Substrate stiffness-dependent activation of Hippo pathway in cancer associated fibroblasts.

The tumor microenvironment (TME) comprises a heterogenous cell population within a complex three-dimensional (3D) extracellular matrix (ECM). Stromal cells within this TME are altered by signaling cues from cancer cells to support uncontrolled tumor growth and invasion events. Moreover, the ECM also plays a fundamental role in tumor development through pathological remodeling, stiffening and interaction with TME cells. In healthy tissues, Hippo signaling pathway actively contributes to tissue growth, cell proliferation and apoptosis. However, in cancer, the Hippo signaling pathway is highly dysregulated, leading to nuclear translocation of the YAP/TAZ complex, which directly contributes to uncontrolled cell proliferation and tissue growth, and ECM remodeling and stiffening processes. Here, we compare the effect of increasing cell culture substrate stiffness, derived from tumor progression, upon the dysregulation of the Hippo signaling pathway in colorectal cancer-associated fibroblasts (CAFs) and normal colorectal fibroblasts (NFs). We correlate the dysregulation of Hippo pathway with the magnitude of the traction forces exerted by healthy and malignant stromal cells. We found that ECM stiffening is crucial in Hippo pathway dysregulation in CAFs, but not in normal fibroblasts.

Oncogenic Ras deregulates cell-substrate interactions during mitotic rounding and respreading to alter cell division orientation.

Oncogenic Ras has been shown to change the way cancer cells divide by increasing the forces generated during mitotic rounding. In this way, RasV12 enables cancer cells to divide across a wider range of mechanical environments than normal cells. Here, we identify a further role for oncogenic Ras-ERK signaling in division by showing that RasV12 expression alters the shape, division orientation, and respreading dynamics of cells as they exit mitosis. Many of these effects appear to result from the impact of RasV12 signaling on actomyosin contractility, because RasV12 induces the severing of retraction fibers that normally guide spindle positioning and provide a memory of the interphase cell shape. In support of this idea, the RasV12 phenotype is reversed by inhibition of actomyosin contractility and can be mimicked by the loss of cell-substrate adhesion during mitosis. Finally, we show that RasV12 activation also perturbs division orientation in cells cultured in 2D epithelial monolayers and 3D spheroids. Thus, the induction of oncogenic Ras-ERK signaling leads to rapid changes in division orientation that, along with the effects of RasV12 on cell growth and cell-cycle progression, are likely to disrupt epithelial tissue organization and contribute to cancer dissemination.

Dynamics of cell rounding during detachment.

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

The role of RAS oncogenes in controlling epithelial mechanics.

Mutations in RAS are key oncogenic drivers and therapeutic targets. Oncogenic Ras proteins activate a network of downstream signalling pathways, including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K), promoting cell proliferation and survival. However, there is increasing evidence that RAS oncogenes also alter the mechanical properties of both individual malignant cells and transformed tissues. Here we discuss the role of oncogenic RAS in controlling mechanical cell phenotypes and how these mechanical changes promote oncogenic transformation in single cells and tissues. RAS activation alters actin organisation and actomyosin contractility. These changes alter cell rheology and impact mechanosensing through changes in substrate adhesion and YAP/TAZ-dependent mechanotransduction. We then discuss how these changes play out in cell collectives and epithelial tissues by driving large-scale tissue deformations and the expansion of malignant cells. Uncovering how RAS oncogenes alter cell mechanics will lead to a better understanding of the morphogenetic processes that underlie tumour formation in RAS-mutant cancers.

Renal tumouroids: challenges of manufacturing 3D cultures from patient derived primary cells.

Recent advancements in 3D in vitro culture have allowed for the development of cancer tissue models which accurately recapitulate the tumour microenvironment. Consequently, there has been increased innovation in therapeutic drug screening. While organoid cultures show great potential, they are limited by the time scale of their growth in vitro and the dependence upon commercial matrices, such as Matrigel, which do not allow for manipulations of their composition or mechanical properties. Here, we show a straightforward approach for the isolation and culture of primary human renal carcinoma cells and matched non-affected kidney. This approach does not require any specific selection for cancer cells, and allows for their direct culture in amenable 3D collagen-based matrices, with the preservation of cancer cells as confirmed by NGS sequencing. This method allows for culture of patient-derived cancer cells in 3D microenvironment, which can be used for downstream experimentation such as investigation of cell-matrix interaction or drug screening.

