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Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin ( Dmd EGFP ), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called “basal sarcolemmal dystrophin units (BSDUs).” These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber—with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle–tendon junctions.

Background Genomic analysis of circulating tumor cells (CTCs) could provide a unique and accessible representation of tumor diversity but remains hindered by technical challenges associated with CTC rarity and heterogeneity. Objective To evaluate CTCs as surrogate samples for genomic analyses in metastatic castration-resistant prostate cancer (mCRPC). Design, setting, and participants Three isolation strategies (filter laser-capture microdissection, self-seeding microwell chips, and fluorescence-activated cell sorting) were developed to capture CTCs with various epithelial and mesenchymal phenotypes and isolate them at the single-cell level. Whole-genome amplification (WGA) and WGA quality control were performed on 179 CTC samples, matched metastasis biopsies, and negative controls from 11 patients. All patients but one were pretreated with enzalutamide or abiraterone. Whole-exome sequencing (WES) of 34 CTC samples, metastasis biopsies, and negative controls were performed for seven patients. Outcome measurements and statistical analysis WES of CTCs was rigorously qualified in terms of percentage coverage at 10× depth, allelic dropout, and uncovered regions. Shared somatic mutations between CTCs and matched metastasis biopsies were identified. A customized approach based on determination of mutation rates for CTC samples was developed for identification of CTC-exclusive mutations. Results and limitations Shared mutations were mostly detected in epithelial CTCs and were recurrent. For two patients for whom a deeper analysis was performed, a few CTCs were sufficient to represent half to one-third of the mutations in the matched metastasis biopsy. CTC-exclusive mutations were identified in both epithelial and nonepithelial CTCs and affected cytoskeleton, invasion, DNA repair, and cancer-driver genes. Some 41% of CTC-exclusive mutations had a predicted deleterious impact on protein function. Phylogenic relationships between CTCs with distinct phenotypes were evidenced. Conclusions CTCs can provide unique insight into metastasis mutational diversity and reveal undiagnosed genomic aberrations in matched metastasis biopsies. Patient summary Our results demonstrate the clinical potential of circulating tumor cells to provide insight into metastatic events that could be critical to target using precision medicine.

BACKGROUND AND OBJECTIVES In head and neck surgery, intraoperative and postoperative evaluation of tumor margins is achieved by histopathological assessment, which is a multistep process. Intraoperative analysis of tumor margins to obtain a preliminary diagnosis is usually carried out on frozen sections. Analysis of frozen sections is challenging due to technical difficulties in processing. Full-field optical coherence tomography (FFOCT) provides ex vivo images of fresh tissue samples at a microscopic scale without tissue processing. The objectives of our study were to define the diagnostic criteria required to interpret head and neck FFOCT images and to evaluate the reliability of a histological diagnosis made on an "optical biopsy" produced by head and neck FFOCT imaging compared with conventional histology. STUDY DESIGN/MATERIALS AND METHODS First, we established an atlas of comparative images (FFOCT/standard histology) and defined the diagnostic criteria based on FFOCT images. Two pathologists subsequently performed a blinded review on 57 FFOCT images (32 patients). Specificity and sensitivity were measured by comparison with the standard histological diagnosis. The primary endpoint was major concordance, defined as two classifications leading to the same therapeutic decision (treatment/no treatment). RESULTS Pathologists identified four main criteria for tissue diagnosis on FFOCT images: heterogeneous cell distribution, stromal reaction, coiling, and keratinization abnormalities. The correlation study showed good results, with sensitivity from 88% to 90% and specificity from 81% to 87%, regardless of whether the FFOCT image review was performed by a pathologist with or without previous experience in optical imaging. CONCLUSIONS Our results demonstrate that FFOCT images can be used by pathologists for differential diagnosis, and that high-resolution FFOCT imaging can provide an assessment of microscopic architecture in head and neck tissues without tissue processing requirements. Lasers Surg. Med. © 2020 Wiley Periodicals, Inc.

