Modeling pre-metastatic lymphvascular niche in the mouse ear sponge assay.

Lymphangiogenesis, the formation of new lymphatic vessels, occurs in primary tumors and in draining lymph nodes leading to pre-metastatic niche formation. Reliable in vivo models are becoming instrumental for investigating alterations occurring in lymph nodes before tumor cell arrival. In this study, we demonstrate that B16F10 melanoma cell encapsulation in a biomaterial, and implantation in the mouse ear, prevents their rapid lymphatic spread observed when cells are directly injected in the ear. Vascular remodeling in lymph nodes was detected two weeks after sponge implantation, while their colonization by tumor cells occurred two weeks later. In this model, a huge lymphangiogenic response was induced in primary tumors and in pre-metastatic and metastatic lymph nodes. In control lymph nodes, lymphatic vessels were confined to the cortex. In contrast, an enlargement and expansion of lymphatic vessels towards paracortical and medullar areas occurred in pre-metastatic lymph nodes. We designed an original computerized-assisted quantification method to examine the lymphatic vessel structure and the spatial distribution. This new reliable and accurate model is suitable for in vivo studies of lymphangiogenesis, holds promise for unraveling the mechanisms underlying lymphatic metastases and pre-metastatic niche formation in lymph nodes, and will provide new tools for drug testing

Cancer du col de l’utérus de stade débutant et dissémination ganglionnaire : analyse informatique détaillée du réseau vasculaire lymphatique global sur tissus cervicaux humains

La dissémination lymphatique est un évènement clé lors de la progression des néoplasies cervicales et des marqueurs tumoraux connexes sont vus comme des facteurs pronostiques potentiels d’extension ganglionnaire. A ce jour, la lymphangiogenèse (formation de nouveaux vaisseaux lymphatiques à partir de vaisseaux préexistants) est décrite comme un processus central favorisant la dissémination des cellules néoplasiques jusqu’aux ganglions locorégionaux et la densité en vaisseaux lymphatiques (LVD) péritumorale est proposée comme un facteur pronostique précoce d’extension ganglionnaire. Cependant, en raison du manque d’objectivité et de reproductibilité qui caractérise la technique de hot-spot, aucun consensus n’a actuellement pu être établi à ce sujet.Dans le but de valider l’éventuelle valeur pronostique de la vascularisation lymphatique sur le risque d’extension ganglionnaire, nous avons mis au point une technique originale d’analyse d’images digitales permettant de réaliser des quantifications objectives, robustes, reproductibles et détaillées du réseau vasculaire dans son intégralité. A l’aide de cette technique, l’évolution de la vascularisation lymphatique au cours de la progression tumorale a été caractérisée, comparée à celle présente au niveau de cols sains et confrontée aux paramètres de dissémination ganglionnaire (statut ganglionnaire et emboles lymphatiques). Nous avons mis en évidence la présence d’une activité lymphangiogène intense, méconnue à ce jour, sous la zone de transformation des cols sains, région au niveau de laquelle les néoplasies cervicales se développent. Au cours de la progression tumorale, nous avons observé que ce microenvironnement spécifique est maintenu. Seule la distribution des vaisseaux par rapport à la jonction « cellules néoplasiques/stroma » est modifiée. Au niveau des lésions de stade FIGO IB1, nous avons démontré que la LVD absolue n’est pas un marqueur de dissémination ganglionnaire, mais que la distribution spatiale des vaisseaux lymphatiques apparaît comme un élément important lors de la dissémination tumorale.En conclusion, nos travaux soutiennent l’intérêt primordial d’une analyse globale de la vascularisation lymphatique pour une meilleure caractérisation des paramètres pouvant être impliqués dans le processus de dissémination tumorale. Ils démontrent également que l’évaluation du risque de dissémination lymphatique ne peut se référer exclusivement à une simple augmentation du nombre de vaisseaux lymphatiques. Ils mettent également en lumière des modifications de l’organisation spatiale du réseau vasculaire lymphatique pouvant jouer un rôle non négligeable dans la progression des tumeurs

Automatic segmentation of glandular epithelium in colorectal tissue images using Deep Learning in order to compartmentalize IHC biomarker quantification

Immunohistochemistry (IHC) is commonly employed for diagnostic and prognostic purposes in histopathology as well as for biomarker validation in clinical research. Whole slide scanning and image analysis tools now enable objective and quantitative evaluation of IHC biomarkers in a whole tissue slide or a specific region of interest delineated by a pathologist. Compartmentalizing the quantitative evaluation of IHC biomarkers in a specific histological structure is often required to provide more relevant and informative measurements for clinical research. For this purpose, pathologists have to annotate thousands of structures present in histological slide series, a long, tedious and potentially biased task that would greatly benefit from automation.We developed an algorithm for automatically annotating glandular epithelium in slide images from colorectal tissue samples submitted to different staining techniques, including haematoxylin-eosin (H&E) as well as IHC. Our approach combines Deep Learning and a new method of data augmentation. The algorithm implements a convolutional neural network, which was first trained and evaluated with regard to the state-of-the-art on H&E images provided by the international GLaS (Gland Segmentation in Colon Histology Images) challenge contest. To apply our method in the context of IHC staining, we created a second dataset by using tissue microarray slides submitted to IHC to evidence the expression of different antigens on colorectal tumour samples. An expert manually annotated the images to delineate the glandular epithelium. We then quantified the IHC staining in and/or out of the glandular epithelium delineated on the basis of the manual or automatic annotations.Our method achieves state-of-the-art performances in epithelium segmentation on the H&E images and provides accurate segmentation on the IHC images, whatever the targeted antigen. Compartmentalized IHC quantification showed high concordance between measurements carried out using either manual or automatic segmentation. In addition to be efficient in terms of epithelium segmentation, our algorithm is very fast and thus relevant for quantitative IHC analysis performed on large series of whole (tissue or TMA) slides, as generally required in clinical research.info:eu-repo/semantics/publishe

