Extracellular matrix stiffness activates mechanosensitive signals but limits breast cancer cell spheroid proliferation and invasion

Abstract

Breast cancer is characterized by physical changes that occur in the tumor microenvironment throughout growth and metastasis of tumors. Extracellular matrix stiffness increases as tumors develop and spread, with stiffer environments thought to correlate with poorer disease prognosis. Changes in extracellular stiffness and other physical characteristics are sensed by integrins which integrate these extracellular cues to intracellular signaling, resulting in modulation of proliferation and invasion. However, the co-ordination of mechano-sensitive signaling with functional changes to groups of tumor cells within 3-dimensional environments remains poorly understood. Here we provide evidence that increasing the stiffness of collagen scaffolds results in increased activation of ERK1/2 and YAP in human breast cancer cell spheroids. We also show that ERK1/2 acts upstream of YAP activation in this context. We further demonstrate that YAP, matrix metalloproteinases and actomyosin contractility are required for collagen remodeling, proliferation and invasion in lower stiffness scaffolds. However, the increased activation of these proteins in higher stiffness 3-dimensional collagen gels is correlated with reduced proliferation and reduced invasion of cancer cell spheroids. Our data collectively provide evidence that higher stiffness 3-dimensional environments induce mechano-signaling but contrary to evidence from 2-dimensional studies, this is not sufficient to promote pro-tumorigenic effects in breast cancer cell spheroids.

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases

“…Similarly, YAP activity is affected by matrix stiffness: like ERK, at high ribose concentrations, spheroids showed more YAP activation and nuclear distribution compared to the citoplasmic compartment ( Figure 1 f). There is evidence that both YAP and ERK activity may play a key role in ECM remodeling to provide a favorable matrix for BC cell migration [ 57 ]. Although the underlying mechanism remains elusive, it is known that the stiffness of the ECM matrix influences cell behavior through cancer metabolism regulation [ 77 , 78 ].…”

“…( f ) Spheroids embedded in a 3D collagen structure show the localization of YAP (green); nuclei are stained blue. Scale bars, 20 μm (adapted from [ 57 ] under the terms of the CC-BY 4.0 publishing license). ( g ) Box representation of doxorubicin effects in the MDA-MB-231 cell line cultured within the 3D biomimetic collagen scaffold, indicating the most significantly altered pathways implicated in DOX resistance (green = up-regulation; red = down-regulation) (adapted from [ 64 ] under the terms of the CC-BY 4.0 publishing license).…”

A Multiparametric Analysis Reveals Differential Behavior of Spheroid Cultures on Distinct Ultra-Low Attachment Plates Types

“…Hippo signaling is critically altered in several imbalance and disease states, such as in wound healing and cancer [26] and it has been intensely addressed as a drug target [26]. Yet, due to its pleiotropic regulation, YAP activity and function were found to be significantly different between adherent and 3D-cell cultures [27][28][29], arguing for further research in that direction. Since YAP nuclear translocation is a major proxy for YAP activity [25,30], determination of the YAP nuclear-to-cytoplasm ratio (N/C ratio) is key to single-cell assays in this field.…”

“…Since YAP nuclear translocation is a major proxy for YAP activity [25,30], determination of the YAP nuclear-to-cytoplasm ratio (N/C ratio) is key to single-cell assays in this field. However, automated analysis of this characteristic in 3D-samples has been hampered by technical issues and it has been achieved only for very small spheroids [28] or by manually checking a few cells in larger spheroids [29].…”

“…Since YAP nuclear translocation is a major proxy for YAP activity [25,30] , determination of the YAP nuclear-to-cytoplasm ratio (N/C ratio) is key to single-cell assays in this field. However, automated analysis of this characteristic in 3D-samples has been hampered by technical issues and it has been achieved only for very small spheroids [28] or by manually checking a few cells in larger spheroids [29] . Thus, to auto-mate detection of cell nuclei and YAP distribution in spheroids of several thousand cells, a novel pipeline of handling spheroids for detailed 3D-morphometric single-cell analysis was needed and established in the present work.…”