Probe-based confocal laser endomicroscopy (pCLE) is a noninvasive and real-time imaging technique allowing acquisition of in situ images of the tissue microarchitecture during oral surgery. We aimed to assess the diagnostic performance of pCLE combined with patent blue V (PB) in improving the management of early oral cavity, oro/hypopharyngeal, and laryngeal cancer by imaging squamous cell carcinoma in vivo. The prospective study enrolled 44 patients with early head and neck lesions. All patients underwent white-light inspection or panendoscopy depending on the lesion’s location, followed by pCLE imaging of the tumor core and its margins after topical application of PB. Each zone imaged by pCLE was interpreted at distance of the exam by three pathologists blinded to final histology. Most imaged zones could be presented to pathologists; the final sensitivity and specificity of pCLE imaging in head and neck cancers was 73.2–75% and 30–57.4%, respectively. During imaging, head and neck surgeons encountered some challenges that required resolving, such as accessing lesions with the flexible optical probe, achieving sufficiently precise imaging on the targeted tissues, and heterogeneous tissue staining by fluorescent dye. Final sensitivity scores were reasonable but final specificity scores were low. pCLE zones used to calculate specificity were acquired in areas of tumor margins, and the poor quality of the images acquired in these areas explains the final low specificity scores. Practical adjustments and technical training are needed to analyze head and neck lesions in various anatomical sites in real-time by pCLE.

Sentinel lymph node identification by near infrared (NIR) fluorescence with indocyanine green (ICG) is recognized in the literature as a useful technique. NIR fluorescence technology could become key in the prevention and management of lymphedema after axillary dissection for breast cancer. Here, we conducted a systematic review focusing on ICG imaging to improve lymphedema prevention and treatment after axillary surgery. A systematic literature review was performed using MEDLINE and Embase to identify articles focused on ICG imaging for breast-cancer-related lymphedema (BCRL). Qualitative analysis was performed to summarize the characteristics of reported ICG procedures. In situ tissue identification and functionality assessment based on fluorescence signal were evaluated. Clinical outcomes were appraised when reported. Studies relating to axillary reverse mapping, lymphography and upper limb supermicrosurgery combined with ICG imaging were identified. We included a total of 33 relevant articles with a total of 2016 patients enrolled. ICG imaging for axillary reverse mapping was safe for all 951 included patients, with identification of arm nodes in 80%-88% of patients with axillary lymph nodes dissection. However, the papers discuss the oncologic safety of the approach and how - regardless of the contrast agent - concerns limit its adoption. ICG lymphography is openly supported in BCRL management, with 1065 patients undergoing this procedure in 26 articles. The technique is reported for lymphedema diagnosis, with high sensitivity and specificity, staging, intraoperative mapping and patency control in lymphaticovenular anastomosis. The substantial advantages/disadvantages of ICG imaging procedures are finally described.

Introduction: Near-infrared (NIR) fluorescence axillary reverse mapping (ARM) procedure is a promising tool to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND). The ARMONIC clinical trial was conducted to validate the technique on a large cohort of patients and to analyse predictive clinical factors for ARM lymph node metastasis. For the first time, the fluorescence signal intensity from the ARM lymph nodes was measured and correlated with clinical findings. Materials and methods: 109 patients with invasive breast cancer with an indication of mastectomy and ALND underwent the NIR fluorescence ARM procedure. Indocyanine green was administered by intradermal injection followed by the intraoperative identification and resection of ARM lymph nodes with NIR fluorescence camera guidance. Fluorescence signal intensity and signal distribution were then measured ex vivo and compared with clinical outcomes.Results: ARM lymph nodes were successfully identified by fluorescence in 94.5% of cases. The mean normalised fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes (p=0.3728). At the microscopic level, fluorescence signal distribution was focally intense in lymphoid tissue areas. Only preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with higher ARM node fluorescence signal intensity (p=0.0253) but not the pathological nodal (pN) status (p=0.8081). Based on an optimal cut-off fluorescence value, the final sensitivity and specificity of NIR fluorescence ARM procedure for ARM lymph node metastatic involvement was 64.7% and 47.3%, respectively.Conclusion: Although our preliminary results did not show fluorescence signal intensity is a reliable diagnostic tool, NIR fluorescence ARM procedure may be useful for ARM lymph node identification. Clinical trial registration: NCT02994225

International audience; Background: Dmdmdx, harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. Methods: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. Results: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. Conclusion: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

We compared the performance of near-infrared imaging using indocyanine green (ICG) with the radioisotope (ISO) method to detect sentinel lymph nodes (SLNs) in breast cancer, to analyze predictive factors for negative ICG identification. The study included 122 patients who underwent sentinel lymph node biopsy (SLNB) using the combined ISO and ICG technique for primary breast cancer. We assessed the putative association between pathologic/clinical variables and ICG failure to detect SLNs. The ISO identification rate was 96.7% and ICG identification 81.9%. Overweight patients or presence of macrometastasis in SLNB were associated with the risk of ICG failing to detect SLNs (P = 0.02).

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One-shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow-up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long-term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long-term biological processes, as in cancer research.

