Reticulated hyaluronan hydrogels: a model for examining cancer cell invasion in 3D.

The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. The roles of HA cell surface receptors and hyaluronidases (HAses) in this process are still controversial. In order to investigate their involvement in cancer pathogenesis, we developed a reticulated HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied thirteen cell lines, from primary tumors or metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. The number of colonies, which reflects tumor cell invasiveness, ranged from 7 to 193 after 5 days of culture. Invasion was dependent on the production of HAse as well as other factors. Optimal colonization occurred when cells released HAse, lacked HA-binding sites and did not secrete HA. Moreover, we describe for the first time a HAse activity at physiological pH that may be responding to the confinement of the enzyme in a three-dimensional structure. We show here that this reticulated matrix provides a three-dimensional model for investigating mechanisms involved in malignant invasion.     

Hyaluronan (HA) Interacting Proteins RHAMM and Hyaluronidase Impact Prostate Cancer Cell Behavior and Invadopodia Formation in 3D HA-Based Hydrogels

To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues.     

Quantitative analysis of the effect of cancer invasiveness and collagen concentration on 3D matrix remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness.     

A Highlights from MBoC Selection: Soft matrix is a natural stimulator for cellular invasiveness

Directional mesenchymal cell invasion in vivo is understood to be a stimulated event and to be regulated by cytokines, chemokines, and types of extracellular matrix (ECM). Instead, by focusing on the cellular response to ECM stiffness, we found that soft ECM (low stiffness) itself is sufficient to prevent stable cell-to-cell adherens junction formation, up-regulate matrix metalloproteinase (MMP) secretion, promote MMP activity, and induce invadosome-like protrusion (ILP) formation. Consistently, similar ILP formation was also detected in a three-dimensional directional invasion assay in soft matrix. Primary human fibroblasts spontaneously form ILPs in a very narrow range of ECM stiffness (0.1–0.4 kPa), and such ILP formation is Src family kinase dependent. In contrast, spontaneous ILP formation in malignant cancer cells and fibrosarcoma cells occurs across a much wider range of ECM stiffness, and these tumor cell ILPs are also more prominent at lower stiffness. These findings suggest that ECM softness is a natural stimulator for cellular invasiveness.     

A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology

Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM) in three dimensional (3D) space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA) scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM) hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.     

Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens

Migration and tube formation of endothelial cells are important in angiogenesis and require a coordinated response to the extra-cellular matrix (ECM) and growth factor. Since focal adhesion kinase (FAK) integrates signals from both ECM and growth factor, we investigated its role in angiogenesis. Type I and II collagens are fibril-forming collagens and stimulate human umbilical vein endothelial cells (HUVECs) to form tube structure. Although knockdown of FAK restrained cell motility and resulted in inhibition of tube formation, FAK degradation and tube formation occurred simultaneously after incubation with fibril-forming collagens. The compensation for the FAK degradation by a calpain inhibitor or transient over-expression of FAK resulted in disturbance of tube formation. These phenomena are specific to fibril-forming collagens and mediated via α2β1 integrin. In conclusion, our data indicate that FAK is functioning in cell migration, but fibril-forming collagen-induced FAK degradation is necessary for endothelial tube formation.     

Defining the role of matrix compliance and proteolysis in three-dimensional cell spreading and remodeling

Recent studies have identified extracellular matrix (ECM) compliance as an influential factor in determining the fate of anchorage-dependent cells. We explore a method of examining the influence of ECM compliance on cell morphology and remodeling in three-dimensional culture. For this purpose, a biological ECM analog material was developed to pseudo-independently alter its biochemical and physical properties. A set of 18 material variants were prepared with shear modulus ranging from 10 to 700 Pa. Smooth muscle cells were encapsulated in these materials and time-lapse video microscopy was used to show a relationship between matrix modulus, proteolytic biodegradation, cell spreading, and cell compaction of the matrix. The proteolytic susceptibility of the matrix, the degree of matrix compaction, and the cell morphology were quantified for each of the material variants to correlate with the modulus data. The initial cell spreading into the hydrogel matrix was dependent on the proteolytic susceptibility of the materials, whereas the extent of cell compaction proved to be more correlated to the modulus of the material. Inhibition of matrix metalloproteinases profoundly affected initial cell spreading and remodeling even in the most compliant materials. We concluded that smooth muscle cells use proteolysis to form lamellipodia and tractional forces to contract and remodel their surrounding microenvironment. Matrix modulus can therefore be used to control the extent of cellular remodeling and compaction. This study further shows that the interconnection between matrix modulus and proteolytic resistance in the ECM may be partly uncoupled to provide insight into how cells interpret their physical three-dimensional microenvironment.     

