Liver Cancer Vascularity Driven by Extracellular Matrix Stiffness: Implications for Imaging Research.

BACKGROUND: Extracellular matrix stiffness represents a barrier to effective local and systemic drug delivery. Increasing stiffness disrupts newly formed vessel architecture and integrity, leading to tumor-like vasculature. The resulting vascular phenotypes are manifested through different cross-sectional imaging features. Contrast-enhanced studies can help elucidate the interplay between liver tumor stiffness and different vascular phenotypes. PURPOSE: This study aims to correlate extracellular matrix stiffness, dynamic contrast-enhanced computed tomography, and dynamic contrast-enhancement ultrasound imaging features of 2 rat hepatocellular carcinoma tumor models. METHODS AND MATERIALS: Buffalo-McA-RH7777 and Sprague Dawley (SD)-N1S1 tumor models were used to evaluate tumor stiffness by 2-dimensional shear wave elastography, along with tumor perfusion by dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography. Atomic force microscopy was used to calculate tumor stiffness at a submicron scale. Computer-aided image analyses were performed to evaluate tumor necrosis, as well as the percentage, distribution, and thickness of CD34+ blood vessels. RESULTS: Distinct tissue signatures between models were observed according to the distribution of the stiffness values by 2-dimensional shear wave elastography and atomic force microscopy ( P < 0.05). Higher stiffness values were attributed to SD-N1S1 tumors, also associated with a scant microvascular network ( P ≤ 0.001). Opposite results were observed in the Buffalo-McA-RH7777 model, exhibiting lower stiffness values and richer tumor vasculature with predominantly peripheral distribution ( P = 0.03). Consistent with these findings, tumor enhancement was significantly greater in the Buffalo-McA-RH7777 tumor model than in the SD-N1S1 on both dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography ( P < 0.005). A statistically significant positive correlation was observed between tumor perfusion on dynamic contrast-enhanced ultrasonography and contrast-enhanced computed tomography in terms of the total area under the curve and % microvessel tumor coverage ( P < 0.05). CONCLUSIONS: The stiffness signatures translated into different tumor vascular phenotypes. Two-dimensional shear wave elastography and dynamic contrast-enhanced ultrasonography adequately depicted different stromal patterns, which resulted in unique imaging perfusion parameters with significantly greater contrast enhancement observed in softer tumors.

Noncanonical Wnt/Ror2 signaling regulates cell-matrix adhesion to prompt directional tumor cell invasion in breast cancer.

Cell-extracellular matrix (ECM) interactions represent fundamental exchanges during tumor progression, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic spread during metastasis remains elusive. We demonstrate that the noncanonical Wnt receptor, Ror2, regulates tumor cell-driven matrix remodeling and invasion in breast cancer. Ror2 loss-of-function (LOF) triggers the disruption of E-cadherin within tumor cells, accompanied by an increase in tumor cell invasion and collagen realignment in three-dimensional cultures. RNA sequencing of Ror2-deficient organoids further uncovered alterations in actin cytoskeleton, cell adhesion, and collagen cross-linking gene expression programs. Spatially, we pinpoint the up-regulation and redistribution of α5 and ß3 integrins together with the production of fibronectin in areas of invasion downstream of Ror2 loss. Wnt/ß-catenin-dependent and Wnt/Ror2 alternative Wnt signaling appear to regulate distinct functions for tumor cells regarding their ability to modify cell-ECM exchanges during invasion. Furthermore, blocking either integrin or focal adhesion kinase (FAK), a downstream mediator of integrin-mediated signal transduction, abrogates the enhanced migration observed upon Ror2 loss. These results reveal a critical function for the alternative Wnt receptor, Ror2, as a determinant of tumor cell-driven ECM exchanges during cancer invasion and metastasis.

Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis.

