Malate-aspartate shuttle promotes l-lactate oxidation in mitochondria.

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD+ /NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.

A Course-Based Undergraduate Research Experience in Biofluid Mechanics.

Course-based undergraduate research experiences (CURE) are a valuable tool to increase research exposure for larger undergraduate cohorts. We implemented a CURE within a senior-level biofluid mechanics course that was primarily taught using a flipped classroom approach. Due to the large class size, the students analyzed data that was publicly available and produced by one of our laboratories. Student teams then developed hypotheses based on the data analysis and designed a set of in vitro and in vivo experiments to test those hypotheses. The hypotheses and experiments that were most highly rated by the class were then tested in our laboratory. At the end of the class, student gains were assessed by self-report and compared to those self-reported by students engaging in a traditional freshman undergraduate summer research experience. While the students in the CURE reported moderate gains in self-assessment of research-based skills, their self-reported gains were statistically significantly lower than those reported by students who participated in the traditional research experience. We believe that the CURE could be improved through implementation in a lower level class, enabling students to observe laboratory experiments, and providing additional feedback throughout the hypothesis development and experimental design process. Overall, the CURE is an innovative way to expand research experiences, in particular for engineering students who often do not participate in hypothesis-driven research during their undergraduate education.

Interstitial Fluid Flow Increases Hepatocellular Carcinoma Cell Invasion through CXCR4/CXCL12 and MEK/ERK Signaling.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer (~80%), and it is one of the few cancer types with rising incidence in the United States. This highly invasive cancer is very difficult to detect until its later stages, resulting in limited treatment options and low survival rates. There is a dearth of knowledge regarding the mechanisms associated with the effects of biomechanical forces such as interstitial fluid flow (IFF) on hepatocellular carcinoma invasion. We hypothesized that interstitial fluid flow enhanced hepatocellular carcinoma cell invasion through chemokine-mediated autologous chemotaxis. Utilizing a 3D in vitro invasion assay, we demonstrated that interstitial fluid flow promoted invasion of hepatocellular carcinoma derived cell lines. Furthermore, we showed that autologous chemotaxis influences this interstitial fluid flow-induced invasion of hepatocellular carcinoma derived cell lines via the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12) signaling axis. We also demonstrated that mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling affects interstitial fluid flow-induced invasion; however, this pathway was separate from CXCR4/CXCL12 signaling. This study demonstrates, for the first time, the potential role of interstitial fluid flow in hepatocellular carcinoma invasion. Uncovering the mechanisms that control hepatocellular carcinoma invasion will aid in enhancing current liver cancer therapies and provide better treatment options for patients.

EMT Transition Alters Interstitial Fluid Flow-Induced Signaling in ERBB2-Positive Breast Cancer Cells.

UNLABELLED: A variety of biophysical forces are altered in the tumor microenvironment (TME) and these forces can influence cancer progression. One such force is interstitial fluid flow (IFF)-the movement of fluid through the tissue matrix. IFF was previously shown to induce invasion of cancer cells, but the activated signaling cascades remain poorly understood. Here, it is demonstrated that IFF induces invasion of ERBB2/HER2-expressing breast cancer cells via activation of phosphoinositide-3-kinase (PI3K). In constitutively activate ERBB2-expressing cells that have undergone epithelial-to-mesenchymal transition (EMT), IFF-mediated invasion requires the chemokine receptor CXCR4, a gradient of its ligand CXCL12, and activity of the PI3K catalytic subunits p110α and ß. In wild-type ERBB2-expressing cells, IFF-mediated invasion is chemokine receptor-independent and requires only p110α activation. To test whether cells undergoing EMT alter their signaling response to IFF, TGFß1 was used to induce EMT in wild-type ERBB2-expressing cells, resulting in IFF-induced invasion dependent on CXCR4 and p110ß. IMPLICATIONS: This study identifies a novel signaling mechanism for interstitial flow-induced invasion of ERBB2-expressing breast cancer cells, one that depends on EMT and acts through a CXCR4-PI3K pathway. These findings suggest that the response of cancer cells to interstitial flow depends on EMT status and malignancy.

