Blueprints from plane to space: outlook of next-generation three-dimensional histopathology.

Here, we summarize the literature relevant to recent advances in three-dimensional (3D) histopathology in relation to clinical oncology, highlighting serial sectioning, tissue clearing, light-sheet microscopy, and digital image analysis with artificial intelligence. We look forward to a future where 3D histopathology expands our understanding of human pathophysiology and improves patient care through cross-disciplinary collaboration and innovation.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

The Helicobacter pylori CagA oncoprotein disrupts Wnt/PCP signaling and promotes hyperproliferation of pyloric gland base cells.

Helicobacter pylori strains that deliver the oncoprotein CagA into gastric epithelial cells are the major etiologic agents of upper gastric diseases including gastric cancer. CagA promotes gastric carcinogenesis through interactions with multiple host proteins. Here, we show that CagA also disrupts Wnt-dependent planar cell polarity (Wnt/PCP), which orients cells within the plane of an epithelium and coordinates collective cell behaviors such as convergent extension to enable epithelial elongation during development. Ectopic expression of CagA in Xenopus laevis embryos impaired gastrulation, neural tube formation, and axis elongation, processes driven by convergent extension movements that depend on the Wnt/PCP pathway. Mice specifically expressing CagA in the stomach epithelium had longer pyloric glands and mislocalization of the tetraspanin proteins VANGL1 and VANGL2 (VANGL1/2), which are critical components of Wnt/PCP signaling. The increased pyloric gland length was due to hyperproliferation of cells at the gland base, where Lgr5+ stem and progenitor cells reside, and was associated with fewer differentiated enteroendocrine cells. In cultured human gastric epithelial cells, the N terminus of CagA interacted with the C-terminal cytoplasmic tails of VANGL1/2, which impaired Wnt/PCP signaling by inducing the mislocalization of VANGL1/2 from the plasma membrane to the cytoplasm. Thus, CagA may contribute to the development of gastric cancer by subverting a Wnt/PCP-dependent mechanism that restrains pyloric gland stem cell proliferation and promotes enteroendocrine differentiation.

Lipopolysaccharides and Cellular Senescence: Involvement in Atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the vascular walls related to aging. Thus far, the roles of cellular senescence and bacterial infection in the pathogenesis of atherosclerosis have been speculated to be independent of each other. Some types of macrophages, vascular endothelial cells, and vascular smooth muscle cells are in a senescent state at the sites of atherosclerotic lesions. Likewise, bacterial infections and accumulations of lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, have also been observed in the atherosclerotic lesions of patients. This review introduces the integration of these two potential pathways in atherosclerosis. Previous studies have suggested that LPS directly induces cellular senescence in cultured monocytes/macrophages and vascular cells. In addition, LPS enhances the inflammatory properties (senescence-associated secretory phenotype [SASP]) of senescent endothelial cells. Thus, LPS derived from Gram-negative bacteria could exaggerate the pathogenesis of atherosclerosis by inducing and enhancing cellular senescence and the SASP-associated inflammatory properties of specific vascular cells in atherosclerotic lesions. This proposed mechanism can provide novel approaches to preventing and treating this common age-related disease.

