Direct Cell-cell Interactions Research Articles

Bone-stromal cells up-regulate tumourigenic markers in a tumour-stromal 3D model of prostate cancer

BackgroundThe cellular and molecular mechanisms that mediate interactions between tumour cells and the surrounding bone stroma are to date largely undetermined in prostate cancer (PCa) progression. The purpose of this study was to evaluate the role of alpha 6 and beta 1 integrin subunits in mediating tumour-stromal interactions.MethodsUtilising 3D in vitro assays we evaluated and compared 1. Monocultures of prostate metastatic PC3, bone stromal derived HS5 and prostate epithelial RWPE-1 cells and 2. Tumour-stromal co-cultures (PC3 + HS5) to ascertain changes in cellular phenotype, function and expression of metastatic markers.ResultsIn comparison to 3D monocultures of PC3 or HS5 cells, when cultured together, these cells displayed up-regulated invasive and proliferative qualities, along with altered expression of epithelial-to-mesenchymal and chemokine protein constituents implicated in metastatic dissemination. When co-cultured, HS5 cells were found to re-express N-Cadherin and chemokine receptor CXCR7. Alterations in N-Cadherin expression were found to be mediated by soluble factors secreted by PC3 tumour cells, while chemokine receptor re-expression was dependent on direct cell-cell interactions. We have also shown that integrins beta 1 and alpha 6 play an integral role in maintaining cell homeostasis and mediating expression of E-Cadherin, N-Cadherin and vimentin, in addition to chemokine receptor CXCR7.ConclusionsCollectively our results suggest that both PC3 and HS5 cells provide a “protective” and reciprocal milieu that promotes tumour growth. As such 3D co-cultures may serve as a more complex and valid biological model in the drug discovery pipeline.

Abstract 130: Secretion of Angiogenic and Anti-apoptotic Factors Accompanies Mesenchymal Stem Cell-mediated Enhancement of Contractile Function in Engineered Cardiac Tissues

Previous studies using three-dimensional engineered cardiac tissues (ECT) demonstrated beneficial effects of mesenchymal stem cells (MSC) on contractile function, recapitulating findings in animal studies and in clinical trials. But the mechanisms by which MSC effect this functional enhancement remain unclear. This study tested the hypothesis that MSC-mediated enhancement of ECT function involves paracrine signaling, independent of direct cell-cell interaction with cardiomyocytes (CM). To create ECT, CM were isolated from neonatal rat ventricles, mixed with 2.0 mg/ml bovine type I collagen and 0.9 mg/ml Matrigel, and pipetted into a custom elastomeric mold with integrated force sensing end-posts. Two types of ECT were created: one containing 1.5 million CM (“CM-only”) and the other containing 1.5 million CM supplemented with 150,000 MSC (“MSC-CM hybrid”). Contractile function of CM-only ECT was then assessed during 2-Hz pacing either in the presence or absence of hybrid tissues sharing the same media. Media surrounding the tissues was also collected and analyzed via protein microarray (RayBiotech) combined with Ingenuity Pathway Analysis software. CM-only ECT cultured for 5 days showed aligned and compacted structure with a cross-sectional area of 0.39 ± 0.03 mm 2 and a twitch force of 4.7 ± 0.7 N (mean ± SD; n = 6). In CM-only tissues co-cultured for 5 days in shared media alongside hybrid tissues, cross-sectional area was unchanged (0.42 ± 0.02 mm 2 , p = 0.17, n=3), but twitch force increased two-fold to 9.8 ± 6.5 N (p = 0.08). Threshold voltage for electrical pacing of CM-only ECT also decreased from 0.54 ± 0.12 V/mm to 0.39 ± 0.07 V/mm when co-cultured with hybrid ECT (p = 0.097). Protein microarray analysis of the shared co-culture conditioned media showed enrichment (relative to CM-only conditioned media) of the angiogenic factors VEGF (1240x) and IL-1 (8.6x) as well as the cardiac anti-apoptotic/pro-survival factors TNF (194x), bFGF (55x), and bNGF (14.9x). In conclusion, these findings support paracrine signaling, independent of direct cell-cell contact, as one mechanism of MSC-mediated enhancement of CM function in rat ECT. Identification and isolation of defined cardiotropic factors may lead to novel molecular therapies for cardiac repair.

Myeloproliferative Neoplasia Remodels the Endosteal Bone Marrow Niche into a Self-Reinforcing Leukemic Niche

Multipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function, and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for thrombopoietin, CCL3, and direct cell-cell interactions in driving OBC expansion,and for changes in TGF-β, Notch, and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of MPN-affected patients and prevention of myelofibrosis.

