Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.

The ability to generate transgenic animals sparked a wave of research committed to implementing such technology in a wide variety of model organisms.Building a solid base of ubiquitous and tissue-specific reporter lines has set the stage for later interrogations of individual cells or genetic elements. Compared to other widely used model organisms such as mice, zebrafish and fruit flies, there are only a few transgenic lines available in the laboratory axolotl (Ambystoma mexicanum), although their number is steadily expanding. In this review, we discuss a brief history of the transgenic methodologies in axolotl and their advantages and disadvantages. Next, we discuss available transgenic lines and insights we have been able to glean from them. Finally, we list challenges when developing transgenic axolotl, and where further work is needed in order to improve their standing as both a developmental and regenerative model.

ObjectiveThe prognosis of patients with advanced stage mucinous epithelial ovarian cancer remains poor due to a modest response to platinum-based chemotherapy and the absence of therapeutic alternatives. As targeted approaches may help to overcome these limitations, the present study evaluates biomarkers indicative of potential immune-checkpoint inhibitor therapy response.MethodsAll patients who underwent primary cytoreductive surgery from January 2001 to December 2020 and for whom formalin-fixed paraffin-embedded tissue samples were available were included (n=35; 12 International Federation of Gynecology and Obstetrics (FIGO) stage ≥IIb). To define sub-groups potentially suitable for checkpoint inhibition, expression of programmed death-ligand 1 (PD-L1), tumor-infiltrating lymphocytes (CD3+, CD8+, CD20+, CD45+, CD68+, FoxP3+), and AT-rich interactive domain-containing protein 1A (ARID1A) immunostaining were evaluated in whole tissue sections and compared with clinicopathologic parameters and next-generation sequencing results, where available (n=11). Survival analyses were performed to assess whether identified sub-groups were associated with specific clinical outcomes.ResultsIn total, 34.3% (n=12/35) of tumors were PD-L1 positive. PD-L1 expression was associated with infiltrative histotype (p=0.027) and correlated with higher CD8+ (r=0.577, p<0.001) and CD45+ (r=0.424, p=0.011), but reduced ARID1A expression (r=−4.39, p=0.008). CD8+ expression was associated with longer progression-free survival (hazard ratio (HR) 0.85 (95% CI 0.72 to 0.99), p=0.047) and disease-specific survival (HR 0.85 (95% CI 0.73 to 1.00), p=0.044) in the sub-group with FIGO stage ≥IIb. Three (8.6%) samples demonstrated high PD-L1 expression at a combined positive score of >10, which was associated with increased CD8+ expression (p=0.010) and loss of ARID1A expression (p=0.034). Next-generation sequencing, which was available for all samples with a combined positive score of >10, showedKRASmutations,BRCAwild-type status, and mismatch repair proficiency in all cases, but did not reveal genetic alterations potentially associated with a pro-immunogenic tumor environment.ConclusionsA sub-group of mucinous ovarian cancers appear to demonstrate a pro-immunogenic tumor environment with high PD-L1 expression, decreased ARID1A expression, and characteristic tumor-infiltrating lymphocyte infiltration patterns. Further clinical validation of anti-PD-L1/PD-1 targeting in selected mucinous ovarian cancers appears promising.