Collagen remodeling contributes to many physiologic and pathologic processes. In primary tumors, the linearization of collagen fibers promotes cancer cell invasion and metastasis and is indicative of poor prognosis. However, it remains unknown whether there are endogenous inhibitors of collagen linearization that could be exploited therapeutically. Here, we show that collagen linearization is controlled by two secreted matricellular proteins with antagonistic functions. Specifically, WISP1 was secreted by cancer cells, bound to type I collagen (Col I), and linearized Col I via its cysteine-rich C-terminal (CT) domain. In contrast, WISP2, which lacks a CT domain, inhibited Col I linearization by preventing WISP1-Col I binding. Analysis of patient data revealed that WISP2 expression is lower in most solid tumors, in comparison with normal tissues. Consequently, genetic or pharmacologic restoration of higher WISP2 levels impaired collagen linearization and prevented tumor cell invasion and metastasis in vivo in models of human and murine breast cancer. Thus, this study uncovers WISP2 as the first inhibitor of collagen linearization ever identified and reveals that collagen architecture can be normalized and metastasis inhibited by therapeutically restoring a high WISP2:WISP1 ratio. SIGNIFICANCE: Two secreted factors, WISP1 and WISP2, antagonistically regulate collagen linearization, and therapeutically increasing the WISP2:WISP1 ratio in tumors limits collagen linearization and inhibits metastasis.See related commentary by Barcus and Longmore, p. 5611.

Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues.

Extrusion is a mechanism used to eliminate unfit, excess, or dying cells from epithelial tissues. The initial events guiding which cells will be selectively extruded from the epithelium are not well understood. Here, we induced damage in a subset of epithelial cells in the developing zebrafish and used time-lapse imaging to examine cell and cytoskeletal dynamics leading to extrusion. We show that cell extrusion is preceded by actomyosin contractions that are pulsatile. Our data show that pulsatile contractions are induced by a junctional to medial re-localization of myosin. Analysis of cell area during contractions revealed that cells pulsing with the longest duration and highest amplitude undergo progressive area loss and extrude. Although pulses were driven by local increases in tension, damage to many cells promoted an overall decrease in the tensile state of the epithelium. We demonstrate that caspase activation leads to sphingosine-1-phosphate enrichment that controls both tissue tension and pulses to dictate areas of extrusion. These data suggest that the kinetics of pulsatile contractions define a key behavioral difference between extruding and non-extruding cells and are predictive of extrusion. Altogether, our study provides mechanistic insight into how localized changes in physical forces are coordinated to remove defective cells for homeostatic maintenance of living epithelial tissues.

Mechanically tunable coaxial electrospun models of YAP/TAZ mechanoresponse and IGF-1R activation in osteosarcoma.

Current in vitro methods for assessing cancer biology and therapeutic response rely heavily on monolayer cell culture on hard, plastic surfaces that do not recapitulate essential elements of the tumor microenvironment. While a host of tumor models exist, most are not engineered to control the physical properties of the microenvironment and thus may not reflect the effects of mechanotransduction on tumor biology. Utilizing coaxial electrospinning, we developed three-dimensional (3D) tumor models with tunable mechanical properties in order to elucidate the effects of substrate stiffness and tissue architecture in osteosarcoma. Mechanical properties of coaxial electrospun meshes were characterized with a series of macroscale testing with uniaxial tensile testing and microscale testing utilizing atomic force microscopy on single fibers. Calculated moduli in our models ranged over three orders of magnitude in both macroscale and microscale testing. Osteosarcoma cells responded to decreasing substrate stiffness in 3D environments by increasing nuclear localization of Hippo pathway effectors, YAP and TAZ, while downregulating total YAP. Additionally, a downregulation of the IGF-1R/mTOR axis, the target of recent clinical trials in sarcoma, was observed in 3D models and heralded increased resistance to combination chemotherapy and IGF-1R/mTOR targeted agents compared to monolayer controls. In this study, we highlight the necessity of incorporating mechanical cues in cancer biology investigation and the complexity in mechanotransduction as a confluence of stiffness and culture architecture. Our models provide a versatile, mechanically variable substrate on which to study the effects of physical cues on the pathogenesis of tumors. STATEMENT OF SIGNIFICANCE: The tumor microenvironment plays a critical role in cancer pathogenesis. In this work, we engineered 3D, mechanically tunable, coaxial electrospun environments to determine the roles of the mechanical environment on osteosarcoma cell phenotype, morphology, and therapeutic response. We characterize the effects of varying macroscale and microscale stiffnesses in 3D environments on the localization and expression of the mechanoresponsive proteins, YAP and TAZ, and evaluate IGF-1R/mTOR pathway activation, a target of recent clinical trials in sarcoma. Increased nuclear YAP/TAZ was observed as stiffness in 3D was decreased. Downregulation of the IGF-1R/mTOR cascade in all 3D environments was observed. Our study highlights the complexity of mechanotransduction in 3D culture and represents a step towards controlling microenvironmental elements in in vitro cancer investigations.

