Biological Applications

In Vivo Multivesicular Body and Exosome Secretion in the Intestinal Epithelial Cells of Turtles During Hibernation by WA Vistro, Y Huang, X Bai, P Yang, A Haseeb, H Chen, Y Liu, Z Yue, I Tarique, Q Chen, Microsc Microanal | doi:10.1017/ S1431927619015071

We investigated the in vivo biological processes of multive-

sicular bodies (MVBs) and exosomes in mitochondria-rich cells (MRCs), goblet cells (GCs), and absorptive cells (ACs) in turtle intestines during hibernation. Te exosome markers cluster of differentiation 63 (CD63) and tumor susceptibility gene 101 (TSG101) were positively expressed in intestinal villi during hibernation. Te distribution and formation processes of MVBs and exosomes in turtle MRCs, GCs, and ACs were further con- firmed by transmission electron microscopy (see figure). During hibernation, MRCs and ACs abundantly secreted early endo- somes (ees), late endosomes (les) and many “heterogeneous” MVBs. Interestingly, the ees, les, and MVBs were detected in the cytoplasm of GCs during hibernation; however, they were absent when turtles were not hibernating. In addition, the num- ber of different MVBs with intraluminal vesicles and heteroge- neous endosome–MVB–exosome complexes was significantly increased in MRCs, GCs, and ACs during hibernation. Tese findings indicate that intestinal epithelial cells potentially per- form a role in the secretion of MVBs and exosomes, which are essential for mucosal immunity during hibernation.

Absorptive cells (ACs) secreted more multivesicular bodies (arrows) in the intes- tine of Chinese soft-shelled turtles during hibernation period. The most MVBs were localized near the apical cytoplasmic region of ACs in the preparation for fusion with the plasma membrane and the release of exosomes. Scale bar = 1 micron.


Quantitative Assessment of Anti-Cancer Drug Efficacy from Coregistered Mass Spectrometry and Fluorescence Microscopy Images of Multicellular Tumor Spheroids by J Michálek, K Štěpka, M Kozubek, J Navrátilová, B Pavlatovská, M Machálková, J Preisler, A Pruška, Microsc Microanal | doi:10.1017/S1431927619014983

Spheroids—three-dimensional aggregates grown from a

cancer cell line—represent a model of living tissue for chemother- apy investigation. Distribution of chemotherapeutics in spheroid sections was determined using matrix-assisted laser desorption/ ionization mass spectrometry imaging (MALDI MSI). Prolif- erating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images (see figure). To allow com- parison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes, we introduced a common diffusion-related coordinate,

the distance from the

spheroid boundary. In a procedure referred to as “peeling,” we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. Tis approach makes it possible to dif- ferentiate between peripheral cells that died due to therapy and the innermost cells that died naturally.

40 doi:10.1017/S1551929519001184

(Top row) Fiducial markers (i.e., three white dots) are registered onto MALDI MS images (red dashed arrows) to align the LSCM and MALDI spheroid images (center). (Middle row) Spheroid “peels” denote regions over which average drug concentration (right) and cell abundance (left) will be calculated. (Bottom row) Mean values of drug concentration and proliferating cell abundance. • 2020 January

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