Peritoneal carcinomatosis is a metastatic stage aggravating abdominal and pelvic cancer dissemination. The preoperative evaluation of lesions remains difficult today. Probe-based confocal laser endomicroscopy (pCLE) provides dynamic images of tissue architecture and cellular details. This technology allows in vivo histological interpretation of tissue. The main limitation of pCLE for adoption in the clinic is the unavailability of fluorescent contrast agents. The aim of our study was to evaluate the staining performance of indocyanine green and patent blue V for histological diagnosis of pCLE images of pathological and non-pathological peritoneal tissue. We performed a correlative study with the histological gold standard on ex vivo human specimens from 25 patients operated for peritoneal carcinomatosis; 70 specimens were stained by topical application with ICG or patent blue V and then imaged with a probe-based confocal laser endomicroscope. A total of 350 pCLE images and 70 corresponding histological sections were randomly and blindly interpreted by two pathologists (PT1 and PT2). The images were first classified into two categories, tumoral versus non-tumoral, and a refined histological diagnosis was then given. All presented images were interpreted by PT1 (who received prior training on PCLE image reading) and PT2 (no training). 100 % sensitivity for PT1 and PT2 was noticed with tissues stained with ICG to differentiate tumoral and non-tumoral tissue. Global scores were always better for PT1 (major concordance between 86 and 94 %) than for PT2 (major concordance between 77 and 89 %) independently of the fluorescent dye when histological diagnosis was done on pCLE images. In conclusion, the pair ICG-pCLE offers the best combination for a non-trained pathologist for the interpretation of pCLE images from peritoneum.

New optical technologies enhancing localization or assessing viability of parathyroid glands (PG) during endocrine surgery have been reported in clinical studies. These technologies could become complementary to the surgeon's eyes and may improve surgical outcomes in thyroidectomy and parathyroidectomy. Here, we conducted a systematic review focusing on PG identification and functional assessment using optical methods to enhance surgery. A systematic literature review was performed using MEDLINE and Embase database. Two authors selected studies and extracted data; qualitative analysis was performed to summarize the characteristics of reported optical tools for thyroidectomy or parathyroidectomy. Identification and vascularisation of PG during surgery were evaluated. Clinical and biochemical outcomes were appraised when reported. Studies relating to parathyroidectomy or thyroidectomy combined with autofluorescence, fluorescent methylene blue, 5-aminolevulinic acid, indocyanine green (ICG), optical coherence tomography, laser speckle contrast imaging, dynamic optical contrast imaging and Raman spectroscopy were identified with MEDLINE and Embase. We included a total of 47 relevant articles with a total of 1615 patients enrolled. Each optical technique is described and appreciated related to its surgical purpose. Autofluorescence and ICG imaging of PG are the most widely reported optical technologies for identification and assessment of vascularisation of PG. Results are mainly based on observational studies and argue for the feasibility of both techniques in endocrine surgery but prospective randomized studies have not been performed. In vivo applications are still limited for the other methods and further investigations correlating these techniques with post-operative parathormone measurements are still needed before considering these technologies in clinical practice.

Background Circulating tumor cell (CTC)-filtration methods capture high numbers of CTCs in non-small-cell lung cancer (NSCLC) and metastatic prostate cancer (mPCa) patients, and hold promise as a non-invasive technique for treatment selection and disease monitoring. However filters have drawbacks that make the automation of microscopy challenging. We report the semi-automated microscopy method we developed to analyze filtration-enriched CTCs from NSCLC and mPCa patients. Methods Spiked cell lines in normal blood and CTCs were enriched by ISET (isolation by size of epithelial tumor cells). Fluorescent staining was carried out using epithelial (pan-cytokeratins, EpCAM), mesenchymal (vimentin, N-cadherin), leukocyte (CD45) markers and DAPI. Cytomorphological staining was carried out with Mayer-Hemalun or Diff-Quik. ALK-, ROS1-, ERG-rearrangement were detected by filter-adapted-FISH (FA-FISH). Microscopy was carried out using an Ariol scanner. Results Two combined assays were developed. The first assay sequentially combined four-color fluorescent staining, scanning, automated selection of CD45− cells, cytomorphological staining, then scanning and analysis of CD45− cell phenotypical and cytomorphological characteristics. CD45− cell selection was based on DAPI and CD45 intensity, and a nuclear area >55 μm2. The second assay sequentially combined fluorescent staining, automated selection of CD45− cells, FISH scanning on CD45− cells, then analysis of CD45− cell FISH signals. Specific scanning parameters were developed to deal with the uneven surface of filters and CTC characteristics. Thirty z-stacks spaced 0.6 μm apart were defined as the optimal setting, scanning 82 %, 91 %, and 95 % of CTCs in ALK-, ROS1-, and ERG-rearranged patients respectively. A multi-exposure protocol consisting of three separate exposure times for green and red fluorochromes was optimized to analyze the intensity, size and thickness of FISH signals. Conclusions The semi-automated microscopy method reported here increases the feasibility and reliability of filtration-enriched CTC assays and can help progress towards their validation and translation to the clinic. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2461-4) contains supplementary material, which is available to authorized users.