Oncogenic RAS instructs morphological transformation of human epithelia via differential tissue mechanics.

The loss of epithelial homeostasis and the disruption of normal tissue morphology are hallmarks of tumor development. Here, we ask how the uniform activation oncogene RAS affects the morphology and tissue mechanics in a normal epithelium. We found that inducible induction of HRAS in confined epithelial monolayers on soft substrates drives a morphological transformation of a 2D monolayer into a compact 3D cell aggregate. This transformation was initiated by the loss of monolayer integrity and formation of two distinct cell layers with differential cell-cell junctions, cell-substrate adhesion, and tensional states. Computational modeling revealed how adhesion and active peripheral tension induces inherent mechanical instability in the system, which drives the 2D-to-3D morphological transformation. Consistent with this, removal of epithelial tension through the inhibition of actomyosin contractility halted the process. These findings reveal the mechanisms by which oncogene activation within an epithelium can induce mechanical instability to drive morphological tissue transformation.

Development of Cell-Derived Matrices for Three-Dimensional In Vitro Cancer Cell Models.

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

Activation of synovial fibroblasts from patients at revision of their metal-on-metal total hip arthroplasty.

BACKGROUND: The toxicity of released metallic particles generated in metal-on-metal (MoM) total hip arthroplasty (THA) using cobalt chromium (CoCr) has raised concerns regarding their safety amongst both surgeons and the public. Soft tissue changes such as pseudotumours and metallosis have been widely observed following the use of these implants, which release metallic by-products due to both wear and corrosion. Although activated fibroblasts, the dominant cell type in soft tissues, have been linked to many diseases, the role of synovial fibroblasts in the adverse reactions caused by CoCr implants remains unknown. To investigate the influence of implants manufactured from CoCr, the periprosthetic synovial tissues and synovial fibroblasts from patients with failed MoM THA, undergoing a revision operation, were analysed and compared with samples from patients undergoing a primary hip replacement, in order to elucidate histological and cellular changes. RESULTS: Periprosthetic tissue from patients with MoM implants was characterized by marked fibrotic changes, notably an increase in collagen content from less than 20% to 45-55%, an increase in α-smooth muscle actin positive cells from 4 to 9% as well as immune cells infiltration. Primary cell culture results demonstrated that MoM synovial fibroblasts have a decreased apoptosis rate from 14 to 6% compared to control synovial fibroblasts. In addition, synovial fibroblasts from MoM patients retained higher contractility and increased responsiveness to chemotaxis in matrix contraction. Their mechanical properties at a single cell level increased as observed by a 60% increase in contraction force and higher cell stiffness (3.3 kPa in MoM vs 2.18 kPa in control), as measured by traction force microscopy and atomic force microscopy. Further, fibroblasts from MoM patients promoted immune cell invasion by secreting monocyte chemoattractant protein 1 (MCP-1, CCL2) and induced monocyte differentiation, which could also be associated with excess accumulation of synovial macrophages. CONCLUSION: Synovial fibroblasts exposed in vivo to MoM THA implants that release CoCr wear debris displayed dramatic phenotypic alteration and functional changes. These findings unravelled an unexpected effect of the CoCr alloy and demonstrated an important role of synovial fibroblasts in the undesired tissue reactions caused by MoM THAs.

Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling. METHODS: We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a "healthy" or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling. RESULTS: CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin). CONCLUSIONS: These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.

The anti-angiogenic tyrosine kinase inhibitor Pazopanib kills cancer cells and disrupts endothelial networks in biomimetic three-dimensional renal tumouroids.

Pazopanib is a tyrosine kinase inhibitor used to treat renal cell carcinoma. Few in vitro studies investigate its effects towards cancer cells or endothelial cells in the presence of cancer. We tested the effect of Pazopanib on renal cell carcinoma cells (CAKI-2,786-O) in two-dimensional and three-dimensional tumouroids made of dense extracellular matrix, treated in normoxia and hypoxia. Finally, we engineered complex tumouroids with a stromal compartment containing fibroblasts and endothelial cells. Simple CAKI-2 tumouroids were more resistant to Pazopanib than 786-O tumouroids. Under hypoxia, while the more 'resistant' CAKI-2 tumouroids showed no decrease in viability, 786-O tumouroids required higher Pazopanib concentrations to induce cell death. In complex tumouroids, Pazopanib exposure led to a reduction in the overall cell viability (p < 0.0001), disruption of endothelial networks and direct killing of renal cell carcinoma cells. We report a biomimetic multicellular tumouroid for drug testing, suitable for agents whose primary target is not confined to cancer cells.