Membrane curvature sensing of the lipid-anchored K-Ras small GTPase.

Plasma membrane (PM) curvature defines cell shape and intracellular organelle morphologies and is a fundamental cell property. Growth/proliferation is more stimulated in flatter cells than the same cells in elongated shapes. PM-anchored K-Ras small GTPase regulates cell growth/proliferation and plays key roles in cancer. The lipid-anchored K-Ras form nanoclusters selectively enriched with specific phospholipids, such as phosphatidylserine (PS), for efficient effector recruitment and activation. K-Ras function may, thus, be sensitive to changing lipid distribution at membranes with different curvatures. Here, we used complementary methods to manipulate membrane curvature of intact/live cells, native PM blebs, and synthetic liposomes. We show that the spatiotemporal organization and signaling of an oncogenic mutant K-Ras G12V favor flatter membranes with low curvature. Our findings are consistent with the more stimulated growth/proliferation in flatter cells. Depletion of endogenous PS abolishes K-Ras G12V PM curvature sensing. In cells and synthetic bilayers, only mixed-chain PS species, but not other PS species tested, mediate K-Ras G12V membrane curvature sensing. Thus, K-Ras nanoclusters act as relay stations to convert mechanical perturbations to mitogenic signaling.

The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

Collagen linearization is a hallmark of aggressive tumors and a key pathogenic event that promotes cancer cell invasion and metastasis. Cell-generated mechanical tension has been proposed to contribute to collagen linearization in tumors, but it is unknown whether other mechanisms play prominent roles in this process. Here, we show that the secretome of cancer cells is by itself able to induce collagen linearization independently of cell-generated mechanical forces. Among the tumor cell-secreted factors, we find a key role in this process for the matricellular protein WISP1 (CCN4). Specifically, WISP1 directly binds to type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors. Consequently, WISP1-induced type I collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis, without significantly affecting gene expression. Furthermore, higher WISP1 expression in tumors from cancer patients correlates with faster progression to metastatic disease and poor prognosis. Altogether, these findings reveal a conceptually novel mechanism whereby pro-metastatic collagen linearization critically depends on a cancer cell-secreted factor.

Sucrose modulation of radiofrequency-induced heating rates and cell death.

BACKGROUND: Applied radiofrequency (RF) energy induces hyperthermia in tissues, facilitating vascular perfusion This study explores the impact of RF radiation on the integrity of the luminal endothelium, and then predominately explores the impact of altering the conductivity of biologically-relevant solutions on RF-induced heating rates and cell death. The ability of cells to survive high sucrose (i.e. hyperosmotic conditions) to achieve lower conductivity as a mechanism for directing hyperthermia is evaluated. METHODS: RF radiation was generated using a capacitively-coupled radiofrequency system operating at 13.56 MHz. Temperatures were recorded using a FLIR SC 6000 infrared camera. RESULTS: RF radiation reduced cell-to-cell connections among endothelial cells and altered cell morphology towards a more rounded appearance at temperatures reported to cause in vivo vessel deformation. Isotonic solutions containing high sucrose and low levels of NaCl displayed low conductivity and faster heating rates compared to high salt solutions. Heating rates were positively correlated with cell death. Addition of sucrose to serum similarly reduced conductivity and increased heating rates in a dose-dependent manner. Cellular proliferation was normal for cells grown in media supplemented with 125 mM sucrose for 24 hours or for cells grown in 750 mM sucrose for 10 minutes followed by a 24 h recovery period. CONCLUSIONS: Sucrose is known to form weak hydrogen bonds in fluids as opposed to ions, freeing water molecules to rotate in an oscillating field of electromagnetic radiation and contributing to heat induction. The ability of cells to survive temporal exposures to hyperosmotic (i.e. elevated sucrose) conditions creates an opportunity to use sucrose or other saccharides to selectively elevate heating in specific tissues upon exposure to a radiofrequency field.