SAT0029 Mesenchymal stem cells modulate in vitro fibroblast activity in systemic sclerosis

BackgroundBone marrow (BM)-derived mesenchymal stem cells (MSC) are currently under investigation for their potential role in the management of some autoimmune diseases. Besides their regenerative effects and ability to induce...

Ozone exposed epithelial cells modify cocultured natural killer cells

Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs.

CD28-Mediated Pro-Survival Signaling in Multiple Myeloma

AbstractAbstract 941Multiple myeloma (MM) is an incurable neoplasm of plasma cells which is critically dependent on the bone marrow for its survival. The microenvironment is able to support MM cells through a variety of mechanisms, including direct cell-cell interactions. Therefore, identifying and targeting these interactions or their downstream pathways would represent a novel mechanism for the treatment of MM. Unfortunately, many of these interactions remain poorly characterized. However, it stands to reason that any receptor or ligand which correlates with worse prognosis or disease progression could be a pro-survival molecule. One such receptor is CD28. Although best known as the prototypic T cell costimulatory receptor, CD28 has been shown to be expressed on normal plasma cells as well as MM cells. Additionally, we have previously shown that CD28 signaling is critical for both plasma cell and MM cell survival.To determine if CD28 signaling is important in the context of complex cell-cell interactions, we cultured MM cell line cells (MM1S) with monocyte-derived human dendritic cells (DC). We and others have previously shown that DCs infiltrate myelomatous portions of bone marrow and contribute to the clonogenicity of MM cells. Importantly, DCs also present the ligands for CD28, CD80 and CD86. To examine whether DCs can protect MM cells in a CD28-dependent fashion, we co-cultured the cells in the presence of melphalan, a clinically relevant chemotherapy. DCs were able to significantly increase the survival of MM cells (p<0.01) compared to MM cells alone; however, when CD28 was blocked (using a blocking antibody, CD28.6) or when CD80 and CD86 were blocked (using the fusion protein CTLA4-Ig), DC-mediated protection was completely abrogated (figure 1). These results demonstrate that even in complex MM-DC interactions, CD28 is the critically important pro-survival signaling molecule.In T cell biology, one of the critical signaling pathways downstream of CD28 is via PI3K and Akt. We have previously shown in MM cells that activating CD28 with an antibody causes the phosphorylation of PI3K. To determine whether or not this activation is indeed linked to survival, we cultured MM1S cells with a CD28 activating antibody ± serum and ± increasing doses of the PI3K inhibitor LY294002. As expected, when MM cells were cultured with a CD28 activating antibody, they survived the no-serum conditions. However, that survival was abrogated by LY294002 in a dose-dependent fashion, indicating that CD28 is mediating survival via PI3K activation. To determine whether Akt was the involved downstream molecule, the above experiment was repeated with an Akt inhibitor. Again, we observed that MM cells were protected by CD28 in the absence of serum, but that protection was abrogated by inhibiting Akt in a dose-dependent fashion. These experiments demonstrate that CD28 is delivering a pro-survival signal via PI3K-Akt.It has been well established in the myeloma literature that the balance of the pro-apoptotic molecule Bim determines apoptosis in MM cells. Importantly, Bim is transcriptionally regulated by the PI3K-Akt-FoxO3a axis. When FoxO3a is phosphorylated by Akt, it is excluded from the nucleus and is unable to upregulate Bim transcripts. To determine if CD28 was regulating FoxO3a, we cultured MM cells with a CD28 activating antibody and examined phospho-FoxO3a via western blot. Indeed, we observed that CD28 activation increases levels of phospho-FoxO3a. We next wanted to test whether or not CD28 activation had any effect on Bim transcript levels. We have observed via western blot and RT-PCR that CD28 activation suppresses Bim expression, and CD28 blockade increases Bim expression. To confirm that this plays a functional role in MM biology, we knocked down Bim using siRNA and cultured the cells without serum + CTLA4-Ig. Consistent with our expectations, when we culture control cells in the absence serum plus CD28 blockade, they die. However, when we knock down Bim, we see that the cells are resistant to CD28 blockade-mediated cell death. These data suggest that CD28 is able to regulate Bim via the PI3K-Akt-FoxO3a axis, and that Bim is critical for MM cell death or survival (figure 2).Taken together, these data demonstrate that CD28 is the critical pro-survival molecule even in the complex cell-cell interactions between MM cells and DCs. CD28 is mediating that prosurvival signal in a PI3K-Akt-FoxO3a-Bim dependent manner. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds

Stem cells obtained from amniotic fluid show high proliferative capacity in culture and multilineage differentiation potential. Because of the lack of significant immunogenicity and the ability of the amniotic fluid-derived stem (AFS) cells to modulate the inflammatory response, we investigated whether they could augment wound healing in a mouse model of skin regeneration. We used bioprinting technology to treat full-thickness skin wounds in nu/nu mice. AFS cells and bone marrow-derived mesenchymal stem cells (MSCs) were resuspended in fibrin-collagen gel and "printed" over the wound site. At days 0, 7, and 14, AFS cell- and MSC-driven wound closure and re-epithelialization were significantly greater than closure and re-epithelialization in wounds treated by fibrin-collagen gel only. Histological examination showed increased microvessel density and capillary diameters in the AFS cell-treated wounds compared with the MSC-treated wounds, whereas the skin treated only with gel showed the lowest amount of microvessels. However, tracking of fluorescently labeled AFS cells and MSCs revealed that the cells remained transiently and did not permanently integrate in the tissue. These observations suggest that the increased wound closure rates and angiogenesis may be due to delivery of secreted trophic factors, rather than direct cell-cell interactions. Accordingly, we performed proteomic analysis, which showed that AFS cells secreted a number of growth factors at concentrations higher than those of MSCs. In parallel, we showed that AFS cell-conditioned media induced endothelial cell migration in vitro. Taken together, our results indicate that bioprinting AFS cells could be an effective treatment for large-scale wounds and burns.

Cross talk between cardiac myocytes and fibroblasts: from multiscale investigative approaches to mechanisms and functional consequences

The heart is comprised of a syncytium of cardiac myocytes (CM) and surrounding nonmyocytes, the majority of which are cardiac fibroblasts (CF). CM and CF are highly interspersed in the myocardium with one CM being surrounded by one or more CF. Bidirectional cross talk between CM and CF plays important roles in determining cardiac mechanical and electrical function in both normal and diseased hearts. Genetically engineered animal models and in vitro studies have provided evidence that CM and CF can regulate each other's function. Their cross talk contributes to structural and electrical remodeling in both atria and ventricles and appears to be involved in the pathogenesis of various heart diseases that lead to heart failure and arrhythmia disorders. Mechanisms of CM-CF cross talk, which are not yet fully understood, include release of paracrine factors, direct cell-cell interactions via gap junctions and potentially adherens junctions and nanotubes, and cell interactions with the extracellular matrix. In this article, we provide an overview of the existing multiscale experimental and computational approaches for the investigation of cross talk between CM and CF and review recent progress in our understanding of the functional consequences and underlying mechanisms. Targeting cross talk between CM and CF could potentially be used therapeutically for the modulation of the cardiac remodeling response in the diseased heart and may lead to new strategies for the treatment of heart failure or rhythm disturbances.

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells.

Cadherin-related protein 24 induces morphological changes and partial cell polarization by facilitating direct cell-cell interactions

Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.

Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.

The Eph/Ephrin family in cancer metastasis: communication at the service of invasion

Cancer cells rely on intercellular communication throughout the different stages of their transformation and progression into metastasis. They do so by co-opting different processes such as cell-cell junctions, growth factors, receptors, and vesicular release. Initially characterized in neuronal and vascular tissues, Ephs and Ephrins, the largest family of receptor tyrosine kinases, comprised of two classes (i.e., A and B types), is increasingly scrutinized by cancer researchers. These proteins possess the particular features of both the receptors and ligands being membrane-bound which, via mandatory direct cell-cell interactions, undergo a bidirectional signal transduction initiated from both the receptor and the ligand. Following cell-cell interactions, Ephs/Ephrins behave as guidance molecules which trigger both repulsive and attractive signals, so as to direct the movement of cells through their immediate microenvironment. They also direct processes which include sorting and positioning and cytoskeleton rearrangements, thus making them perfect candidates for the control of the metastatic process. In fact, the role of Ephs and Ephrins in cancer progression has been demonstrated for many of the family members and they, surprisingly, have both tumor promoter and suppressor functions in different cellular contexts. They are also able to coordinate between multiple processes including cell survival, proliferation, differentiation, adhesion, motility, and invasion. This review is an attempt to summarize the data available on these Ephs/Ephrins' biological functions which contribute to the onset of aggressive cancers. I will also provide an overview of the factors which could explain the functional differences demonstrated by Ephs and Ephrins at different stages of tumor progression and whose elucidation is warranted for any future therapeutic targeting of this signaling pathway in cancer metastasis.