A Novel Three-Dimensional Imaging System Based on Polysaccharide Staining for Accurate Histopathological Diagnosis of Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Tissue-clearing and three-dimensional (3D) imaging techniques aid clinical histopathological evaluation; however, further methodological developments are required before use in clinical practice. METHODS: We sought to develop a novel fluorescence staining method based on the classical periodic acid-Schiff stain. We further attempted to develop a 3D imaging system based on this staining method and evaluated whether the system can be used for quantitative 3D pathological evaluation and deep learning-based automatic diagnosis of inflammatory bowel diseases. RESULTS: We successfully developed a novel periodic acid-FAM hydrazide (PAFhy) staining method for 3D imaging when combined with a tissue-clearing technique (PAFhy-3D). This strategy enabled clear and detailed imaging of the 3D architectures of crypts in human colorectal mucosa. PAFhy-3D imaging also revealed abnormal architectural changes in crypts in ulcerative colitis tissues and identified the distributions of neutrophils in cryptitis and crypt abscesses. PAFhy-3D revealed novel pathological findings including spiral staircase-like crypts specific to inflammatory bowel diseases. Quantitative analysis of crypts based on 3D morphologic changes enabled differential diagnosis of ulcerative colitis, Crohn's disease, and non-inflammatory bowel disease; such discrimination could not be achieved by pathologists. Furthermore, a deep learning-based system using PAFhy-3D images was used to distinguish these diseases The accuracies were excellent (macro-average area under the curve = 0.94; F1 scores = 0.875 for ulcerative colitis, 0.717 for Crohn's disease, and 0.819 for non-inflammatory bowel disease). CONCLUSIONS: PAFhy staining and PAFhy-3D imaging are promising approaches for next-generation experimental and clinical histopathology.

A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues.

Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.

Multiresolution nondestructive 3D pathology of whole lymph nodes for breast cancer staging.

SIGNIFICANCE: For breast cancer patients, the extent of regional lymph node (LN) metastasis influences the decision to remove all axillary LNs. Metastases are currently identified and classified with visual analysis of a few thin tissue sections with conventional histology that may underrepresent the extent of metastases. AIM: We sought to enable nondestructive three-dimensional (3D) pathology of human axillary LNs and to develop a practical workflow for LN staging with our method. We also sought to evaluate whether 3D pathology improves staging accuracy in comparison to two-dimensional (2D) histology. APPROACH: We developed a method to fluorescently stain and optically clear LN specimens for comprehensive imaging with multiresolution open-top light-sheet microscopy. We present an efficient imaging and data-processing workflow for rapid evaluation of H&E-like datasets in 3D, with low-resolution screening to identify potential metastases followed by high-resolution localized imaging to confirm malignancy. RESULTS: We simulate LN staging with 3D and 2D pathology datasets from 10 metastatic nodes, showing that 2D pathology consistently underestimates metastasis size, including instances in which 3D pathology would lead to upstaging of the metastasis with important implications on clinical treatment. CONCLUSIONS: Our 3D pathology method may improve clinical management for breast cancer patients by improving staging accuracy of LN metastases.

Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling.

Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

Perspective: Extending the Utility of Three-Dimensional Organoids by Tissue Clearing Technologies.

An organoid, a self-organizing organ-like tissue developed from stem cells, can exhibit a miniaturized three-dimensional (3D) structure and part of the physiological functions of the original organ. Due to the reproducibility of tissue complexity and ease of handling, organoids have replaced real organs and animals for a variety of uses, such as investigations of the mechanisms of organogenesis and disease onset, and screening of drug effects and/or toxicity. The recent advent of tissue clearing and 3D imaging techniques have great potential contributions to organoid studies by allowing the collection and analysis of 3D images of whole organoids with a reasonable throughput and thus can expand the means of examining the 3D architecture, cellular components, and variability among organoids. Genetic and histological cell-labeling methods, together with organoid clearing, also allow visualization of critical structures and cellular components within organoids. The collected 3D data may enable image analysis to quantitatively assess structures within organoids and sensitively/effectively detect abnormalities caused by perturbations. These capabilities of tissue/organoid clearing and 3D imaging techniques not only extend the utility of organoids in basic biology but can also be applied for quality control of clinical organoid production and large-scale drug screening.

Activation of Sympathetic Signaling in Macrophages Blocks Systemic Inflammation and Protects against Renal Ischemia-Reperfusion Injury.