Parathyroid glands (PGs) can be particularly hard to distinguish from surrounding tissue and thus can be damaged or removed during thyroidectomy. Postoperative hypoparathyroidism is the most common complication after thyroidectomy. Very recently, it has been found that the parathyroid tissue shows near-infrared (NIR) auto-fluorescence which could be used for intraoperative detection, without any use of contrast agents. The work described here presents a histological validation ex vivo of the NIR imaging procedure and evaluates intraoperative PG detection by NIR auto-fluorescence using for the first time to our knowledge a commercially available clinical NIR imaging device. Ex vivo study on resected operative specimens combined with a prospective in vivo study of consecutive patients who underwent total or partial thyroid, or parathyroid surgery at a comprehensive cancer center. During surgery, any tissue suspected to be a potential PG by the surgeon was imaged with the Fluobeam 800 ® system. NIR imaging was compared to conventional histology (ex vivo) and/or visual identification by the surgeon (in vivo). We have validated NIR auto-fluorescence with an ex vivo study including 28 specimens. Sensitivity and specificity were 94.1 and 80 %, respectively. Intraoperative NIR imaging was performed in 35 patients and 81 parathyroids were identified. In 80/81 cases, the fluorescence signal was subjectively obvious on real-time visualization. We determined that PG fluorescence is 2.93 ± 1.59 times greater than thyroid fluorescence in vivo. Real-time NIR imaging based on parathyroid auto-fluorescence is fast, safe, and non-invasive and shows very encouraging results, for intraoperative parathyroid identification.

Background and aimsMultidrug resistance (MDR) limits effectiveness in treating malignancy by modifying internalisation and/or externalisation of drugs through cancer cell membranes. In this study we describe an assay to monitor patients’ responses to chemotherapy.MethodsThe assay is based on the fluorescent properties of doxorubicin alone as well as in combination with methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). The slide-based cell imaging technique was first optimised using a panel of breast and urothelial cancer cell lines and then extended to fine needle breast aspiration biopsy and urine cytology.ResultsThe drug fluorescence behaviour observed on smears of clinical specimens is identical to that obtained using fixed cultured cells. The fluorescence of sensitive cells to chemotherapy is mainly localised in the nucleus, whereas resistant cells show a weak fluorescence signal localised in the cytoplasm. The difference in terms of fluorescence intensity is also highlighted through fluorescence spectra. ConclusionsThe results suggest that the assay provides clinically valuable information in predicting responses to doxorubicin and/or MVAC therapy. Originally set up on a confocal microscope, the assay was also effective using a standard epifluorescence microscope; as such it is technically simple, reliable and inexpensive.

The prevailing concept is that immediate mobilization of bone marrow–derived circulating endothelial progenitor cells (CEP) is a key mechanism mediating tumor resistance to vascular-disrupting agents (VDA). Here, we show that administration of VDA to tumor-bearing mice induces 2 distinct peaks in CEPs: an early, unspecific CEP efflux followed by a late yet more dramatic tumor-specific CEP burst that infiltrates tumors and is recruited to vessels. Combination with antiangiogenic drugs could not disrupt the early peak but completely abrogated the late VDA-induced CEP burst, blunted bone marrow–derived cell recruitment to tumors, and resulted in striking antitumor efficacy, indicating that the late CEP burst might be crucial to tumor recovery after VDA therapy. CEP and circulating endothelial cell kinetics in VDA-treated patients with cancer were remarkably consistent with our preclinical data. These findings expand the current understanding of vasculogenic “rebounds” that may be targeted to improve VDA-based strategies. Significance: Our findings suggest that resistance to VDA therapy may be strongly mediated by late, rather than early, tumor-specific recruitment of CEPs, the suppression of which resulted in increased VDA-mediated antitumor efficacy. VDA-based therapy might thus be significantly enhanced by combination strategies targeting late CEP mobilization. Cancer Discov; 2(5); 434–49. ©2012 AACR. Read the Commentary on this article by De Palma and Nucera, p. 395. This article is highlighted in the In This Issue feature, p. 377.

