Investigating the Role of Extracellular Matrix Stiffness in Modulating the Ferroptosis Process in Hepatocellular Carcinoma Cells via Scanning Electrochemical Microscopy.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

Matrix stiffness-dependent microglia activation in response to inflammatory cues: in situ investigation by scanning electrochemical microscopy.

Microglia play a crucial role in maintaining the homeostasis of the central nervous system (CNS) by sensing and responding to mechanical and inflammatory cues in their microenvironment. However, the interplay between mechanical and inflammatory cues in regulating microglia activation remains elusive. In this work, we constructed in vitro mechanical-inflammatory coupled microenvironment models of microglia by culturing BV2 cells (a murine microglial cell line) on polyacrylamide gels with tunable stiffness and incorporating a lipopolysaccharide (LPS) to mimic the physiological and pathological microenvironment of microglia in the hippocampus. Through characterization of activation-related proteins, cytokines, and reactive oxygen species (ROS) levels, we observed that the LPS treatment induced microglia on a stiff matrix to exhibit overexpression of NOX2, higher levels of ROS and inflammatory factors compared to those on a soft matrix. Additionally, using scanning electrochemical microscopy (SECM), we performed in situ characterization and discovered that microglia on a stiff matrix promoted extracellular ROS production, leading to a disruption in their redox balance and increased susceptibility to LPS-induced ROS production. Furthermore, the respiratory activity and migration behavior of microglia were closely associated with their activation process, with the stiff matrix-LPS-induced microglia demonstrating the most pronounced changes in respiratory activity and migration ability. This work represents the first in situ and dynamic monitoring of microglia activation state alterations under a mechanical-inflammatory coupled microenvironment using SECM. Our findings shed light on matrix stiffness-dependent activation of microglia in response to an inflammatory microenvironment, providing valuable insights into the mechanisms underlying neuroinflammatory processes in the CNS.

In Situ and Quantitatively Monitoring the Dynamic Process of Ferroptosis in Single Cancer Cells by Scanning Electrochemical Microscopy.

Ferroptosis, as a promising therapeutic strategy for cancers, has aroused great interest. Quantifying the quick dynamic changes in key parameters during the early course of ferroptosis can provide insights for understanding the underlying mechanisms of ferroptosis and help the development of therapies targeting ferroptosis. However, in situ and quantitatively monitoring the quick responses of living cancer cells to ferroptosis at the single-cell level remains technically challenging. In this work, we selected HuH7 cells (hepatocellular carcinoma (HCC) cells) as a cell model and Erastin as a typical ferroptosis inducer. We utilized scanning electrochemical microscopy (SECM) to quantitatively and in situ monitor the early course of ferroptosis in HuH7 cells by characterizing the three key parameters of cell ferroptosis (i.e., cell membrane permeability, respiratory activity, and the redox state). The SECM results show that the membrane permeability of ferroptotic HuH7 cells continuously increased from 0 to 8.1 × 10-5 m s-1, the cellular oxygen consumption was continuously reduced by half, and H2O2 released from the cells exhibited periodic bursts during the early course of ferroptosis, indicating the gradually destroyed cell membrane structure and intensified oxidative stress. Our work realizes, for the first time, the in situ and quantitative monitoring of the cell membrane permeability, respiratory activity, and H2O2 level of the early ferroptosis process of a single living cancer cell with SECM, which can contribute to the understanding of the physiological process and underlying mechanisms of ferroptosis.

Correction: In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy.

[This corrects the article DOI: 10.1039/D2SC02708A.].

In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy.

Extracellular matrix (ECM) stiffness affects the drug resistance behavior of cancer cells, while multidrug resistance protein 1 (MRP1) on the cell membrane confers treatment resistance via actively transporting drugs out of cancer cells. However, the relationship between ECM stiffness and MRP1 functional activity in cancer cells remains elusive, mainly due to the technical challenge of in situ monitoring. Herein, we engineered in vitro cancer cell models using breast cancer cells (MCF-7 and MDA-MB-231 cells) as the reprehensive cells on polyacrylamide (PA) gels with three stiffness, mimicking different developmental stages of cancer. We in situ characterized the functional activity of MRP1 and investigated the effect of ECM stiffness on MRP1 of cancer cells before and after vincristine treatment using scanning electrochemical microscopy (SECM) with ferrocenecarboxylic acid (FcCOOH) as the redox mediator and endogenous glutathione (GSH) as the indicator. The SECM results show that the functional activity of MRP1 is enhanced with increasing ECM stiffness, and the MRP1-mediated vincristine efflux activity of MCF-7 cells is more affected by ECM stiffness than that of MDA-MB-231 cells. This work, for the first time, applied SECM to in situ and quantitatively monitor the functional activity of MRP1 in cancer cells in different tumor mechanical microenvironments, which could help to elucidate the mechanism of matrix stiffness-dependent drug resistance behavior in cancer cells.