Autophagy,not apoptosis,plays a role in lumen formation of eccrine gland organoids

Background:Sweat secreted by eccrine sweat glands is transported to the skin surface through the lumen.The eccrine sweat gland develops from the initial solid bud to the final gland structure with a lumen,but how the lumen is formed and the mechanism of lumen formation have not yet been fully elucidated.This study aimed to investigate the mechanism of lumen formation of eccrine gland organoids(EGOs).Methods:Human eccrine sweat glands were isolated from the skin for tissue culture,and the primary cultured cells were collected and cultured in Matrigel for 14 daysin vitro.EGOs at different development days were collected for hematoxylin and eosin(H&E)staining to observe morphological changes and for immunofluorescence staining of proliferation marker Ki67,cellular motility marker filamentous actin(F-actin),and autophagy marker LC3B.Western blotting was used to detect the expression of Ki67,F-actin,and LC3B.Moreover,apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)apoptosis assay kit,and the expression of poly(ADP-ribose)polymerase and Caspase-3 was detected by Western blot.In addition,3-methyladenine(3MA)was used as an autophagy inhibitor to detect whether the formation of sweat glands can be effectively inhibited.Results:The results showed that a single gland cell proliferated rapidly and formed EGOs on day 4.The earliest lumen formation was observed on day 6.From day 8 to day 14,the rate of lumen formation in EGOs increased significantly.The immunofluorescence and Western blot analyses showed that the expression of Ki67 gradually decreased with the increase in days,while the F-actin expression level did not change.Notably,the expression of autophagy marker LC3B was detected in the interior cells of EGOs as the apoptosis signal of EGOs was negative.Compared with the control group,the autophagy inhibitor 3MA can effectively limit the formation rate of the lumen and reduce the inner diameter of EGOs.Conclusion:Using our model of eccrine gland 3D-reconstruction in Matrigel,we determined that autophagy rather than apoptosis plays a role in the lumen formation of EGOs.