Emad Moeendarbary

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.

Actin kinetics shapes cortical network structure and mechanics.

Cells adjust their macroscopic mechanical properties by tuning the actin protomer concentration and activity of actin nucleators. The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

A Chemomechanical Model for Nuclear Morphology and Stresses during Cell Transendothelial Migration

It is now evident that the cell nucleus undergoes dramatic shape changes during important cellular processes such as cell transmigration through extracellular matrix and endothelium. Recent experimental data suggest that during cell transmigration the deformability of the nucleus could be a limiting factor, and the morphological and structural alterations that the nucleus encounters can perturb genomic organization that in turn influences cellular behavior. Despite its importance, a biophysical model that connects the experimentally observed nuclear morphological changes to the underlying biophysical factors during transmigration through small constrictions is still lacking. Here, we developed a universal chemomechanical model that describes nuclear strains and shapes and predicts thresholds for the rupture of the nuclear envelope and for nuclear plastic deformation during transmigration through small constrictions. The model includes actin contraction and cytosolic back pressure that squeeze the nucleus through constrictions and overcome the mechanical resistance from deformation of the nucleus and the constrictions. The nucleus is treated as an elastic shell encompassing a poroelastic material representing the nuclear envelope and inner nucleoplasm, respectively. Tuning the chemomechanical parameters of different components such as cell contractility and nuclear and matrix stiffnesses, our model predicts the lower bounds of constriction size for successful transmigration. Furthermore, treating the chromatin as a plastic material, our model faithfully reproduced the experimentally observed irreversible nuclear deformations after transmigration in lamin-A/C-deficient cells, whereas the wild-type cells show much less plastic deformation. Along with making testable predictions, which are in accord with our experiments and existing literature, our work provides a realistic framework to assess the biophysical modulators of nuclear deformation during cell transmigration.

Cdc42EP3/BORG2 and Septin Network Enables Mechano-transduction and the Emergence of Cancer-Associated Fibroblasts

Summary Cancer-associated fibroblasts (CAFs) are non-cancerous cells found in solid tumors that remodel the tumor matrix and promote cancer invasion and angiogenesis. Here, we demonstrate that Cdc42EP3/BORG2 is required for the matrix remodeling, invasion, angiogenesis, and tumor-growth-promoting abilities of CAFs. Cdc42EP3 functions by coordinating the actin and septin networks. Furthermore, depletion of SEPT2 has similar effects to those of loss of Cdc42EP3, indicating a role for the septin network in the tumor stroma. Cdc42EP3 is upregulated early in fibroblast activation and precedes the emergence of the highly contractile phenotype characteristic of CAFs. Depletion of Cdc42EP3 in normal fibroblasts prevents their activation by cancer cells. We propose that Cdc42EP3 sensitizes fibroblasts to further cues—in particular, those activating actomyosin contractility—and thereby enables the generation of the pathological activated fibroblast state.

Self-organizing actin patterns shape membrane architecture but not cell mechanics.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

In Vitro Modeling of Mechanics in Cancer Metastasis

In addition to a multitude of genetic and biochemical alterations, abnormal morphological, structural, and mechanical changes in cells and their extracellular environment are key features of tumor invasion and metastasis. Furthermore, it is now evident that mechanical cues alongside biochemical signals contribute to critical steps of cancer initiation, progression, and spread. Despite its importance, it is very challenging to study mechanics of different steps of metastasis in the clinic or even in animal models. While considerable progress has been made in developing advanced in vitro models for studying genetic and biological aspects of cancer, less attention has been paid to models that can capture both biological and mechanical factors realistically. This is mainly due to lack of appropriate models and measurement tools. After introducing the central role of mechanics in cancer metastasis, we provide an outlook on the emergence of novel in vitro assays and their combination with advanced measurement technologies to probe and recapitulate mechanics in conditions more relevant to the metastatic disease.

Complex mechanics of the heterogeneous extracellular matrix in cancer

The extracellular matrix (ECM) performs many critical functions, one of which is to provide structural and mechanical integrity, and many of the constituent proteins have clear mechanical roles. The composition and structural characteristics of the ECM are widely variable among different tissues, suiting diverse functional needs. In diseased tissues, particularly solid tumors, the ECM is complex and influences disease progression. Cancer and stromal cells can significantly influence the matrix composition and structure and thus the mechanical properties of the tumor microenvironment (TME). In this review, we describe the interactions that give rise to the structural heterogeneity of the ECM and present the techniques that are widely employed to measure ECM properties and remodeling dynamics. Furthermore, we review the tools for measuring the distinct nature of cell–ECM interactions within the TME.

Engineered Models of Metastasis with Application to Study Cancer Biomechanics

Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types.