BACKGROUND: The sympathetic nervous system regulates immune cell dynamics. However, the detailed role of sympathetic signaling in inflammatory diseases is still unclear because it varies according to the disease situation and responsible cell types. This study focused on identifying the functions of sympathetic signaling in macrophages in LPS-induced sepsis and renal ischemia-reperfusion injury (IRI). METHODS: We performed RNA sequencing of mouse macrophage cell lines to identify the critical gene that mediates the anti-inflammatory effect of ß2-adrenergic receptor (Adrb2) signaling. We also examined the effects of salbutamol (a selective Adrb2 agonist) in LPS-induced systemic inflammation and renal IRI. Macrophage-specific Adrb2 conditional knockout (cKO) mice and the adoptive transfer of salbutamol-treated macrophages were used to assess the involvement of macrophage Adrb2 signaling. RESULTS: In vitro, activation of Adrb2 signaling in macrophages induced the expression of T cell Ig and mucin domain 3 (Tim3), which contributes to anti-inflammatory phenotypic alterations. In vivo, salbutamol administration blocked LPS-induced systemic inflammation and protected against renal IRI; this protection was mitigated in macrophage-specific Adrb2 cKO mice. The adoptive transfer of salbutamol-treated macrophages also protected against renal IRI. Single-cell RNA sequencing revealed that this protection was associated with the accumulation of Tim3-expressing macrophages in the renal tissue. CONCLUSIONS: The activation of Adrb2 signaling in macrophages induces anti-inflammatory phenotypic alterations partially via the induction of Tim3 expression, which blocks LPS-induced systemic inflammation and protects against renal IRI.

CUBIC-Cloud provides an integrative computational framework toward community-driven whole-mouse-brain mapping.

Recent advancements in tissue clearing technologies have offered unparalleled opportunities for researchers to explore the whole mouse brain at cellular resolution. With the expansion of this experimental technique, however, a scalable and easy-to-use computational tool is in demand to effectively analyze and integrate whole-brain mapping datasets. To that end, here we present CUBIC-Cloud, a cloud-based framework to quantify, visualize, and integrate mouse brain data. CUBIC-Cloud is a fully automated system where users can upload their whole-brain data, run analyses, and publish the results. We demonstrate the generality of CUBIC-Cloud by a variety of applications. First, we investigated the brain-wide distribution of five cell types. Second, we quantified Aß plaque deposition in Alzheimer's disease model mouse brains. Third, we reconstructed a neuronal activity profile under LPS-induced inflammation by c-Fos immunostaining. Last, we show brain-wide connectivity mapping by pseudotyped rabies virus. Together, CUBIC-Cloud provides an integrative platform to advance scalable and collaborative whole-brain mapping.

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16high Cells In Vivo.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.

Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.

Whole-organ/body three-dimensional (3D) staining and imaging have been enduring challenges in histology. By dissecting the complex physicochemical environment of the staining system, we developed a highly optimized 3D staining imaging pipeline based on CUBIC. Based on our precise characterization of biological tissues as an electrolyte gel, we experimentally evaluated broad 3D staining conditions by using an artificial tissue-mimicking material. The combination of optimized conditions allows a bottom-up design of a superior 3D staining protocol that can uniformly label whole adult mouse brains, an adult marmoset brain hemisphere, an ~1 cm3 tissue block of a postmortem adult human cerebellum, and an entire infant marmoset body with dozens of antibodies and cell-impermeant nuclear stains. The whole-organ 3D images collected by light-sheet microscopy are used for computational analyses and whole-organ comparison analysis between species. This pipeline, named CUBIC-HistoVIsion, thus offers advanced opportunities for organ- and organism-scale histological analysis of multicellular systems.

The oral hypoxia-inducible factor prolyl hydroxylase inhibitor enarodustat counteracts alterations in renal energy metabolism in the early stages of diabetic kidney disease.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution.

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.

[Recent Developments in a Automated Diagnosis of Pathological Images and Three-dimensional Histopathology].

Rapid improvements in computing power are advancing machine learning technology using neural networks, revolutionizing the field of image analysis and allowing for automated diagnosis of pathological images. In addition, the recent development of tissue clearing technology introduces the possibility of three-dimensional (3D) pathology. The scheme of 3D pathology allows for the collection of the 3D context of tissue and would contribute to improved accuracy of automatic pathological diagnosis by machine learning.

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.

Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.

Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents.

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.