Linking cell mechanical memory and cancer metastasis.

Metastasis causes most cancer-related deaths; however, the efficacy of anti-metastatic drugs is limited by incomplete understanding of the biological mechanisms that drive metastasis. Focusing on the mechanics of metastasis, we propose that the ability of tumour cells to survive the metastatic process is enhanced by mechanical stresses in the primary tumour microenvironment that select for well-adapted cells. In this Perspective, we suggest that biophysical adaptations favourable for metastasis are retained via mechanical memory, such that the extent of memory is influenced by both the magnitude and duration of the mechanical stress. Among the mechanical cues present in the primary tumour microenvironment, we focus on high matrix stiffness to illustrate how it alters tumour cell proliferation, survival, secretion of molecular factors, force generation, deformability, migration and invasion. We particularly centre our discussion on potential mechanisms of mechanical memory formation and retention via mechanotransduction and persistent epigenetic changes. Indeed, we propose that the biophysical adaptations that are induced by this process are retained throughout the metastatic process to improve tumour cell extravasation, survival and colonization in the distant organ. Deciphering mechanical memory mechanisms will be key to discovering a new class of anti-metastatic drugs.

TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs.

BACKGROUND: Aberrant mechanical loading of the spine causes intervertebral disc (IVD) degeneration and low back pain. Current therapies do not target the mediators of the underlying mechanosensing and mechanotransduction pathways, as these are poorly understood. This study investigated the role of the mechanosensitive transient receptor potential vanilloid 4 (TRPV4) ion channel in dynamic compression of bovine nucleus pulposus (NP) cells in vitro and mouse IVDs in vivo. METHODS: Degenerative changes and the expression of the inflammatory mediator cyclooxygenase 2 (COX2) were examined histologically in the IVDs of mouse tails that were dynamically compressed at a short repetitive hyperphysiological regime (vs sham). Bovine NP cells embedded in an agarose-collagen hydrogel were dynamically compressed at a hyperphysiological regime in the presence or absence of the selective TRPV4 antagonist GSK2193874. Lactate dehydrogenase (LDH) and prostaglandin E2 (PGE2) release, as well as phosphorylation of mitogen-activated protein kinases (MAPKs), were analyzed. Degenerative changes and COX2 expression were further evaluated in the IVDs of trpv4-deficient mice (vs wild-type; WT). RESULTS: Dynamic compression caused IVD degeneration in vivo as previously shown but did not affect COX2 expression. Dynamic compression significantly augmented LDH and PGE2 releases in vitro, which were significantly reduced by TRPV4 inhibition. Moreover, TRPV4 inhibition during dynamic compression increased the activation of the extracellular signal-regulated kinases 1/2 (ERK) MAPK pathway by 3.13-fold compared to non-compressed samples. Trpv4-deficient mice displayed mild IVD degeneration and decreased COX2 expression compared to WT mice. CONCLUSIONS: TRPV4 therefore regulates COX2/PGE2 and mediates cell damage induced by hyperphysiological dynamic compression, possibly via ERK. Targeted TRPV4 inhibition or knockdown might thus constitute promising therapeutic approaches to treat patients suffering from IVD pathologies caused by aberrant mechanical stress.

Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective.

Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.

Effects of Early Life Stress on Bone Homeostasis in Mice and Humans.

Bone pathology is frequent in stressed individuals. A comprehensive examination of mechanisms linking life stress, depression and disturbed bone homeostasis is missing. In this translational study, mice exposed to early life stress (MSUS) were examined for bone microarchitecture (µCT), metabolism (qPCR/ELISA), and neuronal stress mediator expression (qPCR) and compared with a sample of depressive patients with or without early life stress by analyzing bone mineral density (BMD) (DXA) and metabolic changes in serum (osteocalcin, PINP, CTX-I). MSUS mice showed a significant decrease in NGF, NPYR1, VIPR1 and TACR1 expression, higher innervation density in bone, and increased serum levels of CTX-I, suggesting a milieu in favor of catabolic bone turnover. MSUS mice had a significantly lower body weight compared to control mice, and this caused minor effects on bone microarchitecture. Depressive patients with experiences of childhood neglect also showed a catabolic pattern. A significant reduction in BMD was observed in depressive patients with childhood abuse and stressful life events during childhood. Therefore, future studies on prevention and treatment strategies for both mental and bone disease should consider early life stress as a risk factor for bone pathologies.

