Holuigue H, Nacci L, Di Chiaro P, Chighizola M, Locatelli I, Schulte C, Alfano M, Diaferia G R, Podestà A
CIMAINA and Dipartimento di Fisica "Aldo Pontremoli", Università degli Studi di Milano, Milano, Italy.
Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milano, Italy.
Nanoscale. 2023 Sep 29;15(37):15382-15395. doi: 10.1039/d3nr01568h.
Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and an appropriate reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components ( collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions (submucosa, detrusor, and adventitia), in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine.
原子力显微镜（AFM）已成功用于对细胞微环境机械传感的定量研究。为此，几种力谱方法旨在测量两个活细胞之间以及细胞与细胞外基质（ECM）适当复制品之间的粘附力，通常利用用ECM的单个成分（胶原蛋白、纤连蛋白）适当功能化的尖端。然而，这些探针只能很差地再现天然细胞微环境的复杂性，进而也无法很好地再现生物相互作用的复杂性。我们开发了一种新方法来制备能忠实地保留ECM结构和生化复杂性的AFM探针；这是通过将经激光显微切割的天然去细胞ECM的微米切片附着到AFM悬臂上来实现的。我们证明这些探针保留了ECM的形态、机械和化学异质性。天然ECM探针可用于旨在靶向细胞 - 微环境相互作用的力谱实验。在此，我们证明了在10皮牛范围内剖析机械转导细胞 - ECM相互作用的可行性。作为原理验证，我们用大鼠膀胱ECM探针检测了AY - 27大鼠膀胱癌细胞系。一方面，我们使用来自相同ECM区域的不同探针获得了可重复的结果；另一方面，我们检测到不同膀胱ECM区域（黏膜下层、逼尿肌和外膜）的粘附模式存在差异，这与这些ECM区域在组成和生物物理性质上的差异一致。我们的结果表明，由器官和组织的患者特异性区域产生的天然ECM探针可用于通过力谱研究细胞 - 微环境相互作用和早期机械转导过程。这为个性化医学领域开辟了新的可能性。