Potentiality of Immobilized Pig Hepatocyte Spheroids in Bioartificial Liver System

Various extracorporeal bioartificial liver (BAL) systems have been developed. To treat fulminant hepatic failure (FHF) patients. Direct cell-cell interaction is one of the major factors influencing the functions of cultured hepatocytes, which increase with progressing of cell aggregation in this study, we investigated the effects of plasma viability and function single and spheroid pig hepatocytes in vitro. Hepatocytes were cultured as spheroids by suspension culture in spinner flasks. We obtained pig plasma from animals in hepatic failure. Immobilized single pig hepatocytes exposed to the toxic pig plasma lost viability and liver function. However, immobilized pig hepatocyte spheroids showed stable ammonia removal functions and urea synthesis and lower lactate dehydrogenase, glutamine oxaloacetate transaminase, and glutamine pyruvate transminase levels during BAL operation. At 5 hours, the ammonia concentration in plasma decreased to 370 and 150 μg/dL by immobilized single and spheroid hepatocytes, respectively, the concentrations at which they were maintained thereafter. The urea concentrations in plasma were 44 versus 72 μg/dL in immobilized single versus spheroid hepatocytes respectively, at 5 hours of operation. Spheroid hepatocytes not only showed in vivo structure, but also maintained high levels of liver-specific functions. The spheroid-based BAL system may be a good candidate to treat FHF patients.

Synthetic Signal Propagation Through Direct Cell-Cell Interaction

Contact-dependent cell communication has the potential to generate elaborate cell patterns, and this occurs in vivo. We used the Delta-Notch signaling system, consisting of the ligand Delta and the receptor Notch, to construct a positive feedback loop between adjacent cells to generate a propagating signal in cultured cells. To amplify the responses of Notch to Delta, we created a cell-cell positive feedback loop using an engineered transcriptional cascade and a Notch positive regulator, Lunatic fringe. We used mathematical modeling to determine the appropriate amount of amplification to enable the induction of Delta to propagate from one cell to its neighboring cells, which generated bistability within the local cell populations and resulted in discrete groups of cells that were either positive or negative for Delta. These results demonstrate the sufficiency of the cell-cell positive feedback loop to generate signal propagation and cell population-level bistability. This study represents a step in engineering more elaborate cell patterns in mammalian cells.

Clinical Perspectives of Mesenchymal Stem Cells

Clinical Perspectives of Mesenchymal Stem Cells

Inflammatory Cytokines Link Obesity and Breast Cancer

The risk of postmenopausal breast cancer is significantly increased by obesity. Further, low grade chronic inflammation, a hallmark of obesity, can contribute to detrimental health effects including high cancer incidence. Our goal is to understand the molecular basis for obesity-breast cancer interactions and dissect the role of inflammatory mediators secreted by adipocytes and breast cancer cells. Accordingly, we developed a three-dimensional (3D) coculture system to facilitate direct cell-cell interactions and also performed media transfer from adipocyte cultures to growing breast cell cultures. The co-culture system will facilitate both adipocyte and breast cell growth in an environment closely mimicking in-vivo tumor microenvironment. Co-cultures of human primary adipocytes obtained from lean and obese women with MCF10A, MCF7 and MDAMB231 breast cells (non-cancerous epithelial cells, cancerous and invasive breast cancer cells, respectively), led to cell type specific changes in secretion of several pro-inflammatory cytokines, such as IL-6 and TNFα, compared to monocultures. Regulation of cytokine secretion of breast cells by adipocytes and vice versa indicates the two-way communication between breast cells and adipocytes. Further, 3D co-culture and adipocyte conditioned media transfer experiments demonstrated that obese adipocytederived conditioned media can promote higher growth of breast cancer cells compared to that from lean adipocytes. Moreover, obese adipocyte conditioned media increased the activation of nuclear factor-κB (NF-κB) family members in breast cells, compared to lean adipocyte-derived media. Our results provide a novel model system to study adipocytebreast cancer cell interactions which may underlie the link between breast cancer and obesity and also demonstrate that inflammatory cytokines and other secreted factors are important in this interaction.

Mesenchymal stem cell-based treatment for cartilage defects in osteoarthritis

Osteoarthritis (OA) is a common disorder and the restoration of the diseased articular cartilage in patients with OA is still a challenge for researchers and clinicians. Currently, a variety of experimental strategies have investigated whether mesenchymal stem cells (MSCs) instead of chondrocytes can be used for the regeneration and maintenance of articular cartilage in OA. MSCs can modulate the immune response of individuals and positively influence the microenvironment of the stem cells already present in the diseased tissue. Through direct cell-cell interaction or the secretion of various factors, MSCs can initiate endogenous regenerative activities in the OA joint. Targeted gene-modified MSC-based therapy might further enhance the cartilage regeneration in OA. Conventionally, delivery of MSCs was attained by graft of engineered constructs derived from cell-seeded scaffolds. However, intra-articular MSCs transplantation without scaffolds is a more attractive option for OA treatment. This article briefly summarizes the current knowledge about MSC-based therapy for prevention or treatment of OA, discussing the direct intra-articular injection of MSCs for the treatment of OA in animal models and in clinical applications, as well as potential future strategies for OA treatment.