Molecular analysis of HIF activation as a potential biomarker for adverse reaction to metal debris (ARMD) in tissue and blood samples.

We aimed to find a biomarker for patients with adverse reaction to metal debris (ARMD) due to a metal-on-metal (MoM) hip implant. First, we compared molecular markers of hypoxia-inducible factor (HIF) pathway activation (BNIP3, GLUT1, HO1, VEGF, and HIF1A) and inflammatory response (IL1B and COX2) in tissue from patients undergoing revision of MoM hip implant with tissue from patients undergoing primary hip replacement (PHR). Second, we compared blood levels of the above molecular markers and additional inflammatory markers: TNFA, IL18, CASPASE1, NFKB or IKB, and TLR1-4 mRNA in patients with non-failed MoM hips. We report the presence of increased expression of HIF-target genes in the periprosthetic tissue in MoM patients when compared to the PHR group. This suggests HIF pathway activation due to MoM debris and the potential of using HIF targets as a predictor of failure. Analysis of blood samples from nonoverlapping, nonfailed, MoM group showed significantly higher expression of COX2 mRNA and significant correlations between HIF1A and GLUT1 mRNA expressions, and between HIF1A mRNA and selection of inflammatory genes, including IL18, IKB, TLR1, and TLR4. HIF pathway activation in the periprosthetic tissue biopsies of patients with hip replacements may represent the first biomarker to identify early ARMD. Further studies investigating blood biomarkers could also prove beneficial in detecting ARMD that could lead to an early intervention and improved patient outcome after hip revision surgery. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1352-1362, 2019.

Cobalt (II) ions and nanoparticles induce macrophage retention by ROS-mediated down-regulation of RhoA expression.

Histological assessments of synovial tissues from patients with failed CoCr alloy hip prostheses demonstrate extensive infiltration and accumulation of macrophages, often loaded with large quantities of particulate debris. The resulting adverse reaction to metal debris (ARMD) frequently leads to early joint revision. Inflammatory response starts with the recruitment of immune cells and requires the egress of macrophages from the inflamed site for resolution of the reaction. Metal ions (Co2+ and Cr3+) have been shown to stimulate the migration of T lymphocytes but their effects on macrophages motility are still poorly understood. To elucidate this, we studied in vitro and in vivo macrophage migration during exposure to cobalt and chromium ions and nanoparticles. We found that cobalt but not chromium significantly reduces macrophage motility. This involves increase in cell spreading, formation of intracellular podosome-type adhesion structures and enhanced cell adhesion to the extracellular matrix (ECM). The formation of podosomes was also associated with the production and activation of matrix metalloproteinase-9 (MMP9) and enhanced ECM degradation. We showed that these were driven by the down-regulation of RhoA signalling through the generation of reactive oxygen species (ROS). These novel findings reveal the key mechanisms driving the wear/corrosion metallic byproducts-induced inflammatory response at non-toxic concentrations. STATEMENT OF SIGNIFICANCE: Adverse tissue responses to metal wear and corrosion products from CoCr alloy implants remain a great challenge to surgeons and patients. Macrophages are the key regulators of these adverse responses to the ions and debris generated. We demonstrated that cobalt, rather than chromium, causes macrophage retention by restructuring the cytoskeleton and inhibiting cell migration via ROS production that affects Rho Family GTPase. This distinctive effect of cobalt on macrophage behaviour can help us understand the pathogenesis of ARMD and the cellular response to cobalt based alloys, which provide useful information for future implant design and biocompatibility testing.

The next level of 3D tumour models: immunocompetence.

The complexity of the tumour microenvironment encompasses interactions between cancer and stromal cells. Moving from 2D cell culture methods into 3D models enables more-accurate investigation of those interactions. Current 3D cancer models focus on cancer spheroid interaction with stromal cells, such as fibroblasts. However, over recent years, the cancer immune environment has been shown to have a major role in tumour progression. This review summarises the state-of-art on immunocompetent 3D cancer models that, in addition to cancer cells, also incorporate immune cells, including monocytes, cancer-associated macrophages, dendritic cells, neutrophils and lymphocytes.

