Deciphering tumour tissue organization by 3D electron microscopy and machine learning

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumour tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also found anatomical connections between the blood capillary and the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for future investigations in the emerging onconanotomy field.

STUDY THE HARMFUL EFFECTS OF SODIUM METABISULFITE ON LIVER, TESTEs, AND LIPID PEROXIDATION IN MALE MICE, (MUS MUSCULUS)

The present study was designed to evaluate the toxic effect of Sodium metabisulfite (SMB) on testes and liver of 8 week- old mouse, which were grouped as control (C) and dose group, SM (500 mg/kg B.W SMB). The Experimental group was injected 0.1ml SM (500 mg/g B.W + 10 ml distilled water) for twenty-one days, while a control group was given 0.1 ml water for the same durations. Recovered testes were subjected to morphological, morphometric, and histological analysis. A Hormone evaluation was also conducted. Methods Dose group SM (500 mg/kg B.W SMB) showed morphological defects like wrinkles on the surface, shrinkage, small size, whereas the testes of control group appeared normal just like control (C). Morphometric analysis of dose group SM (500 mg/g B.W SMB) showed significant decrease in length, width and weight of testis as compared to control group. Blood analysis showed significantly levels of testosterone and glutathione reductions in dose group SM (500 mg/g B.W SMB) as compared to the control group. While the level of Melanodialdehyde (MDA) increased significantly in dose group SM (500 mg/g B.W SMB) as compare to the control group. Histological defects were observed in testes of dose group SM (500 mg/g B.W SMB) like deshaped somniferous tubule, degenerated spermatids, vacuolization, rupturing of germinal epithelium, absence of sperms in the lumen, degenerated peritubular myoid cells, hyalinization, cluster of degenerated spermatozoa in lumen and absence of leydig cells. Histology of the liver showed various defects like mitotic cells, vacuolization, degeneration of hepatic cells and cell shrinkage, broad suicidal spacing, absence of bile canaliculus cells, regenerated kuppfer cell, and interrupted sheet of hepatocytes. Conclusion - The harmful impact of sodium metabisulfite on the liver and reproductive system of male mice. A Sharp decrease in the level of testosterone was formed to be a key factor for studied deformation in morphology, morphometry, and histol

Correlative microscopy and block-face imaging (CoMBI) method for both paraffin-embedded and frozen specimens

Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.

Deciphering Tumour Tissue Organization by 3D Electron Microscopy and machine learning

Despite recent progress in the characterization of tumour components, the tri-dimensional (3D) organization of this pathological tissue and the parameters determining its internal architecture remain elusive. Here, we analysed the spatial organization of patient-derived xenograft tissues generated from hepatoblastoma, the most frequent childhood liver tumour, by serial block-face scanning electron microscopy using an integrated workflow combining 3D imaging, manual and machine learning-based semi-automatic segmentations, mathematics and infographics. By digitally reconstituting an entire hepatoblastoma sample with a blood capillary, a bile canaliculus-like structure, hundreds of tumour cells and their main organelles (e.g. cytoplasm, nucleus, mitochondria), we report unique 3D ultrastructural data about the organization of tumoral tissue. We found that the size of hepatoblastoma cells correlates with the size of their nucleus, cytoplasm and mitochondrial mass. We also discovered that the blood capillary controls the planar alignment and size of tumour cells in their 3D milieu. Finally, a set of tumour cells polarized in the direction of a hot spot corresponding to a bile canaliculus-like structure. In conclusion, this pilot study allowed the identification of bioarchitectural parameters that shape the internal and spatial organization of tumours, thus paving the way for new investigations in an emerging field that we call onconanotomy.

Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis

Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of “cholestatic” signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.