Infection Augments Expression of Mechanosensing Piezo1 Channels in Amyloid Plaque-Reactive Astrocytes

A defining pathophysiological hallmark of Alzheimer’s disease (AD) is the amyloid plaque; an extracellular deposit of aggregated fibrillar Aβ1-42 peptides. Amyloid plaques are hard, brittle structures scattered throughout the hippocampus and cerebral cortex and are thought to cause hyperphosphorylation of tau, neurofibrillary tangles, and progressive neurodegeneration. Reactive astrocytes and microglia envelop the exterior of amyloid plaques and infiltrate their inner core. Glia are highly mechanosensitive cells and can almost certainly sense the mismatch between the normally soft mechanical environment of the brain and very stiff amyloid plaques via mechanosensing ion channels. Piezo1, a non-selective cation channel, can translate extracellular mechanical forces to intracellular molecular signaling cascades through a process known as mechanotransduction. Here, we utilized an aging transgenic rat model of AD (TgF344-AD) to study expression of mechanosensing Piezo1 ion channels in amyloid plaque-reactive astrocytes. We found that Piezo1 is upregulated with age in the hippocampus and cortex of 18-month old wild-type rats. However, more striking increases in Piezo1 were measured in the hippocampus of TgF344-AD rats compared to age-matched wild-type controls. Interestingly, repeated urinary tract infections with Escherichia coli bacteria, a common comorbidity in elderly people with dementia, caused further elevations in Piezo1 channel expression in the hippocampus and cortex of TgF344-AD rats. Taken together, we report that aging and peripheral infection augment amyloid plaque-induced upregulation of mechanoresponsive ion channels, such as Piezo1, in astrocytes. Further research is required to investigate the role of astrocytic Piezo1 in the Alzheimer’s brain, whether modulating channel opening will protect or exacerbate the disease state, and most importantly, if Piezo1 could prove to be a novel drug target for age-related dementia.

Abstract A53: Probing forces and modulation of cancer cell mechanical properties during transendothelial migration

For all types of cancer, the survival rate while the tumor is still localized is significantly higher than when cancer has metastasized. Though different therapeutic methods can be applied to successfully treat primary tumors, treatment of metastasised cancer is a great challenge due to its complex and systemic nature. A deeper understanding of how cancer initiates, grows and migrates is essential for creating successful therapies. Particularly, extravasation — a process during which streaming tumor cells (TCs) adhere to the blood vessels and traverse through the vascular endothelium into the surrounding tissue — is one of the crucial steps of cancer metastasis. While cancer research has mostly focused on the initial processes of metastasis, little is known about mechanisms of transmigration of tumor cells through the vascular wall. We found that during transendothelial migration (TEM), cancers cells undergo substantial shape changes transforming from semi-spherical (with a 2D contact with the endothelium) to a fully spread 3D morphology inside the ECM. This significant morphological change occurs due to forces generated intracellularly and distributed externally. During these morphological changes in transmigration, the cytoskeleton provides the force for shape change but the rate at which shape change occurs is dictated by the degree of deformability of the tumor cell, endothelial cells (ECs) and subendothelial tissue. Hence, the strength and distribution of forces, as well as mechanical properties of the cells and ECM, play a central role in the process of TEM. In this project, we investigate modulation of TCs and ECs rheological properties and the role of forces generated by the cytoskeleton and their transmission to adjacent cells and the ECM during extravasation. To address the role of mechanical modulation during TEM, we have developed in vitro assays and combed them with optical/confocal microscopy, Traction Force Microscopy (TFM) and Brillouin Confocal Microscopy (BCM). In particular, we recently designed a simple system in which ECs are cultured on a thin layer of collagen. Using a combination of different coating treatments on glass to produce surface hydrophobicity and maintain strong adhesion, we created a thin (~80 μm) layer of gel. Following seeding by ECs (HUVECs) to produce a tight and uniform EC monolayer we introduced TCs on top of the EC monolayer and monitored extravasation in real-time with high spatiotemporal resolution. Measurements of HUVEC monolayer permeability in this system show physiologically relevant values (8±3 10-8 cm2/s). We further tested our system by using different cancer cells with different invasiveness potentials and investigated their extravasation efficiencies. A major advantage of this platform is that it allows integration of TFM and BCM so it enabled us to characterize forces and modulation of cell mechanical properties during TEM. In particular, we demonstrated the feasibility of our system for application of BCM on cancer cells extravasating through a monolayer. BCM is an optical technique in which the mechanical information can be read out optically via spectral analysis of the scattered light; thanks to the interaction of light with intrinsic mechanical vibrations (phonons) of the material. Our BCM measurements indicate a significant decrease in elasticity of TCs at different stages; from 260±20 Pa for TCs that were in the process of transmigration to 195±17 Pa for those that were fully transmigrated. Our results shed a new light on the fundamental understanding of the extravasation mechanics. Furthermore, we anticipate that these studies will potentially enhance our ability to identify and screen for new therapies to inhibit the tendency for metastatic spread of disease. Citation Format: Emad Moeendarbary, Roger Kamm, Giuliano Scarcelli. Probing forces and modulation of cancer cell mechanical properties during transendothelial migration. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A53.