A cost-effective method to immobilize hydrated soft-tissue samples for atomic force microscopy.

Immobilizing hydrated soft tissue specimens for atomic force microscopy (AFM) is a challenge. Here, we describe a simple and very cost-effective immobilization method, based on the use of transglutaminase in an aqueous environment, and successfully apply it to AFM characterization of human native Wharton's Jelly (nWJ), the gelatinous connective tissue matrix of the umbilical cord. A side-by-side comparison with a widely used polyphenolic protein-based tissue adhesive (Corning Cell-Tak), which is known to bind strongly to virtually all inorganic and organic surfaces in aqueous environments, shows that both adhesives successfully immobilize nWJ in its physological hydrated state. The cost of transglutaminase, however, is over 3000-fold lower than that of Cell-Tak, making it a very attractive method for immobilizing soft tissues for AFM characterization.

Use of non-contact hopping probe ion conductance microscopy to investigate dynamic morphology of live platelets.

Circulating platelets are anucleated and multi-functional cells that participate in hemostasis and arterial thrombosis. Multiple ligands and mechanical forces activate platelets, leading to cytoskeletal rearrangement and dramatic shape-changes. Such dramatic changes in platelets membrane structures are commonly detected by optical and electron microscopy after platelets are fixed. We have recently developed a method to study the membrane morphology of live platelets using Hopping Probe Ion Conductance Microscopy (HPICM). We have successfully used this technology to study the process of platelet microvesiculation upon exposure to selective agonists. Here, we further discussed technical details of using HPICM to study platelet biology and compared results from HPICM to those from conventional atomic force microscopy and scanning electron microscopy. This method offers several advantages over current technologies. First, it monitors morphological changes of platelets in response to agonists in real time. Second, platelets can be repeatedly scanned over time without damages brought by heat and prolong light exposure. Third, there is no direct contact with platelet surface so that there will no or minimal mechanical damages brought by a cantilever of a conventional atomic force microscopy. Finally, it offers the potential to study platelet membrane ion channels, which have been technically challenging up-to-date. Our data show that HPICM has high-resolution in delineating changes of platelet morphology in response to stimulations and could help to unravel the complex role of platelet in thrombus formation.

Contact- and agonist-regulated microvesiculation of human platelets.

After exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets.We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbß3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.

Biological Atomic Force Microscopy for Imaging Gold-Labeled Liposomes on Human Coronary Artery Endothelial Cells.

Although atomic force microscopy (AFM) has been used extensively to characterize cell membrane structure and cellular processes such as endocytosis and exocytosis, the corrugated surface of the cell membrane hinders the visualization of extracellular entities, such as liposomes, that may interact with the cell. To overcome this barrier, we used 90 nm nanogold particles to label FITC liposomes and monitor their endocytosis on human coronary artery endothelial cells (HCAECs) in vitro. We were able to study the internalization process of gold-coupled liposomes on endothelial cells, by using AFM. We found that the gold-liposomes attached to the HCAEC cell membrane during the first 15-30 min of incubation, liposome cell internalization occurred from 30 to 60 min, and most of the gold-labeled liposomes had invaginated after 2 hr of incubation. Liposomal uptake took place most commonly at the periphery of the nuclear zone. Dynasore monohydrate, an inhibitor of endocytosis, obstructed the internalization of the gold-liposomes. This study showed the versatility of the AFM technique, combined with fluorescent microscopy, for investigating liposome uptake by endothelial cells. The 90 nm colloidal gold nanoparticles proved to be a noninvasive contrast agent that efficiently improves AFM imaging during the investigation of biological nanoprocesses.