Interstitial fluid flow in cancer: implications for disease progression and treatment.

As cancer progresses, a dynamic microenvironment develops that creates and responds to cellular and biophysical cues. Increased intratumoral pressure and corresponding increases in interstitial flow from the tumor bulk to the healthy stroma is an observational hallmark of progressing cancers. Until recently, the role of interstitial flow was thought to be mostly passive in the transport and dissemination of cancer cells to metastatic sites. With research spanning the past decade, we have seen that interstitial flow has a promigratory effect on cancer cell invasion in multiple cancer types. This invasion is one mechanism by which cancers can resist therapeutics and recur, but the role of interstitial flow in cancer therapy is limited to the understanding of transport of therapeutics. Here we outline the current understanding of the role of interstitial flow in cancer and the tumor microenvironment through cancer progression and therapy. We also discuss the current role of fluid flow in the treatment of cancer, including drug transport and therapeutic strategies. By stating the current understanding of interstitial flow in cancer progression, we can begin exploring its role in therapeutic failure and treatment resistance.

α-Catenin localization and sarcomere self-organization on N-cadherin adhesive patterns are myocyte contractility driven.

The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.

Three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment (1). Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions (1-2). However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion(3). Matrix density (4), stiffness (5-6), and structure (6-7), interstitial fluid pressure (8), and interstitial fluid flow (8) are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues (8-10). The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 µm s(-1), depending on tumor size and differentiation (9, 11). This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability (12). Interstitial fluid flow has been shown to increase invasion of cancer cells (13-14), vascular fibroblasts and smooth muscle cells (15). This invasion may be due to autologous chemotactic gradients created around cells in 3-D (16) or increased matrix metalloproteinase (MMP) expression (15), chemokine secretion and cell adhesion molecule expression (17). However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts (18), (b) transporting of antigens and other soluble factors to lymph nodes (19), and (c) modulating lymphatic endothelial cell morphogenesis (20). The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion (13-15, 17). By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Regulation of tumor invasion by interstitial fluid flow.

The importance of the tumor microenvironment in cancer progression is undisputed, yet the significance of biophysical forces in the microenvironment remains poorly understood. Interstitial fluid flow is a nearly ubiquitous and physiologically relevant biophysical force that is elevated in tumors because of tumor-associated angiogenesis and lymphangiogenesis, as well as changes in the tumor stroma. Not only does it apply physical forces to cells directly, but interstitial flow also creates gradients of soluble signals in the tumor microenvironment, thus influencing cell behavior and modulating cell-cell interactions. In this paper, we highlight our current understanding of interstitial fluid flow in the context of the tumor, focusing on the physical changes that lead to elevated interstitial flow, how cells sense flow and how they respond to changes in interstitial flow. In particular, we emphasize that interstitial flow can directly promote tumor cell invasion through a mechanism known as autologous chemotaxis, and indirectly support tumor invasion via both biophysical and biochemical cues generated by stromal cells. Thus, interstitial fluid flow demonstrates how important biophysical factors are in cancer, both by modulating cell behavior and coupling biophysical and biochemical signals.

Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts.

Interstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion. Fibroblasts are key constituents of the tumor stroma that modulate the mechanical environment by matrix remodeling and contraction. Here, we explore how interstitial fluid flow affects fibroblast-tumor cell interactions. Using a 3-dimensional invasion assay and MDA-MB-435S cells cocultured with dermal fibroblasts in a collagen matrix, we showed a synergistic enhancement of tumor cell invasion by fibroblasts in the presence of interstitial flow. Interstitial flow also drove transforming growth factor (TGF)-ß1 and collagenase-dependent fibroblast migration, consistent with previously described mechanisms in which flow promotes invasion through autologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cell invasion by matrix priming via Rho-mediated contraction. We propose a model in which interstitial flow promotes fibroblast migration through increased TGF-ß1 activation and collagen degradation, positioning fibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, in turn enhancing tumor cell invasion via mechanotactic cues. This represents a novel mechanism in which interstitial flow causes fibroblast-mediated stromal remodeling that facilitates tumor invasion.