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were "separately" co-cultured with Nc cells without direct cell-cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The "spinal cord-promotion" and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range "Nc suppression" will be determined by future studies. Interestingly, these effects, "Nc suppression" and "SC promotion" were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.

Activation of Notch1 signaling in stromal fibroblasts inhibits melanoma growth by upregulating WISP-1

The tumor microenvironment is emerging as an important target for cancer therapy. Fibroblasts (Fbs) within the tumor stroma are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix and direct cell-cell interaction. In this work, we aim to alter the biological activity of stromal Fbs by modulating the Notch1 signaling pathway. We show that Fbs engineered to constitutively activate the Notch1 pathway significantly inhibit melanoma growth and tumor angiogenesis. We determine that the inhibitory effect of 'Notch-engineered' Fbs is mediated by increased secretion of Wnt-induced secreted protein-1 (WISP-1) as the effects of Notch1 activation in Fbs are reversed by shRNA-mediated blockade of WISP-1. When 'Notch-engineered' Fbs are co-grafted with melanoma cells in SCID mice, shRNA-mediated blockade of WISP-1 reverses the tumor-suppressive phenotype of the 'Notch-engineered' Fbs, significantly increases melanoma growth and tumor angiogenesis. Consistent with these findings, supplement of recombinant WISP-1 protein inhibits melanoma cell growth in vitro. In addition, WISP-1 is modestly expressed in melanoma-activated Fbs but highly expressed in inactivated Fbs. Evaluation of human melanoma skin biopsies indicates that expression of WISP-1 is significantly lower in melanoma nests and surrounding areas filled with infiltrated immune cells than in the adjacent dermis unaffected by the melanoma. Overall, our study shows that constitutive activation of the Notch1 pathway confers Fbs with a suppressive phenotype to melanoma growth, partially through WISP-1. Thus, targeting tumor stromal Fbs by activating Notch signaling and/or increasing WISP-1 may represent a novel therapeutic approach to combat melanoma.

Abstract 529: WISP-1/CCN4 is responsible for mediating the inhibitory effect of Notch-engineered stromal fibroblasts on melanoma growth

Abstract Purpose. The tumor microenvironment plays a pivotal role in influencing melanoma progression. Fibroblasts are major components of tumor stroma and are critically involved in promoting tumor growth and angiogenesis through secretion of soluble factors, synthesis of extracellular matrix, and direct cell-cell interaction. We aimed to alter the biological activity of stromal fibroblasts by modulating a specific signaling pathway to convert them from promoters to inhibitors of melanoma progression. Methods. Enforced activation of the Notch pathway was achieved by transducing human dermal fibroblasts with lentiviral vector encoding activated Notch1 (NIC). The effect of NIC-engineered stromal fibroblasts on melanoma growth was tested by the in vivo tumor-fibroblast co-xenografting approach. The soluble factor produced by NIC-engineered fibroblasts was identified by RT2-PCRArray and validated by Western blot analysis. The biological effect of WISP-1 on melanoma cell growth was tested in vitro by MTT. The function of WISP-1/CCN4 in mediating the inhibitory effect of NIC-engineered stromal fibroblasts on melanoma growth was examined through an siRNA-mediated blockade approach and tested by in vitro cell co-culture and animal models of tumor-fibroblast co-xenografting. Results. NIC-engineered fibroblasts significantly inhibited melanoma growth in vivo by suppressing melanoma cell proliferation and inducing melanoma cell apoptosis. Constitutive activation of Notch1 in fibroblasts up-regulated WISP-1 production. The inhibitory effect of NIC-engineered fibroblasts on melanoma growth was mediated, at least in part, by up-regulating WISP-1 expression, since recombinant WISP-1 was able to inhibit melanoma cell growth in vitro while blockade of WISP-1 production by NIC-engineered fibroblasts liberated their inhibitory effect on melanoma growth in vivo. Conclusions. Activation of Notch pathway in stromal fibroblasts can suppress melanoma growth, partially through WISP-1. Our study provides a novel approach to target the tumor microenvironment through modulating stromal fibroblasts by manipulating Notch signaling, and highlights WISP-1 as a potential therapeutic target in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2011-529