close to conventional histology; a promising clinical tool to assist physicians in examination of upper aerodigestive tract, as long as depth imaging issues can be overcome.

Development of a multi-scale and multi-modality imaging system to characterize tumours and their microenvironment in vivo

In vivo high-resolution imaging of tumor development is possible through dorsal skinfold chamber implantable on mice model. However, current intravital imaging systems are weakly tolerated along time by mice and do not allow multimodality imaging. Our project aims to develop a new chamber for: 1- long-term micro/macroscopic visualization of tumor (vascular and cellular compartments) and tissue microenvironment; and 2- multimodality imaging (photonic, MRI and sonography). Our new experimental device was patented in March 2014 and was primarily assessed on 75 mouse engrafted with 4T1-Luc tumor cell line, and validated in confocal and multiphoton imaging after staining the mice vasculature using Dextran 155KDa-TRITC or Dextran 2000kDa-FITC. Simultaneously, a universal stage was designed for optimal removal of respiratory and cardiac artifacts during microscopy assays. Experimental results from optical, ultrasound (Bmode and pulse subtraction mode) and MRI imaging (anatomic sequences) showed that our patented design, unlike commercial devices, improves longitudinal monitoring over several weeks (35 days on average against 12 for the commercial chamber) and allows for a better characterization of the early and late tissue alterations due to tumour development. We also demonstrated the compatibility for multimodality imaging and the increase of mice survival was by a factor of 2.9, with our new skinfold chamber. Current developments include: 1- defining new procedures for multi-labelling of cells and tissue (screening of fluorescent molecules and imaging protocols); 2- developing ultrasound and MRI imaging procedures with specific probes; 3- correlating optical/ultrasound/MRI data for a complete mapping of tumour development and microenvironment.

Su1759 Ex-Vivo Characterization of Liver and Peritoneal Metastases by Confocal Laser Endomicroscopy: The PERSEE Project

Rationale: In digestive oncology, the rapid evaluation of the local and regional extent of cancer is crucial to determine the best therapeutic treatment for the patient. Indeed, the presence of metastases can significantly worsen the prognosis and change drastically the choice of therapy. For this reason, a minimally invasive surgical exploration that evaluates the local and metastatic extent of cancer is generally required before the tumor resection. Probe-based Confocal Laser Endomicroscopy (pCLE) is a new promising imaging technique enabling microscopic analysis of tissues, at cell resolution, in real time. Objective: First evaluation of pCLE for the discrimination of benign or metastatic nodules in liver and peritoneum.Methods: Several fresh samples of liver and peritoneal nodules were analyzed ex vivo right after the resection using a endomicroscopy system and a UHD confocal miniprobe. Healthy samples of the same tissues were also analyzed for comparison. Indocyanine green (ICG) was topically applied on the specimens (2.5mg/ml). For each sample, side by side comparison with histology was performed. Results. We analyzed and correctly identified metastatic (n=5) and inflammatory (n=4) peritoneal nodules, and metastatic liver nodules before (n=6) and after (n=2) treatment with chemotherapy. In healthy peritoneal CLE images (aquired on 10 samples) the adipocytes, surrounded by the extra-cellular matrix (ECM) composed by a strongly and uniformly fluorescent connective tissue, were clearly recognizable. In healthy liver CLE images (acquired on 7 samples), a compact line-structure of hepatocytes was observed. Following features could also be seen in both liver and peritoneal cancerous nodules: the irregular associations of cells forming tubular structures typically found on adenocarcinoma histology; the strongly inhomogeneity in the fluorescence signal of the ECM compared to healthy tissue. In contrast to the adenocarcinoma, the connective tissue composing the ECM of inflammatory peritoneal nodules seemed to be more abundant than in the healthy tissues and with a uniform fluorescent signal. Lastly, after chemotherapy treatment, the liver metastatic nodule appeared to have a very compact and fluorescent fibrotic tissue that replaces the tubular cell structures typical of the adenocarcinoma. Conclusion: Our preliminary results suggest that pCLE is a promising tool for an immediate identification of metastases both in liver and peritoneal tissues. This study reveals that the structure of the ECM may be an important additional parameter to take into account in order to improve diagnosis. In vivo use of this innovative approach should enable us to directly detect cancer without the need of more invasive and time-demanding biological sampling.

Quelles perspectives pour l’imagerie photonique in vivo en pratique clinique ?

With the increasing molecular understanding of disease processes, there has been a dramatic change, over the past years, in our consideration of non invasive imaging of cancer in humans. The miniaturization of optical devices, and fiber optic coupling greatly improved the ability of in-vivo photon imaging to emphasis pathological pathways at molecular level or to achieve true “optical biopsy”. Innovative fluorescent imaging agents called “smart probes” can provide in-vivo readouts of some of the key activities known to underlie human disease states in oncology. The current contribution of biophotonics in clinical imaging remains limited but diffuse optical tomography of the breast has paved the way. New optical concepts come to age and should soon find their place among the other imaging modalities.RésuméUne meilleure compréhension des processus moléculaires intervenant dans le développement des processus pathologiques a entraîné, depuis quelques années, un changement drastique de notre conception de l’imagerie non invasive des cancers chez l’homme. La miniaturisation des dispositifs optiques et les couplages par fibre optique, ont considérablement amélioré les capacités de l’imagerie photonique in vivo, particulièrement pour l’étude des mécanismes pathologiques à l’échelle moléculaire ou encore pour produire des « biopsies optiques ». Des agents d’imagerie fluorescents innovants ou « sondes intelligentes » peuvent cibler in vivo des activités fonctionnelles spécifiques liées au développement tumoral. L’implication actuelle de la biophotonique en imagerie clinique reste faible, mais l’imagerie tomographique diffuse du sein a ouvert la voie. De nouveaux concepts optiques arrivent à maturité et devraient trouver prochainement leur place parmi les autres modalités d’imagerie.