Claudia T. Mierke

Magnetically Guided Titania Nanotubes for Site-Selective Photocatalysis and Drug Release†

The nanoscale encapsulation of ferromagnetic structures has received a great deal of attention because of the exciting possibilities to use these materials in various applications that range from novel electromagnetic to biomedical devices. For example, nanoscale magnetic entities could be transported and concentrated at pretargeted locations or organs within the human body bymeans of an external magnetic field in order to exert a specific function with high local and temporal precision. Therefore, functionalized magnetic nanodots, nanowires, or nanotubes have a high potential for in vivo applications such as magnetic resonance imaging or siteselective drug delivery systems, if the magnetic property is combined with an appropriate drug loading and release mechanism. TiO2 nanotubes are a highly promising encapsulating material for a magnetic core as a high degree of biocompatibility can be combined with a broad range of other functionalities. Since the pioneering work of Fujishima and Honda in 1971, it has been established that TiO2 is a highly active photocatalyst; this is based on the ability of TiO2 to produce electron–hole pairs upon light irradiation and thereby create highly reactive radical species. This property of TiO2 has been intensively explored in the form of photoelectrodes for the decomposition of various organic pollutants in water and air, and it has been used in self-cleaning, disinfecting, and anticancer materials. The photocatalytic ability of TiO2 can be enhanced by using nanosized TiO2 materials because of their large specific surface area. Herein, we describe a simple way of embedding magnetic properties into TiO2 nanotubes and demonstrate their different site-selective photocatalytic applications. Not only can these tubes be used as a magnetically guided photocatalyst for the decomposition of organic matter but also the photocatalytic mechanism can be exploited to release an active species (a model drug). Among the various synthetic routes used to prepare TiO2 nanotubes, [26–28] anodization approaches have gained significant attention as they lead to highly ordered nanotubular arrangements. During the past few years, our research group has contributed several generations of anodically grown self-organized TiO2 nanotube layers by anodization of Ti in aqueous and organic electrolytes. In our approach, we use nanotube layers (Figure 1a) that were produced in ethylene glycol/NH4F electrolytes [36–38] (see Section S1 and Figure S1 in the Supporting Information). These TiO2 nanotubes were filled with magnetic nanoparticles by sucking a droplet of ferrofluid placed on the top of the nanotube layer using a permanent magnet (see the Supporting Information). Figure 1b shows topand side-view SEM images of the nanotubes that are loaded with the magnetic nanoparticles. It is clear from these images that the majority of the inside tube walls were coated relatively uniformly with the magnetic particles leaving a hollow space inside the tubes. In contrast to other established but time-consuming and

Mechano-coupling and regulation of contractility by the vinculin tail domain.

Vinculin binds to multiple focal adhesion and cytoskeletal proteins and has been implicated in transmitting mechanical forces between the actin cytoskeleton and integrins or cadherins. It remains unclear to what extent the mechano-coupling function of vinculin also involves signaling mechanisms. We report the effect of vinculin and its head and tail domains on force transfer across cell adhesions and the generation of contractile forces. The creep modulus and the adhesion forces of F9 mouse embryonic carcinoma cells (wild-type), vinculin knock-out cells (vinculin −/−), and vinculin −/− cells expressing either the vinculin head domain, tail domain, or full-length vinculin (rescue) were measured using magnetic tweezers on fibronectin-coated super-paramagnetic beads. Forces of up to 10 nN were applied to the beads. Vinculin −/− cells and tail cells showed a slightly higher incidence of bead detachment at large forces. Compared to wild-type, cell stiffness was reduced in vinculin −/− and head cells and was restored in tail and rescue cells. In all cell lines, the cell stiffness increased by a factor of 1.3 for each doubling in force. The power-law exponent of the creep modulus was force-independent and did not differ between cell lines. Importantly, cell tractions due to contractile forces were suppressed markedly in vinculin −/− and head cells, whereas tail cells generated tractions similar to the wild-type and rescue cells. These data demonstrate that vinculin contributes to the mechanical stability under large external forces by regulating contractile stress generation. Furthermore, the regulatory function resides in the tail domain of vinculin containing the paxillin-binding site.

Contractile forces in tumor cell migration

Cancer is a deadly disease primarily because of the ability of tumor cells to spread from the primary tumor, to invade into the connective tissue, and to form metastases at distant sites. In contrast to cell migration on a planar surface where large cell tractions and contractile forces are not essential, tractions and forces are thought to be crucial for overcoming the resistance and steric hindrance of a dense three-dimensional connective tissue matrix. In this review, we describe recently developed biophysical tools, including 2-D and 3-D traction microscopy to measure contractile forces of cells. We discuss evidence indicating that tumor cell invasiveness is associated with increased contractile force generation.

