Autophagy, Not Apoptosis, Plays a Role in Lumen Formation of Eccrine Gland Organoids

Autophagy, Not Apoptosis, Plays a Role in Lumen Formation of Eccrine Gland Organoids

Eccrine sweat glands are essential skin appendages in humans and mammals, playing critical roles in body temperature regulation, secretion, and metabolism. These glands develop from solid buds into mature structures with a lumen, which transports sweat to the skin surface. However, the mechanisms underlying lumen formation in eccrine sweat glands remain poorly understood. This study investigates the role of autophagy and apoptosis in the lumen formation of eccrine gland organoids (EGOs) using a three-dimensional (3D) reconstruction model in Matrigel.

Introduction

Eccrine sweat glands consist of a secretory portion in the dermis and a duct that opens onto the skin surface. During embryonic development, sweat gland progenitors form placodes on the epidermal basal layer, which invaginate into the dermis. These placodes differentiate into luminal and myoepithelial cells, forming the secretory coiled portion. The basal cells in the ducts extend upward to the skin surface, creating openings that connect the coiled portion to the outer environment. While other exocrine glands, such as mammary and salivary glands, form lumens through cavitation or cell bundle hollowing, the exact mechanism in sweat glands remains unclear.

The formation of 3D organ structures is a key focus in regenerative biology, as it allows for the study of organ development and potential medical applications. Previous studies have used 3D models to simulate the development of various glands, including mammary, pancreatic, and salivary glands. These models rely on cell polarization, migration, and differentiation to replicate organ morphology. For eccrine sweat glands, a 3D reconstruction model using Matrigel-embedded cells has been developed to study lumen formation.

Methods

Ethical Approval and Primary Culture of Eccrine Sweat Gland Cells (ESGCs)

The study was conducted with ethical approval from the Hubei University of Medicine. Full-thickness skin specimens were obtained from five patients undergoing plastic surgery. The specimens were processed to isolate eccrine sweat glands, which were then cultured in Matrigel for 14 days. The culture medium was supplemented with essential growth factors, and autophagy was inhibited using 3-methyladenine (3MA).

Reconstruction of Eccrine Gland Organoids (EGOs)

Isolated ESGCs were suspended in Matrigel and cultured in six-well plates. The medium was replaced every 48 hours for the first seven days and daily for the remaining seven days. EGOs were observed using brightfield microscopy, and their morphological changes were analyzed using hematoxylin and eosin (H&E) staining.

Immunofluorescence and Western Blot Analysis

EGOs were fixed, embedded in paraffin, and sectioned for H&E and immunofluorescence staining. Proliferation marker Ki67, cellular motility marker filamentous actin (F-actin), and autophagy marker LC3B were analyzed. Western blotting was performed to quantify the expression of these markers, as well as apoptosis markers poly (ADP-ribose) polymerase (PARP) and cleaved caspase-3.

Detection of Apoptosis and Autophagy

Apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Autophagy was assessed by immunofluorescence staining of LC3B and Western blot analysis. The effects of autophagy inhibition on lumen formation were evaluated by treating EGOs with 3MA.

Results

Morphological Development of EGOs

EGOs developed from single cells into multicellular structures by day 4. By day 6, the earliest signs of lumen formation were observed, and the rate of lumen formation increased significantly from day 8 to day 14. H&E staining revealed that the internal cells of EGOs separated from the surrounding cells, forming a rudimentary lumen. The volume of EGOs increased over time, and the lumen became more recognizable.

Role of Cell Proliferation and Polarization

Immunofluorescence staining showed that Ki67 expression was highest in the early stages of EGO development and gradually decreased over time. Western blot analysis confirmed the reduction in Ki67 expression. F-actin, a marker of cell polarization, was expressed throughout the EGOs, particularly on the surface of the spherical structures. As the lumen formed, F-actin accumulated on the inner membrane of EGOs, and the nuclei of peripheral cells flattened, contributing to lumen expansion.

Autophagy in Lumen Formation

Immunofluorescence and Western blot analysis revealed that autophagy marker LC3B was expressed in the internal cells of EGOs, while apoptosis markers PARP and cleaved caspase-3 were absent. The proportion of autophagic cells increased from day 8 to day 14, coinciding with the period of lumen formation. Treatment with 3MA, an autophagy inhibitor, reduced the expression of LC3B and limited the formation of the lumen, confirming the role of autophagy in this process.

Discussion

This study demonstrates that autophagy, rather than apoptosis, plays a critical role in the lumen formation of eccrine gland organoids. The 3D reconstruction model in Matrigel allowed for the observation of key biological events, including cell proliferation, polarization, and autophagy, during lumen formation. The results suggest that the internal cells of EGOs undergo autophagy due to nutrient deprivation, leading to the formation of a lumen. This process is distinct from the apoptosis-driven lumen formation observed in other exocrine glands.

The findings align with previous studies showing that autophagy contributes to cell survival under stress conditions, such as nutrient deprivation and ischemia. In the 3D model, the polarized outer layer of cells surrounds the internal cells, creating a nutrient-deficient environment that triggers autophagy. This mechanism is consistent with the hollowing process observed in other tubular organs, such as blood vessels and the zebrafish intestine.

The study also highlights the potential of 3D models in regenerative biology. By replicating the development of eccrine sweat glands, these models provide insights into organogenesis and offer a platform for testing therapeutic interventions. The ability to inhibit lumen formation with 3MA underscores the importance of autophagy in glandular development and suggests potential targets for modulating sweat gland function.

Conclusion

In summary, this study establishes that autophagy is essential for lumen formation in eccrine gland organoids. The 3D reconstruction model in Matrigel provides a valuable tool for studying the mechanisms of sweat gland development and offers new avenues for research in regenerative medicine. Further studies are needed to explore the molecular pathways regulating autophagy in eccrine sweat glands and to develop strategies for enhancing glandular function in clinical applications.

doi.org/10.1097/CM9.0000000000001936

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