Biomechanical forces shape the tumor microenvironment.

The importance of the tumor microenvironment in cancer progression is indisputable, yet a key component of the microenvironment--biomechanical forces--remains poorly understood. Tumor growth and progression is paralleled by a host of physical changes in the tumor microenvironment, such as growth-induced solid stresses, increased matrix stiffness, high fluid pressure, and increased interstitial flow. These changes to the biomechanical microenvironment promote tumorigenesis and tumor cell invasion and induce stromal cells--such as fibroblasts, immune cells, and endothelial cells--to change behavior and support cancer progression. This review highlights what we currently know about the biomechanical forces generated in the tumor microenvironment, how they arise, and how these forces can dramatically influence cell behavior, drawing not only upon studies directly related to cancer and tumor cells, but also work in other fields that have shown the effects of these types of mechanical forces vis-à-vis cell behaviors relevant to the tumor microenvironment. By understanding how all of these biomechanical forces can affect tumor cells, stromal cells, and tumor-stromal crosstalk, as well as alter how tumor and stromal cells perceive other extracellular signals in the tumor microenvironment, we can develop new approaches for diagnosis, prognosis, and ultimately treatment of cancer.

A multichamber fluidic device for 3D cultures under interstitial flow with live imaging: development, characterization, and applications.

Interstitial flow is an important biophysical cue that can affect capillary morphogenesis, tumor cell migration, and fibroblast remodeling of the extracellular matrix, among others. Current models that incorporate interstitial flow and that are suitable for live imaging lack the ability to perform multiple simultaneous experiments, for example, to compare effects of growth factors, extracellular matrix composition, etc. We present a nine-chamber radial flow device that allows simultaneous 3D fluidic experiments for relatively long-term culture with live imaging capabilities. Flow velocity profiles were characterized by fluorescence recovery after photobleaching (FRAP) for flow uniformity and estimating the hydraulic conductivity. We demonstrate lymphatic and blood capillary morphogenesis in fibrin gels over 10 days, comparing flow with static conditions as well as the effects of an engineered variant of VEGF that binds fibrin via Factor XIII. We also demonstrate the culture of contractile fibroblasts and co-cultures with tumor cells for modeling the tumor microenvironment. Therefore, this device is useful for studies of capillary morphogenesis, cell migration, contractile cells like fibroblasts, and multicellular cultures, all under interstitial flow.

Strain-dependent recovery behavior of single chondrocytes.

One of the challenges facing researchers studying chondrocyte mechanobiology is determining the range of mechanical forces pertinent to the problems they study. One possible way to deal with this problem is to quantify how the biomechanical behavior of cells varies in response to changing mechanical forces. In this study, the compressibility and recovery behaviors of single chondrocytes were determined as a function of compressive strains from 6 to 63%. Bovine articular chondrocytes from the middle and deep zones were subjected to this range of strains, and digital videocapture was used to track changes in cell dimensions during and after compression. The normalized volume change, apparent Poisson's ratio, residual strain after recovery, cell volume fraction after recovery, and characteristic recovery time constant were analyzed with respect to axial strain. Normalized volume change varied as a function of strain, demonstrating that chondrocytes exhibited compressibility. The mean Poisson's ratio of chondrocytes was found to be 0.29 +/- 0.14, and did not vary with axial strain. In contrast, residual strain, recovered volume fraction, and recovery time constant all depended on axial strain. The dependence of residual strain and recovered volume fraction on axial strain showed a change in behavior around 25-30% strain, opening up the possibility that this range of strains represents a critical value for chondrocytes. Quantifying the mechanical behavior of cells as a function of stress and strain is a potentially useful approach for identifying levels of mechanical stimulation that may be germane to normal cartilage physiology, functional tissue engineering of cartilage, and the etiopathogenesis of osteoarthritis.

Biomechanics of single zonal chondrocytes.

