Heterotypic Cell-cell Interactions Research Articles

Abstract 25: Neutrophil Akt2 Regulates Heterotypic Cell-Cell Interactions During Vascular Inflammation

Introduction: Interactions between platelets, leukocytes and endothelial cells are important during microvascular occlusion. Although the major receptors and counter receptors have been well identified, it remains unclear how heterotypic cell-cell interactions are regulated under inflammatory conditions. Hypothesis: We assessed the hypothesis that AKT, a Ser/Thr protein kinase, plays a regulatory role in platelet-neutrophil-endothelial cell interactions during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy to evaluate mice lacking specific Akt isoform, we demonstrated that Akt2, but not Akt1 and Akt3, regulates neutrophil adhesion and crawling, and platelet-neutrophil interactions on the activated endothelium during TNF-α-induced cremaster venular inflammation. Further, using chimeric mice generated from bone marrow transplants on wild-type (WT) and Akt2 knockout (KO) mice, we found that hematopoietic cell-associated AKT2 is important for the regulatory effect in vivo. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under conditions of shear. In particular, neutrophil AKT2 was critical for intracellular calcium mobilization, β2-talin1 interaction, and membrane translocation of αMβ2 integrin - a crucial receptor for neutrophil-platelet interactions. We observed that the basal phosphorylation levels of AKT isoforms are markedly increased in neutrophils and platelets from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition significantly reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in venules of Berkeley (SCD) mice, thereby improving blood flow rates. Conclusion: Our results provide genetic and pharmacologic evidence that neutrophil AKT2 regulates αMβ2 integrin function and is important for neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD.

Neutrophil AKT2 regulates heterotypic cell-cell interactions during vascular inflammation

Interactions between platelets, leukocytes, and activated endothelial cells are important during microvascular occlusion; however, the regulatory mechanisms of these heterotypic cell-cell interactions remain unclear. Here, using intravital microscopy to evaluate mice lacking specific isoforms of the serine/threonine kinase AKT and bone marrow chimeras, we found that hematopoietic cell-associated AKT2 is important for neutrophil adhesion and crawling and neutrophil-platelet interactions on activated endothelial cells during TNF-α-induced venular inflammation. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under shear conditions. In particular, neutrophil AKT2 was critical for membrane translocation of αMβ2 integrin, β2-talin1 interaction, and intracellular Ca2+ mobilization. We found that the basal phosphorylation levels of AKT isoforms were markedly increased in neutrophils and platelets isolated from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow rates. Our results provide evidence that neutrophil AKT2 regulates αMβ2 integrin function and suggest that AKT2 is important for neutrophil recruitment and neutrophil-platelet interactions under thromboinflammatory conditions such as SCD.

Neutrophil Akt2 Plays a Critical Role In Heterotypic Neutrophil-Platelet Interactions During Vascular Inflammation

Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients' neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors' cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients' neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD. Disclosures:No relevant conflicts of interest to declare.

Abstract A112: The role of microenvironment in age-related breast cancers

Abstract Aside from being a woman, age is the greatest risk factor for developing breast cancer, but there is little understanding how the aging process modifies cancer susceptibility. Multipotent mammary epithelial progenitors are putative roots of some breast cancers, thus expansion combined with increased vulnerability of these cells to transformation could potentiate cancer progression. We have shown in humans that with age, the proportion of multipotent progenitors accumulates, whereas myoepithelial cells decrease, and that these shifts have a basis in altered progenitor function. Progenitors from post-menopausal women gave rise to incompletely differentiated myoepithelial cells and to basal-like luminal cells that expressed markers normally associated with myoepithelial cells in young women. Because epithelial progenitors are thought to be cells-of-origin for a number of breast cancers, and myoepithelial cells are thought to be tumor suppressive, we hypothesize that these tissue-level changes make post-menopausal women more vulnerable to malignant progression. Age-related tissue changes arise in parallel with age-dependent gene expression patterns that have unknown functional consequences and unknown origins. Here we demonstrate that utilizing biomimetic substrata with tuned physical and molecular properties to probe primary human mammary progenitors from women aged 16 to 91 years can reveal age-related functional consequences. As increased breast density is associated with greater breast cancer risk and stiff mechanical microenvironments are known to exacerbate malignant cell behavior, we examined how normal progenitors responded to changes in elastic modulus. Age-related changes in Rho and HIPPO pathway mechano-transduction and transcriptional regulation contributed to accumulation of progenitors by preventing normal differentiation in response to mechanical stimuli. Young progenitors gave rise to more luminal cells on compliant substrata and to more myoepithelial cells on stiffer substrata. However, post-menopausal progenitors were insensitive to physiological mechanical changes and differentiated stochastically instead. To better understand the genesis of the gene expression patterns underlying these age-related functional states, molecular analyses of co-cultures established with primary myoepithelial and luminal epithelial lineages from combinations of young and old individuals were preformed. Age-related gene expression states in luminal cells were imposed through heterotypic cell-cell interactions. Changes in gene expression were coincident with changes in DNA promoter methylation and expression of histone modifying enzymes. We propose that aging results in a continuum of microenvironmental changes that impose epigenetically regulated metastable gene expression states, which are the basis of age-related cell functions that ultimately lead to vulnerable tissue-level phenotypes. Citation Format: Fanny A. Pelissier, Masaru Miyano, James C. Garbe, Martha R. Stampfer, Mark A. LaBarge. The role of microenvironment in age-related breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A112.

