Journal of Histochemistry & Cytochemistry

Ultrastructural analysis of healthy, diseased, or experimental tissues is essential in diagnostic and investigative pathology. Evaluation of large tissue areas with suborganelle resolution is challenging because biological structures ranging from several millimeters to nanometers in size need to be identified and imaged while maintaining context over multiple scales. Imaging with field emission scanning electron microscopes (FE-SEMs) is uniquely suited for this task. We describe an efficient workflow for the preparation and unobstructed multiscale imaging of tissue sections with backscattered electron scanning electron microscopy (BSE-SEM) for applications in ultrastructural pathology. We demonstrate that a diverse range of tissues, processed by conventional electron microscopy protocols and avoiding the use of mordanting agents, can be imaged on standard glass slides over multiple scales, from the histological to the ultrastructural level, without any visual obstructions. Our workflow takes advantage of the very large scan fields possible with modern FE-SEMs that allow for the acquisition of wide-field overview images which can be explored at the ultrastructural level by digitally zooming into the images. Examples from applications in pulmonary research and neuropathology demonstrate the versatility and efficiency of this method. This BSE-SEM-based multiscale imaging procedure promises to substantially simplify and accelerate ultrastructural tissue analysis in pathology.

Triple-negative breast cancer (TNBC) is a heterogeneous group of breast cancer and is characterized by aggressiveness and poor prognosis. MicroRNA represents a new class of biomarkers, and accumulating evidence indicates that microRNAs contribute to tumorigenesis and cancer metastasis. It has been described that miR-210 is highly expressed in TNBC, and its overexpression had been linked to poor prognosis. In a previous work, we showed that in TNBC miR-210 is expressed in tumor cells and also in the tumor microenvironment (TME), particularly in inflammatory CD45-LCA positive cells. However, the exact identity of these cells remained unknown. In this study, we performed in situ hybridization and immunohistochemistry using validated antibodies for the different specific immune cell markers on adjacent sections of 23 TNBC infiltrated with immune cells. We found that miR-210 expressing cells in the TME were stained positive with CD79a, a B-cell lineage marker. These tumor-infiltrating cells were negative for CD20 and Ki-67 but positive for MUM1 and CD38 and also expressed immunoglobulins, indicating that they are immunoglobulin-producing plasma cells (PCs). To the best of our knowledge, this is the first study demonstrating miR-210 expression in tumor-infiltrating PCs.

Glioblastoma is the most aggressive primary brain tumor. Slowly dividing and therapy-resistant glioblastoma stem cells (GSCs) reside in protective peri-arteriolar niches and are held responsible for glioblastoma recurrence. Recently, we showed similarities between GSC niches and hematopoietic stem cell (HSC) niches in bone marrow. Acute myeloid leukemia (AML) cells hijack HSC niches and are transformed into therapy-resistant leukemic stem cells (LSCs). Current clinical trials are focussed on removal of LSCs out of HSC niches to differentiate and to become sensitized to chemotherapy. In the present study, we elaborated further on these similarities by immunohistochemical analyses of 17 biomarkers in paraffin sections of human glioblastoma and human bone marrow. We found all 17 biomarkers to be expressed both in hypoxic peri-arteriolar HSC niches in bone marrow and hypoxic peri-arteriolar GSC niches in glioblastoma. Our findings implicate that GSC niches are being formed in glioblastoma as a copy of HSC niches in bone marrow. These similarities between HSC niches and GSC niches provide a theoretic basis for the development of novel strategies to force GSCs out of their niches, in a similar manner as in AML, to induce GSC differentiation and proliferation to render them more sensitive to anti-glioblastoma therapies.

Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues. The changes in localization and the roles of tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR) during cytokinesis are not well known, although new tricellular tight junctions form at the flank of the midbody during cytokinesis. In this study, we investigated the changes in localization and the role of LSR at the midbody and centrosome during cytokinesis using human endometrial carcinoma cell line Sawano, comparing the tricellular tight junction molecule tricellulin; bicellular tight junction molecules occludin, claudin-7, zonula occludens-1, and cingulin; and the epithelial polarized related molecules apoptosis-stimulating of p53 protein 2, PAR3, and yes-associated protein. During cytokinesis induced by treatment with taxol, the epithelial barrier was maintained and the tricellular tight junction molecules LSR and tricellulin were concentrated at the flank of the acetylated tubulin–positive midbody and in γ-tubulin-positive centrosomes with the dynein adaptor Hook2, whereas the other molecules were localized there as well. All the molecules disappeared by knockdown using small interfering RNAs. Furthermore, by the knockdown of Hook2, the epithelial barrier was maintained and most of the molecules disappeared from the centrosome. These findings suggest that LSR may play crucial roles not only in barrier function but also in cytokinesis.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin’s Solution, Carnoy’s Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson’s Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy’s Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson’s Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.