Articular cartilage has a distinct zonal architecture, and previous work has shown that chondrocytes from different zones exhibit variations in gene expression and biosynthesis. In this study, the material properties of single chondrocytes from the superficial and middle/deep zones of bovine distal metatarsal articular cartilage were determined using unconfined compression and digital videocapture. To determine the viscoelastic properties of zonal chondrocytes, unconfined creep compression experiments were performed and the resulting creep curves of individual cells were fit using a standard linear viscoelastic solid model. In the model, a fixed value of the Poisson's ratio was used, determined optically from direct compression of middle/deep chondrocytes. The two approaches used in this study yielded the following average material properties of single chondrocytes: Poisson's ratio of 0.26+/-0.08, instantaneous modulus of 1.06+/-0.82 kPa, relaxed modulus of 0.78+/-0.58 kPa, and apparent viscosity of 4.08+/-7.20 kPa s. Superficial zone chondrocytes were found to be significantly stiffer than middle/deep zone chondrocytes. Attachment time did not affect the stiffness of the cells. The zonal variation in viscoelastic properties may result from the distinct mechanical environments experienced by the cells in vivo. Identifying intrinsic differences in the biomechanics of superficial and middle/deep zone chondrocytes is an important component in understanding how biomechanics influence articular cartilage health and disease.

Creep indentation of single cells.

An apparatus for creep indentation of individual adherent cells was designed, developed, and experimentally validated. The creep cytoindentation apparatus (CCA) can perform stress-controlled experiments and measure the corresponding deformation of single anchorage-dependent cells. The apparatus can resolve forces on the order of 1 nN and cellular deformations on the order of 0.1 micron. Experiments were conducted on bovine articular chondrocytes using loads on the order of 10 nN. The experimentally observed viscoelastic behavior of these cells was modeled using the punch problem and standard linear solid. The punch problem yielded a Young's modulus of 1.11 +/- 0.48 kPa. The standard linear solid model yielded an instantaneous elastic modulus of 8.00 +/- 4.41 kPa, a relaxed modulus of 1.09 +/- 0.54 kPa, an apparent viscosity of 1.50 +/- 0.92 kPa-s, and a time constant of 1.32 +/- 0.65 s. To our knowledge, this is the first time that stress-controlled indentation testing has been applied at the single cell level. This methodology represents a new tool in understanding the mechanical nature of anchorage-dependent cells and mechanotransductional pathways.

Principles of cell mechanics for cartilage tissue engineering.

The critical importance of mechanical signals to the health and maintenance of articular cartilage has been well demonstrated. Tissue engineers have taken a cue from normal cartilage physiology and incorporated the use of mechanical stimulation into their attempts to engineer functional cartilage. However, the specific types of mechanical stimulation that are most beneficial, and the mechanisms that allow a chondrocyte to perceive and respond to those forces, have yet to be elucidated. To develop a better understanding of these processes, it is necessary to examine the mechanical behavior of the single chondrocyte. This paper reviews salient topics related to chondrocyte biomechanics and mechanotransduction, and attempts to put this information into a context both appropriate and useful to cartilage tissue engineering. It also describes the directions this exciting field is taking, and lays out a vision for future studies that could have a significant impact on our understanding of cartilage health and disease.

Biomechanics of single chondrocytes and osteoarthritis.

Osteoarthritis is a significant, debilitating disease that afflicts millions of Americans, yet its etiology is poorly understood. However, there is substantial evidence that biomechanical factors play a role in the development and progression of osteoarthritis. Previous work has demonstrated that biomechanical factors such as an acute insult or the cumulative effects of repetitive loads can induce degenerative changes in joints, cartilage explants, and isolated chondrocytes. Nevertheless, all of these studies suffer from the limitation that the precise nature of the mechanical loads experienced by individual cells is not well defined. Implementation of a single-cell approach, employing existing cell mechanics methodologies and molecular techniques such as single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), offers an exciting new means to identify which biomechanical factors precipitate pathological changes in chondrocytes indicative of osteoarthritis. This article reviews the particular methods used in mechanical studies of single cells with emphasis on techniques that have been used to investigate chondrocytes and similar anchorage-dependent cell types.The fundamentals of RT-PCR and its application at the single-cell level are also discussed.