Ion currents induced by ATP and angiotensin II in cultured follicular cells of Xenopus laevis.

Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at -60 mV revealed that ATP (20-250 µM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ~60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ~5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at -60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F(in) and S(in)-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using whole-cell recordings, 1 µM Ang II yielded smooth inward currents of 157 ± 45 pA in ~16% of the FC held at -60 mV. The net Ang II response, mediated by the activation of the AT(1) receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes.

Modulation of syndecan-1 shedding after hemorrhagic shock and resuscitation.

The early use of fresh frozen plasma as a resuscitative agent after hemorrhagic shock has been associated with improved survival, but the mechanism of protection is unknown. Hemorrhagic shock causes endothelial cell dysfunction and we hypothesized that fresh frozen plasma would restore endothelial integrity and reduce syndecan-1 shedding after hemorrhagic shock. A prospective, observational study in severely injured patients in hemorrhagic shock demonstrated significantly elevated levels of syndecan-1 (554±93 ng/ml) after injury, which decreased with resuscitation (187±36 ng/ml) but was elevated compared to normal donors (27±1 ng/ml). Three pro-inflammatory cytokines, interferon-γ, fractalkine, and interleukin-1ß, negatively correlated while one anti-inflammatory cytokine, IL-10, positively correlated with shed syndecan-1. These cytokines all play an important role in maintaining endothelial integrity. An in vitro model of endothelial injury then specifically examined endothelial permeability after treatment with fresh frozen plasma orlactated Ringers. Shock or endothelial injury disrupted junctional integrity and increased permeability, which was improved with fresh frozen plasma, but not lactated Ringers. Changes in endothelial cell permeability correlated with syndecan-1 shedding. These data suggest that plasma based resuscitation preserved endothelial syndecan-1 and maintained endothelial integrity, and may help to explain the protective effects of fresh frozen plasma after hemorrhagic shock.

Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy.

TNF-α (tumor necrosis factor-α) is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL) in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1) mechanical properties, employing atomic force microscopy; 2) cytoskeletal organization, through fluorescence microscopy; and 3) membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h), for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells); the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with controls. Collectively, these results demonstrate that sufficiently high levels of circulating TNF-α have similar effects on different endothelial districts, and provide additional information for unraveling the possible correlations between circulating pro-inflammatory cytokines and systemic vascular dysfunction.

Cellular association and assembly of a multistage delivery system.

The realization that blood-borne delivery systems must overcome a multiplicity of biological barriers has led to the fabrication of a multistage delivery system (MDS) designed to temporally release successive stages of particles or agents to conquer sequential barriers, with the goal of enhancing delivery of therapeutic and diagnostic agents to the target site. In its simplest form, the MDS comprises stage-one porous silicon microparticles that function as carriers of second-stage nanoparticles. Cellular uptake of nontargeted discoidal silicon microparticles by macrophages is confirmed by electron and atomic force microscopy (AFM). Using superparamagnetic iron oxide nanoparticles (SPIONs) as a model of secondary nanoparticles, successful loading of the porous matrix of silicon microparticles is achieved, and retention of the nanoparticles is enhanced by aminosilylation of the loaded microparticles with 3-aminopropyltriethoxysilane. The impact of silane concentration and reaction time on the nature of the silane polymer on porous silicon is investigated by AFM and X-ray photoelectron microscopy. Tissue samples from mice intravenously administered the MDS support co-localization of silicon microparticles and SPIONs across various tissues with enhanced SPION release in spleen, compared to liver and lungs, and enhanced retention of SPIONs following silane capping of the MDS. Phantom models of the SPION-loaded MDS display negative contrast in magnetic resonance images. In addition to forming a cap over the silicon pores, the silane polymer provides free amines for antibody conjugation to the microparticles, with both VEGFR-2- and PECAM-specific antibodies leading to enhanced endothelial association. This study demonstrates the assembly and cellular association of a multiparticle delivery system that is biomolecularly targeted and has potential for applications in biological imaging.