Segmentation of glandular epithelium in colorectal tumours to automatically compartmentalise IHC biomarker quantification: a deep learning approach

In this paper, we propose a method for automatically annotating slide images from colorectal tissue samples. Our objective is to segment glandular epithelium in histological images from tissue slides submitted to different staining techniques, including usual haematoxylin-eosin (H&E) as well as immunohistochemistry (IHC). The proposed method makes use of Deep Learning and is based on a new convolutional network architecture. Our method achieves better performances than the state of the art on the H&E images of the GlaS challenge contest, whereas it uses only the haematoxylin colour channel extracted by colour deconvolution from the RGB images in order to extend its applicability to IHC. The network only needs to be fine-tuned on a small number of additional examples to be accurate on a new IHC dataset. Our approach also includes a new method of data augmentation to achieve good generalisation when working with different experimental conditions and different IHC markers. We show that our methodology enables to automate the compartmentalisation of the IHC biomarker analysis, results concurring highly with manual annotations.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A Novel Approach for Quantifying Cancer Cells Showing Hybrid Epithelial/Mesenchymal States in Large Series of Tissue Samples: Towards a New Prognostic Marker

In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e. which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients.info:eu-repo/semantics/publishe

Persistent vs Transient Alteration of Folliculogenesis and Estrous Cycle After Neonatal vs Adult Exposure to Bisphenol A.

Exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is known to produce variable effects on female puberty and ovulation. This variability of effects is possibly due to differences in dose and period of exposure. Little is known about the effects of adult exposure to environmentally relevant doses of this EDC and the differences in effect after neonatal exposure. This study sought to compare the effects of neonatal vs adult exposure to a very low dose or a high dose of BPA for 2 weeks on ovulation and folliculogenesis and to explore the hypothalamic mechanisms involved in such disruption by BPA. One-day-old and 90-day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25 ng/kg/d or 5 mg/kg/d) for 15 days. Neonatal exposure to both BPA doses significantly disrupted the estrous cycle and induced a decrease in primordial follicles. Effects on estrous cyclicity and folliculogenesis persisted into adulthood, consistent with a disruption of organizational mechanisms. During adult exposure, both doses caused a reversible decrease in antral follicles and corpora lutea. A reversible disruption of the estrous cycle associated with a delay and a decrease in the amplitude of the LH surge was also observed. Alterations of the hypothalamic expression of the clock gene Per1 and the reproductive peptide phoenixin indicated a disruption of the hypothalamic control of the preovulatory LH surge by BPA

Activational and organizational disruption of folliculogenesis and estrous cycle caused by exposure to Bisphenol A (BPA) during early postnatal or adult life

Our previous studies have shown that an early postnatal exposure to a very low dose of bisphenol A (BPA) disrupts sexual maturation and pubertal timing. However, the long-term effects of such low dose exposure as well as the effects of adult exposure have not been studied. One day-old and 90 day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25ng/kg/d or 5mg/kg/d) for 15 days. The early postnatal exposure to both BPA doses significantly decreased the percentage of females with a regular cycle (BPA-25ng: 51±15%; BPA-5mg: 7±7%; OIL: 86±2%). The estrus cycle alterations were characterized by a decrease in time spent in proestrus (BPA-25ng: 13±3%; BPA-5mg: 12±3%; OIL: 18±3%). During adult exposure, both doses caused a disruption of the estrous cycle characterized by a significant decrease in the average time spent in proestrus (BPA-25ng: 19±2%; BPA-5mg: 17±1%; OIL: 23±1%). This effect was transient as the exposed females showed a regular cycle one month after the last dose of BPA. After adult exposure, we also observed a disruption of folliculogenesis characterized by a significant decrease of antral follicles (BPA-25ng: 21±2%; BPA-5mg: 21±2%; OIL: 36±2%) and increase of atretic follicles (BPA-25ng: 24±4%; BPA-5mg: 26±6%; OIL: 15±1%). GnRH secretion measured ex vivo 24h after adult exposure was moderately affected by BPA. Indeed, GnRH interpulse interval was significantly different when comparing animals exposed to the high or low dose of BPA but not when comparingexposed animals to the control group (BPA-25ng: 42.6±0.5; BPA-5mg: 40.2±0.6%; OIL: 41.1±0,2minutes±SEM). In conclusion, while exposure to BPA produces persistent alterations of the estrous cycle after early postnatal exposure, exposure during adulthood appears to cause activational non-persistent alternations of both the estrous cycle and folliculogenesis