Cajal-Retzius Cells Instruct Neuronal Migration by Coincidence Signaling between Secreted and Contact-Dependent Guidance Cues

Cajal-Retzius (CR) cells are a transient cell population of the CNS that is critical for brain development. In the neocortex, CR cells secrete reelin to instruct the radial migration of projection neurons. It has remained unexplored, however, whether CR cells provide additional molecular cues important for brain development. Here, we show that CR cells express the immunoglobulin-like adhesion molecule nectin1, whereas neocortical projection neurons express its preferred binding partner, nectin3. We demonstrate that nectin1- and nectin3-mediated interactions between CR cells and migrating neurons are critical for radial migration. Furthermore, reelin signaling to Rap1 promotes neuronal Cdh2 function via nectin3 and afadin, thus directing the broadly expressed homophilic cell adhesion molecule Cdh2 toward mediating heterotypic cell-cell interactions between neurons and CR cells. Our findings identify nectins and afadin as components of the reelin signaling pathway and demonstrate that coincidence signaling between CR cell-derived secreted and short-range guidance cues direct neuronal migration.

Biofabrication of Layered Membrane Systems by Using Human Hepatocytes and Endothelial Cells: A Comparative Study

Strategies to overcome in vitro dedifferentiation of hepatocytes preserving key features of the liver phenotype in vitro constitute a major goal in medical applications. The realization of biohybrid membrane systems, utilizing primary human hepatocytes in co-culture with non-parenchymal cells and polymeric membranes with suitable properties, offered interesting opportunities for the design of bioartificial liver. This study was aimed to investigate the morphological and functional behavior of primary human hepatocytes with endothelial cells (ECs) in two different layered co-culture membrane systems, which were realized by using a synthetic polymeric membrane and two different sequential cell seeding methods. We evaluated the heterotypic cell-cell interactions in the different layered membrane systems in order to understand if the variation of the heterotypic interface between hepatocytes and ECs and of cell type interacting with membrane may influence the performance of functional biofabricated hepatocellular constructs. Overall results demonstrated a strong influence of the heterotypic interactions on the performance of the tissue constructs in terms of albumin secretion, urea synthesis and drug biotransformation functions. Keywords: Biohybrid membrane systems, endothelial cells, hepatocytes, hepatocellular constructs, heterotypic interactions, membranes.

Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model

Solid tumors consist of genetically and phenotypically diverse subpopulations of cancer cells with unique capacities for growth, differentiation, and invasion. While the molecular and microenvironmental bases for heterogeneity are increasingly appreciated, the outcomes of such intratumor heterogeneity, particularly in the context of tumor invasion and metastasis, remain poorly understood. To study heterotypic cell-cell interactions and elucidate the biological consequences of intratumor heterogeneity, we developed a tissue-engineered multicellular spheroid (MCS) co-culture model that recapitulates the cellular diversity and fully three-dimensional cell-cell and cell-matrix interactions that characterize human carcinomas. We found that "invasion-competent" malignant cells induced the collective invasion of otherwise "invasion-incompetent" epithelial cells, and that these two cell types consistently exhibited distinct leader and follower roles during invasion. Analysis of extracellular matrix (ECM) microarchitecture revealed that malignant cell invasion was accompanied by extensive ECM remodeling including matrix alignment and proteolytic track-making. Inhibition of cell contractility- and proteolysis-mediated matrix reorganization prevented leader-follower behavior and malignant cell-induced epithelial cell invasion. These results indicate that heterogeneous subpopulations within a tumor may possess specialized roles during tumor progression and suggest that complex interactions among the various subpopulations of cancer cells within a tumor may regulate critical aspects of tumor biology and affect clinical outcome.