Importance of the HIF pathway in cobalt nanoparticle-induced cytotoxicity and inflammation in human macrophages.

Recent, unexpected high failure rates of metal-on-metal hip implants have reintroduced the issue of cobalt toxicity. An adverse reaction to cobalt ions and cobalt-induced lung injury occurs during environmental exposure and is now strictly controlled. Currently adverse reaction occurs to cobalt nanoparticles during wear and tear of metal-on-metal hip implants of which the underlying mechanism is not fully understood. The putative role of the hypoxia-inducible factor (HIF) pathway in the mechanism of cobalt nanoparticle (Co-NPs) toxicity was examined using the U937 cell line, human alveolar macrophages and monocyte-derived macrophages. Co-NPs (5-20 µg/ml)-induced cytotoxicity (viability ranged from 75% to <20% of control, respectively) and reactive oxygen species (ROS), whereas a comparable concentration of cobalt ions (Co(II); up to 350 µM) did not. Co-NPs induced HIF-1α stabilization. Addition of ascorbic acid (100 µM) and glutathione (1 mM) both prevented the increased ROS. However, only treatment with ascorbic acid reduced HIF-1α levels and prevented cell death, indicating that a ROS-independent pathway is involved in Co-NPs-induced cytotoxicity. Replenishing intracellular ascorbate, which is crucial in preventing HIF pathway activation, modified Co-induced HIF target gene expression and the inflammatory response, by decreasing interleukin-1 beta (IL-1ß) mRNA and protein expression. Addition of glutathione had no effect on Co-NPs-induced HIF target gene expression or inflammatory response. Thus, Co-NPs induce the HIF pathway by depleting intracellular ascorbate, leading to HIF stabilization and pathway activation. This suggests a strong, ROS-independent role for HIF activation in Co-NPs-induced cytotoxicity and a possible role for HIF in metal-on-metal hip implant pathology.

A 3D in vitro cancer model as a platform for nanoparticle uptake and imaging investigations.

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue-engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X-ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.

A novel tissue engineered three-dimensional in vitro colorectal cancer model.

The interactions of cancer cells within a solid mass with the surrounding reactive stroma are critical for growth and progression. The surrounding vasculature is recruited into the periphery of the growing tumour to supply cancer cells with nutrients and O2. This study focuses on developing a novel three-dimensional (3-D) in vitro biomimetic colorectal cancer model using colorectal cancer cells and connective tissue cells. The 3-D model comprises a dense artificial cancer mass, created by partial plastic compression of collagen type I containing HT29 colorectal cancer cells, nested in a non-dense collagen type I gel populated by fibroblasts and/or endothelial cells. HT29 cells within the dense mass proliferate slower than when cultured in a two-dimensional system. These cells form tumour spheroids which invade the surrounding matrix, away from the hypoxic conditions in the core of the construct, measured using real time O2 probes. This model is also characterized by the release of vascular endothelial growth factor (VEGF) by HT29 cells, mainly at the invading edge of the artificial cancer mass. This characterization is fundamental in establishing a reproducible, complex model that could be used to advance our understanding of cancer pathology and will facilitate therapeutic drug testing.

3D tumour models: novel in vitro approaches to cancer studies.

3D in vitro models have been used in cancer research as a compromise between 2-dimensional cultures of isolated cancer cells and the manufactured complexity of xenografts of human cancers in immunocompromised animal hosts. 3D models can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario in which they are found. These 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Approaches to create more biomimetic 3D models of cancer include, but are not limited to, (i) providing the appropriate matrix components in a 3D configuration found in vivo, (ii) co-culturing cancer cells, endothelial cells and other associated cells in a spatially relevant manner, (iii) monitoring and controlling hypoxia- to mimic levels found in native tumours and (iv) monitoring the release of angiogenic factors by cancer cells in response to hypoxia. This article aims to overview current 3D in vitro models of cancer and review strategies employed by researchers to tackle these aspects with special reference to recent promising developments, as well as the current limitations of 2D cultures and in vivo models. 3D in vitro models provide an important alternative to both complex in vivo whole organism approaches, and 2D culture with its spatial limitations. Here we review current strategies in the field of modelling cancer, with special reference to advances in complex 3D in vitro models.