3D Traction forces in cancer cell invasion

Cell invasion through a dense three-dimensional (3D) matrix is believed to depend on the ability of cells to generate traction forces. To quantify the role of cell tractions during invasion in 3D, we present a technique to measure the elastic strain energy stored in the matrix due to traction-induced deformations. The matrix deformations around a cell were measured by tracking the 3D positions of fluorescent beads tightly embedded in the matrix. The bead positions served as nodes for a finite element tessellation. From the strain in each element and the known matrix elasticity, we computed the local strain energy in the matrix surrounding the cell. We applied the technique to measure the strain energy of highly invasive MDA-MB-231 breast carcinoma and A-125 lung carcinoma cells in collagen gels. The results were compared to the strain energy generated by non-invasive MCF-7 breast and A-549 lung carcinoma cells. In all cases, cells locally contracted the matrix. Invasive breast and lung carcinoma cells showed a significantly higher contractility compared to non-invasive cells. Higher contractility, however, was not universally associated with higher invasiveness. For instance, non-invasive A-431 vulva carcinoma cells were the most contractile cells among all cell lines tested. As a universal feature, however, we found that invasive cells assumed an elongated spindle-like morphology as opposed to a more spherical shape of non-invasive cells. Accordingly, the distribution of strain energy density around invasive cells followed patterns of increased complexity and anisotropy. These results suggest that not so much the magnitude of traction generation but their directionality is important for cancer cell invasion.     

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.     

Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear–centrosomal orientation and lumen initiation during 3D epithelial morphogenesis.     

Nanoscale imaging and quantification of local proteolytic activity

Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7-clone) were seeded on fluorescent gelatin or collagen-IV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFM-tip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, three-dimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (-76.6%), matrix volume (-46.6%), and height (-38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFM-scanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagen-IV by A7-cells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples.     

Engineering strategies to recapitulate epithelial morphogenesis within synthetic three-dimensional extracellular matrix with tunable mechanical properties

The mechanical properties (e.g. stiffness) of the extracellular matrix (ECM) influence cell fate and tissue morphogenesis and contribute to disease progression. Nevertheless, our understanding of the mechanisms by which ECM rigidity modulates cell behavior and fate remains rudimentary. To address this issue, a number of two and three-dimensional (3D) hydrogel systems have been used to explore the effects of the mechanical properties of the ECM on cell behavior. Unfortunately, many of these systems have limited application because fiber architecture, adhesiveness and/or pore size often change in parallel when gel elasticity is varied. Here we describe the use of ECM-adsorbed, synthetic, self-assembling peptide (SAP) gels that are able to recapitulate normal epithelial acini morphogenesis and gene expression in a 3D context. By exploiting the range of viscoelasticity attainable with these SAP gels, and their ability to recreate native-like ECM fibril topology with minimal variability in ligand density and pore size, we were able to reconstitute normal and tumor-like phenotypes and gene expression patterns in nonmalignant mammary epithelial cells. Accordingly, this SAP hydrogel system presents the first tunable system capable of independently assessing the interplay between ECM stiffness and multi-cellular epithelial phenotype in a 3D context.     

Effects of epidermal growth factor on fibroblast migration through biomimetic hydrogels

We have previously reported on the development and use of synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study the mechanisms of migration. These biomimetic hydrogels consist of bioinert poly(ethylene glycol) diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesion peptide sequences grafted into the network. Cells adhere to the hydrogel via interaction between the grafted adhesion ligands and receptors on the cell surface. The cells migrate through the three-dimensional system by secreting the appropriate proteolytic enzymes, which are involved in cell migration and are targeted to the peptide sequences incorporated in the backbone of the polymer. It was observed that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. In this study, we demonstrate that we can covalently attach epidermal growth factor (EGF) to PEG and graft them into the hydrogels. It was observed that EGF when tethered maintained mitogenic activity. It was also observed that fibroblast migration significantly increased in the presence of the grafted EGF through the collagenase-sensitive hydrogels. In addition, the increase in migration was found to be independent from the proliferative response of the cells. These synthetic ECM analogues allow one to systematically control identities and concentrations of biomolecules and are useful tools to study mechanisms of cell migration.     

Signaling by discoidin domain receptor 1 in cancer metastasis

ABSTRACT

Collagen is the most abundant component of tumor extracellular matrix (ECM). ECM collagens are known to directly interact with the tumor cells via cell surface receptor and play crucial role in tumor cell survival and promote tumor progression. Collagen receptor DDR1 is a member of receptor tyrosine kinase (RTK) family with a unique motif in the extracellular domain resembling Dictyostelium discoideum protein discoidin-I. DDR1 displays delayed and sustained activation upon interaction with collagen and recent findings have demonstrated that DDR1-collagen signaling play important role in cancer progression. In this review, we discuss the current knowledge on the role of DDR1 in cancer metastasis and possibility of a potential therapeutic approach of DDR1 targeted therapy in cancer.     

Matrix hyaluronan alters epidermal growth factor receptor-dependent cell morphology

EGFR, a critical regulator of oncogenic signaling during cancer progression, is capable of integrating multireceptor signaling pathways that promote metastasis. EGFR is subject to regulatory cues from the extracellular matrix (ECM), of which hyaluronan (HA) is a major component. In mammary tumors, HA is deposited in the ECM where it functions in biomechanical support and modulates intracellular signaling. We utilized a 3D collagen system in which HA is either polymerized in collagen matrix or provided soluble in the media (sHA). Here we report that collagen-embedded HA (eHA) inhibits EGFR activation, filopodia formation, and cell spreading on a collagen matrix. These findings demonstrate a novel role for eHA as a protective molecule when encountered in the collagen matrix during cancer progression.     

Contractile forces contribute to increased glycosylphosphatidylinositol-anchored receptor CD24-facilitated cancer cell invasion

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.     

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.