Reconstruction of hepatic stellate cell-incorporated liver capillary structures in small hepatocyte tri-culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC-incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri-culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell-cell interactions, which are essential to form HSC-EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d, l-lactide-co-glycolide) (PLGA) microporous membranes in SH-HSC-EC tri-culture to reconstruct the HSC-incorporated liver capillary structures in vitro. First, the formation of EC capillary-like structures was induced on Matrigel-coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary-like structures. This process was mediated in part by platelet-derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary-like structures with their long cytoplasmic processes. In the HSC-incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro.

Effect of pericytes on melanoma development.

83 Background: Melanoma-associated stroma cells regulate tumor establishment, invasion, and dissemination through paracrine and heterotypic cell-cell interactions with cancer cells. Pericytes constitute a major noninflammatory component of the melanoma-associated stroma. Our study is designed to evaluate the role of pericytes in melanoma development. Methods: Tumors induced in GFP+/SCID mice with GFP-B16 cells were harvested after 14 days, enzyme digested, labeled against mesenchymal markers, and analyzed by flow cytometry (FC). The distribution of stromal subpopulations in tumors was investigated by immunofluorescence microscopy (IFM). Stromal pericytes from tumors and adipose tissue were FACS sorted, mixed with B16 cells at a 3:1 ratio, and injected into SCID mice. Tumor diameters were taken periodically with a scientific caliper, and used to determine tumor volumes using the ellipsoid formula. Results: FC analysis showed that the melanoma stroma was composed of CD45+ inflammatory cells, CD31+ endothelial cells, FAP+ myofibroblasts and CD146+ cells. IFM analysis showed that the CD146+ cells are perivascular CD31-/CD34-/α-SMA+ stromal pericytes that associated with TGF-β immunoreactive CD271+/CD146+B16 cells in perivascular tumor niches. Co-induction experiments were performed to investigate the effect of pericytes on melanoma development. We observed faster growth rates in the size of tumors co-induced with pericytes, compared with tumors induced with B16 cells alone. These co-induced tumors had bigger masses, more volume, were hypercellular, and had increase vascularization compared with control tumors. In addition, pericytes increased the B16 cell proliferation rate, as determined by higher expression of Ki-67 in the tumor fraction of co-induced tumors. Conclusions: Pericytes promote melanoma development by inducing angiogenesis and melanoma cell proliferation. Our results suggest that pericytes could be utilized as a therapeutic target for melanoma treatment.

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

Vascular Morphogenesis of Adipose-Derived Stem Cells is Mediated by Heterotypic Cell-Cell Interactions

Adipose-derived stromal/stem cells (ASCs) are a promising cell source for vascular-based approaches to clinical therapeutics, as they have been shown to give rise to both endothelial and perivascular cells. While it is well known that ASCs can present a heterogeneous phenotypic profile, spontaneous interactions among these subpopulations that result in the formation of complex tissue structures have not been rigorously demonstrated. Our study reports the novel finding that ASCs grown in monolayers in the presence of angiogenic cues are capable of self-assembling into complex, three-dimensional vascular structures. This phenomenon is only apparent when the ASCs are seeded at a high density (20,000 cells/cm(2)) and occur through orchestrated interactions among three distinct subpopulations: CD31-positive cells (CD31+), α-smooth muscle actin-positive cells (αSMA+), and cells that are unstained for both these markers (CD31-/αSMA-). Investigations into the kinetics of the process revealed that endothelial vessel-like structures initially arose from individual CD31+ cells through proliferation and their interactions with CD31-/αSMA- cells. During this period, αSMA+ cells proliferated and appeared to migrate toward the vessel structures, eventually engaging in cell-cell contact with them after 1 week. By 2 weeks, the lumen-containing CD31+ vessels grew greater than a millimeter in length, were lined with vascular basement membrane proteins, and were encased within a dense, three-dimensional cluster of αSMA+ and CD31-/αSMA- cells. The recruitment of αSMA+ cells was largely due to platelet-derived growth factor (PDGF) signaling, as the inhibition of PDGF receptors substantially reduced αSMA+ cell growth and vessel coverage. Additionally, we found that while hypoxia increased endothelial gene expression and vessel width, it also inhibited the growth of the αSMA+ population. Together, these findings underscore the potential use of ASCs in forming mature vessels in vitro as well as the need for a further understanding of the heterotypic interactions among ASC subpopulations.

N-Cadherin in Neuroblastoma Disease: Expression and Clinical Significance

One of the first and most important steps in the metastatic cascade is the loss of cell-cell and cell-matrix interactions. N-cadherin, a crucial mediator of homotypic and heterotypic cell-cell interactions, might play a central role in the metastasis of neuroblastoma (NB), a solid tumor of neuroectodermal origin. Using Reverse Transcription Quantitative PCR (RT-qPCR), Western blot, immunocytochemistry and Tissue MicroArrays (TMA) we demonstrate the expression of N-cadherin in neuroblastoma tumors and cell lines. All neuroblastic tumors (n = 356) and cell lines (n = 10) expressed various levels of the adhesion protein. The N-cadherin mRNA expression was significantly lower in tumor samples from patients suffering metastatic disease. Treatment of NB cell lines with the N-cadherin blocking peptide ADH-1 (Exherin, Adherex Technologies Inc.), strongly inhibited tumor cell proliferation in vitro by inducing apoptosis. Our results suggest that N-cadherin signaling may play a role in neuroblastoma disease, marking involvement of metastasis and determining neuroblastoma cell viability.

Effects of endothelial cells on human mesenchymal stem cell activity in a three-dimensional in vitro model

An increasing body of data suggest that mesenchymal stem cells (MSCs) reside in a perivascular niche. To more closely mimic this in vivo microenvironment and for better understanding of its complexity, and the factors that regulate the MSC activity, human umbilical vein endothelial cells (HUVECs) were co-cultured with human bone marrow MSCs--using a novel three-dimensional (3D) spheroid co-culture system. Using confocal microscopy of fluorescently labelled cells, we observed HUVECs and MSCs to self-assemble and form organised structures with segregated cell-type partitioning. Under osteogenic conditions, the rate and extent of differentiation in MSC/HUVEC spheroids was significantly elevated compared to 3D co-cultures of MSCs and human dermal fibroblast controls as shown by alkaline phosphatase staining. Conversely, HUVECs inhibited adipogenic differentiation and the proliferation of MSCs in 3D co-cultures indicating that HUVECs suppressed MSC cycling and selectively promoted osteogenic differentiation in 3D. We have also shown that HUVECs enhanced activation of endogenous Wnt signalling and bone morphogenetic protein (BMP) signalling as shown by increased levels of active nuclear β-catenin and pSmad 1/5/8 immunopositivity respectively. These data suggest strongly that endothelial cells regulate the MSC activity in simulated in vivo conditions, by maintaining quiescence and facilitating niche exit via osteogenic differentiation following appropriate cues. Our findings also underline the importance of 3D heterotypic cell-cell interactions in the regulation of MSC behaviour, suggesting that multicellular cocktails and/or 3D-based delivery strategies may be beneficial for bone repair.

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.

3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.

Novel in vitro co-culture methodology to investigate heterotypic cell-cell interactions

Cell-cell interactions are of crucial importance for the formation of tissues, homeostasis and regeneration processes as well as reactions on foreign bodies including implants. So far, however, the importance of heterotypic cell-cell interactions in the in vitro evaluation of implant surfaces has been largely neglected. This work aims to develop an in vitro methodology that enables the in-depth investigation of heterotypic cell-cell interactions in a mixed co-culture system, and to validate it with a primary adult human bone-derived osteoblast cells (HBCs) - abdominal fibroblasts (HAFs) system. The methodology proposed combines a simple live labelling step, semiautomated fluorescence image acquisition and analysis to characterize the interactions between different cell types (cell population dynamics) in co-culture in terms of cell proliferation and cell spatial distribution of each cell type. In this co-culture system, direct cell-cell contacts between the two cell types were permitted while the determination of cell-type specific responses could still be elucidated. We could show that HAF proliferation was reduced in a way negatively correlated with the seeding HBC/HAF ratio, i.e., a high proportion of HBC in the co-culture had an inhibitory effect on HAF proliferation. In all cultures segregation was found after 4 and 7 days of co-culture. HBCs were segregated at low ratios while HAFs were segregated at high ratios. Cell-cell distances depended on the total cell number in the co-culture but the dependence was different for each cell type.

Abstract LB-240: Smad7 blocks melanoma invasion by suppressing n-cadherin cleavage and preserving heterotypic cell-cell interactions in vivo.

Abstract The number of TGF-ß-related proteins involved in non-canonical signaling continues to grow. Examples include receptor-Smads directing micro-RNA processing, as well as inhibitory-Smads, such as Smad7, influencing cell adhesion. Our past work has shown Smad7 overexpression blocks MMP secretion and subsequent tumorigenesis using a metastatic melanoma model. Employing live-animal fluorescent imaging and in vivo human skin grafts, we have found 1205Lu metastatic melanoma cells expressing Smad7 position themselves proximal to the epidermal-dermal junction and appear stationary within the dermis, while Smad7-deficient controls readily invade. Localization of Smad7-expressing cells within the graft correlated with absences in tumorigenesis, invasion and metastasis. Smad7-deficient controls formed dense, ulcerative tumors indicative of the aggressive nature of the cell line. Cell adhesion assays efficiently reproduced the graft and identified a strong interaction (p<0.001) between Smad7-expressing cells and primary dermal fibroblasts, suggestive of the formation of heterotypic cell-cell interactions. Smad7-deficient cells and fibroblasts were incapable of heterotypic interaction as no adhesions were observed. We explored the mechanism behind the inhibited tumorigenesis and found Smad7 stabilized the cell adhesion-related proteins -catenin and N-cadherin. Immunoprecipitation revealed Smad7 directly bound -catenin and permitted a significantly increased association with membranous N-cadherin. More importantly, -catenin linkage via Smad7 resulted in loss of N-cadherin processing, exhibited by the absence of unique N-cadherin cleavage products seen only in Smad7-deficient controls. Additionally, N-cadherin skin graft immunohistochemistry showed preserved N-cadherin expression at the surface of both Smad7-expressing cells and dermal fibroblasts indicating that stable cell-cell interactions via N-cadherin are maintained in vivo. Conversely, both Smad7-deficient cells and tumor-associated fibroblasts lacked N-cadherin expression, while interestingly, fibroblasts beyond the tumor border retained N-cadherin. Previous work has established that stable cell adhesions via cadherins are detrimental to cell migration. We propose Smad7 functions to revert the invasive phenotype of these metastatic melanoma cells by stabilizing heterotypic cell-cell interactions between primary dermal fibroblasts and Smad7-expressing 1205Lu's, a mechanism not previously reported to occur in response to Smad7. We find these cell-cell interactions are mediated through N-cadherin and, in cooperation with loss of MMP 2 and 9, result in mitigated tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-240.

1105 再生医療を目指した初代肝細胞スフェロイドアレイの作製と機能評価(OS13-(6)オーガナイズドセッション《生体医工学及びバイオマテリアル》)

In this study, we aimed to construct the in vitro culture method with in vivo function for tissue engineering and biomedical application, and we confirmed effects of spheroid culture system and co-culture system. It is noted that spheroids significantly express a stable level of liver-specific functions (albumin secretion), compared to the usual cell monolayer. Co-cultivation of hepatocyte with endothelial cells was further essential to stabilize hepatocyte viability and liver-specific function. These results suggested that heterotypic cell-cell interaction within spheroid play important roles to maintain hepatocyte viability and liver-specific functions, which is more effective for BAEC than for HUVEC. This co-culture system is highly useful for tissue engineering and biomedical application.

Apoptotic Lymphoma Cells Evoke a Pro-Senescent Stromal Signal That Limits Lymphoma Development.

Abstract 2392Poster Board II-369 Introduction:Cancer entities frequently exhibiting constitutive Myc expression, such as aggressive B-cell lymphomas, typically display significant amounts of apoptotic cell death. So far, cellular senescence as another cell-autonomous oncogene-inducible safeguard program has been recognized in RAS/BRAF-driven scenarios but not as a bona fide Myc-evoked anti-cancer mechanism. Understanding how oncogenic Myc may provoke not only apoptosis but cellular senescence as a failsafe mechanism to counter tumor development has broad implications for the clinical presentation and therapeutic strategies in frequently Myc-driven lymphoma entities such as Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). Results:Using the Burkitt's like Eμ-myc transgenic mouse lymphoma model, we show here that cellular senescence serves as another crucial anti-neoplastic barrier during Myc-driven tumorigenesis in addition to apoptosis. Eμ-myc lymphomas harbor a substantial fraction of senescent tumor cells, that stain positive for histone H3 lysine 9 (H3K9)-trimethylation. Lymphomas lacking the H3K9 methyltransferase Suv39h1 display no senescence and develop significantly faster, although apoptosis is not affected by Suv39h1 deficiency. While Myc, unlike other Ras-type oncogenes, shows rather modest pro-senescent activity in vitro, we identified the cytostatic cytokine TGF-β as the main paracrine senescence trigger in vivo. When neutralizing TGF-β action during Myc-driven lymphomagenesis utilizing a secretable TGF-β receptor II ecto-domain, senescence is completely blunted and tumor latency is significantly shortened. We identify macrophages, but not lymphoma cells, as the main source of exogenous TGF-β, that is secreted upon phagocytosis of apoptotic lymphoma cells. Lymphomas harboring a Bcl2-mediated apoptotic block presented with a much lower frequency of both infiltrating macrophages and senescent cells in vivo, suggesting that there is a functional link between cell-autonomous Myc-triggered apoptosis and non-cell-autonomous, macrophage-induced senescence. Both pharmacological suppression of TGF-β production in macrophages via the angiotensin-converting enzyme (ACE) inhibitor lisinopril and depletion of macrophages in Eμ-myc lymphoma-harboring mice by systemic exposure to clodronate resulted in a profound reduction of senescence, thereby underscoring the important role for tumor-infiltrating macrophages in TGF-β-mediated senescence in vivo. We recapitulated components of such a mechanism in human aggressive B-cell lymphomas, a frequently Myc-activated entity where TGF-β1 signaling has previously been identified as a component of the prognostically favourable “stromal-1” signature (Lenz-G et al., NEJM, 2008). A panel of 30 DLBCL samples was sub-divided based on Ki67 immunoreactivity into a very high proliferation (Ki67hi; ≥80% Ki67-positive cells) and a lower proliferation (Ki67lo; <80% Ki67-positive cells) group. Ki67lo samples exhibited a higher frequency of H3K9me3-positive cells, indicative of cellular senescence. Importantly, the Ki67lo group also presented with a higher fraction of apoptotic cells, more lymphoma-infiltrating macrophages, and a stronger reactivity for the TGF-β signaling mediator Smad3-P, thereby representing a subgroup in DLBCL that displays features highly reminiscent of the macrophage-derived mechanism of senescence induction. Conclusions:Our study expands the relevance of oncogene-induced senescence to Myc-driven cancers and demonstrates that different tumor suppressor programs - such as apoptosis and senescence - are enforced in an interdependent fashion between the tumor- and non-malignant stroma cells during lymphomagenesis. Utilizing the Eμ-myc transgenic mouse lymphoma model and furthermore supported by evidence from human aggressive B-cell lymphoma samples, this study establishes a novel network of heterotypic cell-cell interactions within a tumor in which apoptotic tumor cells induce a paracrine response in non-malignant bystander cells that limits lymphomagenesis by cellular senescence. Given the anti-cancer relevance of senescence and the demonstrated inducibility of senescence by a non-DNA damaging cytokine, such as TGF-β, these findings open the exciting perspective to utilize Suv39h1/H3K9me3-mimicking approaches for future cancer therapies. Disclosures:No relevant conflicts of interest to declare.

Systems-Level Modeling of Cancer-Fibroblast Interaction

Cancer cells interact with surrounding stromal fibroblasts during tumorigenesis, but the complex molecular rules that govern these interactions remain poorly understood thus hindering the development of therapeutic strategies to target cancer stroma. We have taken a mathematical approach to begin defining these rules by performing the first large-scale quantitative analysis of fibroblast effects on cancer cell proliferation across more than four hundred heterotypic cell line pairings. Systems-level modeling of this complex dataset using singular value decomposition revealed that normal tissue fibroblasts variably express at least two functionally distinct activities, one which reflects transcriptional programs associated with activated mesenchymal cells, that act either coordinately or at cross-purposes to modulate cancer cell proliferation. These findings suggest that quantitative approaches may prove useful for identifying organizational principles that govern complex heterotypic cell-cell interactions in cancer and other contexts.