TRPV4 Inhibition and CRISPR-Cas9 Knockout Reduce Inflammation Induced by Hyperphysiological Stretching in Human Annulus Fibrosus Cells.

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.

Cell-Laden Agarose-Collagen Composite Hydrogels for Mechanotransduction Studies.

The increasing investigation of cellular mechanotransduction mechanisms requires biomaterials combining biofunctionality and suitable mechanical properties. Agarose is a standard biomaterial for cartilage and intervertebral disc mechanobiology studies, but lacks adhesion motifs and the necessary cell-matrix interaction for mechanotransduction. Here, collagen type I was blended at two concentrations (2 and 4.5 mg/mL) with agarose 2% wt/vol. The composite hydrogels were characterized in terms of structural homogeneity, rheological properties and size stability. Nucleus pulposus (NP) cell viability, proliferation, morphology, gene expression, GAG production, adhesion and mechanotransduction ability were further tested. Blended hydrogels presented a homogenous network of the two polymers. While the addition of 4.5 mg/mL collagen significantly decreased the storage modulus and increased the loss modulus of the gels, blended gels containing 2 mg/mL collagen displayed similar mechanical properties to agarose. Hydrogel size was conserved over 21 days for all agarose-based gels. Embedded cells were viable (>80%) and presented reduced proliferation and a round morphology typical of NP cells in vivo. Gene expression of collagen types I and II and aggrecan significantly increased in blended hydrogels from day 1 to 7, further resulting in a significantly superior GAG/DNA ratio compared to agarose gels at day 7. Agarose-collagen hydrogels not only promoted cell adhesion, contrary to agarose gels, but also showed a 5.36-fold higher focal adhesion kinase phosphorylation (pFAK/ß-tubulin) when not compressed, and increased pFAK/FAK values 10 min after compression. Agarose-collagen thus outperforms agarose, mimics native tissues constituted of non-fibrillar matrix and collagens, and allows exploring complex loading in a highly reproducible system.

TRPC6 in simulated microgravity of intervertebral disc cells.

PURPOSE: Prolonged bed rest and microgravity in space cause intervertebral disc (IVD) degeneration. However, the underlying molecular mechanisms are not completely understood. Transient receptor potential canonical (TRPC) channels are implicated in mechanosensing of several tissues, but are poorly explored in IVDs. METHODS: Primary human IVD cells from surgical biopsies composed of both annulus fibrosus and nucleus pulposus (passage 1-2) were exposed to simulated microgravity and to the TRPC channel inhibitor SKF-96365 (SKF) for up to 5 days. Proliferative capacity, cell cycle distribution, senescence and TRPC channel expression were analyzed. RESULTS: Both simulated microgravity and TRPC channel antagonism reduced the proliferative capacity of IVD cells and induced senescence. While significant changes in cell cycle distributions (reduction in G1 and accumulation in G2/M) were observed upon SKF treatment, the effect was small upon 3 days of simulated microgravity. Finally, downregulation of TRPC6 was shown under simulated microgravity. CONCLUSIONS: Simulated microgravity and TRPC channel inhibition both led to reduced proliferation and increased senescence. Furthermore, simulated microgravity reduced TRPC6 expression. IVD cell senescence and mechanotransduction may hence potentially be regulated by TRPC6 expression. This study thus reveals promising targets for future studies. These slides can be retrieved under Electronic Supplementary Material.

