Sezen Serap, Adiguzel Sevin, Zarepour Atefeh, Khosravi Arezoo, Gordon Joseph W, Ghavami Saeid, Zarrabi Ali
Department of Molecular Biology, Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla, Istanbul, Türkiye.
Nanotechnology Research and Application Center, Sabanci University, Tuzla, Istanbul, Türkiye.
Methods Mol Biol. 2025;2879:275-287. doi: 10.1007/7651_2024_538.
Remodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy-dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use of GelMA hydrogels with defined stiffness represents a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells.
细胞外基质（ECM）的重塑最终会导致肿瘤变硬并改变微环境。这种变硬会改变癌细胞中的生物学过程，因为通过细胞表面受体的信号传导发生了改变。自噬是正常细胞和癌细胞中的关键分解代谢过程，被认为参与了机械转导，并且自噬水平可能取决于硬度。在这里，我们提供了一种方法来研究基质硬度对胚胎性横纹肌肉瘤细胞自噬的影响。为了模拟硬度，我们将细胞接种在具有确定硬度的GelMA水凝胶基质上，并评估自噬相关的终点。我们还评估了自噬依赖性途径、细胞凋亡和细胞活力。具体而言，我们利用免疫细胞化学和共聚焦显微镜通过LC3脂化来追踪自噬体的形成。这种方法表明，使用具有确定硬度的GelMA水凝胶是评估自噬在胚胎性横纹肌肉瘤和其他癌细胞中作用的一种新方法。