Une meilleure comprehension des processus moleculaires intervenant dans le developpement des processus pathologiques a entraine, depuis quelques annees, 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 considerablement ameliore les capacites de l’imagerie photonique in vivo, particulierement pour l’etude des mecanismes pathologiques a l’echelle moleculaire ou encore pour produire des « biopsies optiques ». Des agents d’imagerie fluorescents innovants ou « sondes intelligentes » peuvent cibler in vivo des activites fonctionnelles specifiques liees au developpement 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 a maturite et devraient trouver prochainement leur place parmi les autres modalites d’imagerie.

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.

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.

Full-Field OCT (FFOCT) produces optical slices of tissue using white light interferometry providing in-depth 2D images, with an isotropic resolution around 1 micrometer. These optical biopsy images are similar to those obtained with established histological procedures, but without tissue preparation and within few minutes. This technology could be useful when diagnosing a lesion or at the time of its surgical management. Here we evaluate the clinical value of FFOCT imaging in the management of patients with Head and Neck cancers by assessing the accuracy of the diagnosis done on FFOCT images from resected specimen. FFOCT images from Head and Neck samples were first compared to the gold standard (HES-conventional histology). An image atlas dedicated to the training of pathologists was built and diagnosis criteria were identified. Then, we performed a morphological correlative study: both healthy and cancerous samples from patients who undergo Head and Neck surgery of oral cavity, pharynx, and larynx were imaged. Images were interpreted in a random way by two pathologists and the FFOCT based diagnostics were compared with HES (gold standard) of the same samples. Here we present preliminary results showing that FFOCT provides a quick assessment of tissue architecture at microscopic level that could guide surgeons for tumor margin delineation during intraoperative procedure.

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.

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Here we evaluate the clinical value of Full-Field OCT imaging in the management of patients with Head and Neck cancers by making a reliable histological diagnosis on FFOCT images produced during preoperative procedure. FFOCT performs a true "virtual extemporaneous exam" that we want to compare to the gold standard (extemporaneous and conventional histology with H and E staining). This new optical technology could be useful when diagnosing a lesion, cancerous or precancerous, or at the time of its surgical management. Full-Field Optical Coherence Tomography virtually slices the tissue using white light interferometry to produce in-depth 2D images with an isotropic resolution around 1 micrometer. With such a high resolution FFOCT systems produce ”optical biopsy” images that are similar to that obtained with classical histology procedures, but without any staining and in only a few minutes. We imaged freshly excised samples from patients, of mouth, tongue, epiglottis and larynx tissues, both healthy and cancerous. FFOCT images were acquired and later compared with histology of the same samples. Common features were identified and characteristics of each tissue type were matched in order to form an image atlas for pathologist training. We were able to identify indicators of tumors such as heterogeneities in cell distribution, surrounding stroma, anomalous keratinization… In conclusion, FFOCT is a fast, non-invasive, non-destructive imaging tool that can be inserted into the pathology lab workflow and can provide a quick assessment of microscopic tissue architecture and content. Furthermore we are developing a similar system with a rigid endoscopic probe in order to do in vivo and in situ high-resolution imaging. Our probe could thus guide the surgeon in real time before and during excision and ensure a more precise gesture.

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.

Aim Confocal Endomicroscopy (CEM) is a non invasive imaging tool enabling "optical biopsies" of tissues at cellular level. Clinical studies have successfully reported the accuracy of CEM for the characterization of gastrointestinal, dermatologic and ocular diseases. In this study, we assess the potential use of endomicroscopy in combination with fluorophores clinically approved, to characterize premalignant and malignant lesions in human larynx.

Background: 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. Methods: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. Results: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. Conclusion: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation. © 2007 International Society for Analytical Cytology

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.

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.

In the central nervous system (CNS), there are innate obstacles to the modification of neurons: their relative low abundance versus glia and oligodendrocytes, the inaccessibility of certain target populations, and the volume one can inject safely. Our aim in this study was to characterize the in vivo efficacy of a novel viral vector derived from a canine adenovirus (CAV-2). Here we show that CAV-2 preferentially transduced i) rat olfactory sensory neurons; ii) rodent CNS neurons in vitro and in vivo; and, more clinically relevant, iii) neurons in organotypic slices of human cortical brain. CAV-2 also showed a high disposition for retrograde axonal transport in vivo. We examined the molecular basis of neuronal targeting by CAV-2 and suggest that due to CAR (coxsackie adenovirus receptor) expression on neuronal cells-and not oligodendrocytes, glia, myofibers, and nasal epithelial cells-CAV-2 vectors transduced neurons preferentially in these diverse tissues.

Introduction This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. Methods PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. Results We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. Conclusions We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

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