Integrin α5β1 facilitates cancer cell invasion through enhanced contractile forces

Cell migration through connective tissue, or cell invasion, is a fundamental biomechanical process during metastasis formation. Cell invasion usually requires cell adhesion to the extracellular matrix through integrins. In some tumors, increased integrin expression is associated with increased malignancy and metastasis formation. Here, we have studied the invasion of cancer cells with different α5β1 integrin expression levels into loose and dense 3D collagen fiber matrices. Using a cell sorter, we isolated from parental MDA-MB-231 breast cancer cells two subcell lines expressing either high or low amounts of α5β1 integrins (α5β1high or α5β1low cells, respectively). α5β1high cells showed threefold increased cell invasiveness compared to α5β1low cells. Similar results were obtained for 786-O kidney and T24 bladder carcinoma cells, and cells in which the α5 integrin subunit was knocked down using specific siRNA. Knockdown of the collagen receptor integrin subunit α2 also reduced invasiveness, but to a lesser degree than knockdown of integrin subunit α5. Fourier transform traction microscopy revealed that the α5β1high cells generated sevenfold greater contractile forces than α5β1low cells. Cell invasiveness was reduced after addition of the myosin light chain kinase inhibitor ML-7 in α5β1high cells, but not in α5β1low cells, suggesting that α5β1 integrins enhance cell invasion through enhanced transmission and generation of contractile forces.

Vinculin Facilitates Cell Invasion into Three-dimensional Collagen Matrices

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

The role of the tissue microenvironment in the regulation of cancer cell motility and invasion

During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion.

Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.

Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force. (Mol Cancer Res 2008;6(9):1410–20)

Breakdown of the Endothelial Barrier Function in Tumor Cell Transmigration

The ability of tumor cells to metastasize is associated with a poor prognosis for cancer. During the process of metastasis, tumor cells circulating in the blood or lymph vessels can adhere to, and potentially transmigrate through, the endothelium and invade the connective tissue. We studied the effectiveness of the endothelium as a barrier against the invasion of 51 tumor cell lines into a three-dimensional collagen matrix. Only nine tumor cell lines showed attenuated invasion in the presence of an endothelial cell monolayer, whereas 17 cell lines became invasive or showed a significantly increased invasion. Endothelial cells cocultured with invasive tumor cells increased chemokine gene expression of IL-8 and Gro-β. Expression of the IL-8 and Gro-β receptor, CXCR2, was upregulated in invasive tumor cells. Addition of IL-8 or Gro-β increased tumor cell invasiveness by more than twofold. Tumor cell variants selected for high CXCR2 expression were fourfold more invasive in the presence of an endothelial cell layer, whereas CXCR2 siRNA knock-down cells were fivefold less invasive. We demonstrate that Gro-β and IL-8 secreted by endothelial cells, together with CXCR2 receptor expression on invasive tumor cells, contribute to the breakdown of the endothelial barrier by enhancing tumor cell force generation and cytoskeletal remodeling dynamics.

HUMAN INTESTINAL MAST CELLS PRODUCE IL-5 IN VITRO UPON IGE RECEPTOR CROSS-LINKING AND IN VIVO IN THE COURSE OF INTESTINAL INFLAMMATORY DISEASE

IL‐5, known to be produced by T lymphocytes and eosinophils, is a key regulator of intestinal diseases such as parasitosis or eosinophilic gastroenteritis. Here we examined if mast cells contribute to the IL‐5 production in human intestinal mucosa. The number of IL‐5‐positive lamina propria cells was substantially higher in patients with intestinal inflammatory diseases (5.3 ± 4.6 %, n = 17) compared to healthy controls (0.5 ± 0.9 %, n = 8, p < 0.01). In patients, the IL‐5‐positive cells were eosinophils (70 ± 13  %) and mast cells (29 ± 14 %), whereas in controls all IL‐5‐positive cells were eosinophils. IL‐5‐positive T cells were not detected, likely because they do not store IL‐5. In vitro: studies with isolated human intestinal mast cells and eosinophils showed that mast cells do not produce IL‐5 constitutively, but release high amounts of IL‐5 (315 ± 115 pg/106 cells) following IgE receptor cross‐linking, compared to activated eosinophils (24 ± 5 pg/106 cells). Inhibitor studies suggest a regulation of IL‐5 production at the transcriptional level. In conclusion our data demonstrate that activated mast cells are a potent source of IL‐5 in the human intestinal mucosa.

Cancer Cells Regulate Biomechanical Properties of Human Microvascular Endothelial Cells

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1high cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs.