Muriel Abbaci

Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication

The role of gap junctional intercellular communication (GJIC) in regulation of normal growth and differentiation is becoming increasingly recognized as a major cellular function. GJIC consists of intercellular exchange of low molecular weight molecules, and is the only means for direct contact between cytoplasms of adjacent animal cells. Disturbances of GJIC have been associated with many pathological conditions, such as carcinogenesis or hereditary illness. Reliable and accurate methods for the determination of GJIC are therefore important in cell biology studies. There are several methods used successfully in numerous laboratories to measure GJIC both in vitro and in vivo. This review comments on techniques currently used to study cell-to-cell communication, either by measuring dye transfer, as in methods like microinjection, scrape loading, gap-fluorescence recovery after photobleaching (gap-FRAP), the preloading assay, and local activation of a molecular fluorescent probe (LAMP), or by measuring electrical conductance and metabolic cooperation. As we will discuss in this review, these techniques are not equivalent but instead provide complementary information. We will focus on their main advantages and limitations. Although biological applications guide the choice of techniques we describe, we also review points that must be taken into consideration before using a methodology, such as the number of cells to analyze.

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.

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.

Clinical approved fluorescent dyes coupled to endomicroscopy for in vivo diagnostic of peritoneal carcinomatosis

Peritoneal carcinomatosis is metastatic stage aggravating digestive, gynecological or bladder cancer dissemination and the preoperative evaluation of lesions remains difficult. There is therefore a need for minimal invasive innovative techniques to establish a precise preoperative assessment of cancer peritoneal cavity. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of the microarchitecture of tissues during an endoscopy. The PERSEE project proposes new developments in robotics and pCLE for the exploration of the peritoneal cavity during laparoscopy. Two fluorescent dyes, Patent blue V and Indocyanine green have been evaluated on human ex vivo samples to improve the contrast of pCLE images. For a future implementation in clinical study, two topically staining protocols operable in vivo have been validated on 70 specimens from 25 patients with a peritoneal carcinomatosis. The specimens were then imaged by pCLE with an optical probe designed for the application. A histo-morphological correlative study was performed on 350 pCLE images and 70 standard histological preparations. All images were interpreted in a random way by two pathologists. Differential histological diagnostics such as normal peritoneum or pseudomyxoma could be recognized on fluorescence images. The statistical analysis of the correlative study is underway. These dyes already approved for human use are interesting for pCLE imaging because some micromorphological criteria look like to conventional histology and are readable by pathologist. Thus pCLE images using both dyes do not require a specific semiology unlike to what is described in the literature, for pCLE associated with fluorescein for the in vivo imaging of pancreatic cysts.

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.

System identification of the fluorescence recovery after photobleaching in gap junctional intracellular communications

Gap-Fluorescence Recovery After Photobleaching (gap-FRAP) is a technique used to estimate functionality of intercellular connections in biology. Such a technique could potentially be involved in the diagnostic of normal/cancer cells. Discrimination of cell types may be performed directly, by comparing plots of fluorescence kinetics or indirectly by statistical testing applied to model parameters. This paper focuses on the latter model-based approach. Up to now, more than ninety percent of the models used to fit gap-FRAP responses have been derived from diffusion equations (partial differential equation). We propose to simplify the modeling procedure by using behavioral models derived from system identification techniques used in control engineering. To assess in practice the relevance of this concurrent method, two human head and neck carcinoma cell lines (KB and FaDu) were used. The former (KB) expresses connexin proteins (positive line) while the latter (FaDu) does not (negative line). Moreover, each cell line was tested on spheroid (3-D) and monolayer (2-D) slices and in vitro assays were repeated six times. System identification algorithms of the CONTSID Matlab toolbox were used to estimate the model parameters from the in vitro data sets. Results have particularly emphasized there is no need to use complex models to fit the observed gap-FRAP responses. We show that the static gain of the estimated transfer functions is able to discriminate cell types used in this study, which corroborates the relevance of system identification techniques for diagnostic applications based on gap-FRAP analysis.

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.

In vitro characterization of gap junctional intercellular communication by gap-FRAP technique

Gap junctional intercellular communication (GJIC) has been shown to be involved in the carcinogenesis process. Gap-FRAP (Fluorescence Recovery After Photobleaching) technique could be used to estimate gap junctions functionality and their potential involvement for distinguish normal and cancer cells. In this study, the gap-FRAP technique was used to analyse functional gap-junction-mediated communication for cell lines with different GJIC status. Gap-FRAP data and connexin 43 protein expression decreased for FaDu cancer cell line, in contrast to fibroblast and KB positives cell lines. To check the involvement and functionality of gap junctions in the restitution of the fluorescence after photobleaching, we used a gap junction channel inhibition assay with 18 α-glycyrrhetinic acid. Our results indicate that the degree of gap junctional intercellular communication could be estimated by this technique in vitro.