The potential of CRISPR/Cas9 genome editing for the study and treatment of intervertebral disc pathologies.

The CRISPR/Cas9 system has emerged as a powerful tool for mammalian genome engineering. In basic and translational intervertebral disc (IVD) research, this technique has remarkable potential to answer fundamental questions on pathway interactions, to simulate IVD pathologies, and to promote drug development. Furthermore, the precisely targeted CRISPR/Cas9 gene therapy holds promise for the effective and targeted treatment of degenerative disc disease and low back pain. In this perspective, we provide an overview of recent CRISPR/Cas9 advances stemming from/with transferability to IVD research, outline possible treatment approaches for degenerative disc disease, and discuss current limitations that may hinder clinical translation.

Translational cardiac stem cell therapy: advancing from first-generation to next-generation cell types.

Acute myocardial infarction and chronic heart failure rank among the major causes of morbidity and mortality worldwide. Except for heart transplantation, current therapy options only treat the symptoms but do not cure the disease. Stem cell-based therapies represent a possible paradigm shift for cardiac repair. However, most of the first-generation approaches displayed heterogeneous clinical outcomes regarding efficacy. Stemming from the desire to closely match the target organ, second-generation cell types were introduced and rapidly moved from bench to bedside. Unfortunately, debates remain around the benefit of stem cell therapy, optimal trial design parameters, and the ideal cell type. Aiming at highlighting controversies, this article provides a critical overview of the translation of first-generation and second-generation cell types. It further emphasizes the importance of understanding the mechanisms of cardiac repair and the lessons learned from first-generation trials, in order to improve cell-based therapies and to potentially finally implement cell-free therapies.

Cardiac Regenerative Medicine: The Potential of a New Generation of Stem Cells.

Cardiac stem cell therapy holds great potential to prompt myocardial regeneration in patients with ischemic heart disease. The selection of the most suitable cell type is pivotal for its successful application. Various cell types, including crude bone marrow mononuclear cells, skeletal myoblast, and hematopoietic and endothelial progenitors, have already advanced into the clinical arena based on promising results from different experimental and preclinical studies. However, most of these so-called first-generation cell types have failed to fully emulate the promising preclinical data in clinical trials, resulting in heterogeneous outcomes and a critical lack of translation. Therefore, different next-generation cell types are currently under investigation for the treatment of the diseased myocardium. This review article provides an overview of current stem cell therapy concepts, including the application of cardiac stem (CSCs) and progenitor cells (CPCs) and lineage commitment via guided cardiopoiesis from multipotent cells such as mesenchymal stem cells (MSCs) or pluripotent cells such as embryonic and induced pluripotent stem cells. Furthermore, it introduces new strategies combining complementary cell types, such as MSCs and CSCs/CPCs, which can yield synergistic effects to boost cardiac regeneration.

Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment.

More people die annually from cardiovascular diseases than from any other cause. In particular, patients who suffer from myocardial infarction may be affected by ongoing adverse remodeling processes of the heart that may ultimately lead to heart failure. The introduction of stem and progenitor cell-based applications has raised substantial hope for reversing these processes and inducing cardiac regeneration. However, current stem cell therapies using single-cell suspensions have failed to demonstrate long-lasting efficacy due to the overall low retention rate after cell delivery to the myocardium. To overcome this obstacle, the concept of 3D cell culture techniques has been proposed to enhance therapeutic efficacy and cell engraftment based on the simulation of an in vivo-like microenvironment. Of great interest is the use of so-called microtissues or spheroids, which have evolved from their traditional role as in vitro models to their novel role as therapeutic agents. This review will provide an overview of the therapeutic potential of microtissues by addressing primarily cardiovascular regeneration. It will accentuate their advantages compared to other regenerative approaches and summarize the methods for generating clinically applicable microtissues. In addition, this review will illustrate the unique properties of the microenvironment within microtissues that makes them a promising next-generation therapeutic approach.

Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.