Laminin-rich Basement Membrane Research Articles

Interommatidial cells build a tensile collagen network during Drosophila retinal morphogenesis

Drosophila compound eye morphogenesis transforms a simple epithelium into an approximate hollow hemisphere comprised of ∼700 ommatidia, packed as tapering hexagonal prisms between a rigid external array of cuticular lenses and a parallel, rigid internal floor, the fenestrated membrane (FM). Critical to vision, photosensory rhabdomeres are sprung between these two surfaces, grading their length and shape accurately across the eye and aligning them to the optical axis. Using fluorescently tagged collagen and laminin, we show that that the FM assembles sequentially, emerging in the larval eye disc in the wake of the morphogenetic furrow as the original collagen-containing basement membrane (BM) separates from the epithelial floor and is replaced by a new, laminin-rich BM, which advances around axon bundles of newly differentiated photoreceptors as they exit the retina, forming fenestrae in this new, laminin-rich BM. In mid-pupal development, the interommatidial cells (IOCs) autonomously deposit collagen at fenestrae, forming rigid, tension-resisting grommets. In turn, stress fibers assemble in the IOC basal endfeet, where they contact grommets at anchorages mediated by integrin linked kinase (ILK). The hexagonal network of IOC endfeet tiling the retinal floor couples nearest-neighbor grommets into a supracellular tri-axial tension network. Late in pupal development, IOC stress fiber contraction folds pliable BM into a hexagonal grid of collagen-stiffened ridges, concomitantly decreasing the area of convex FM and applying essential morphogenetic longitudinal tension to rapidly growing rhabdomeres. Together, our results reveal an orderly program of sequential assembly and activation of a supramolecular tensile network that governs Drosophila retinal morphogenesis.

Modeling Collective Invasion and Single-Cell Mesenchymal Invasion in Three-Dimensional Matrigel-Collagen I Cultures.

Understanding the modes and mechanisms of tumor cell invasion is key to developing targeted therapies against metastatic disease. In vitro assays modeling tumor progression have primarily been optimized for studying classical single-cell migration through an epithelial-mesenchymal transition (EMT). Although experimental and clinical histopathological evidence has revealed that tumor invasion is plastic and that epithelial carcinomas can invade by a range of modes that vary from single, mesenchyme-like cells, all the way to cohesive, collective units, few in vitro assays have been designed to assess these modes specifically. Thus, we have developed a Matrigel-Collagen I overlay assay that is suitable for identifying and quantifying both collective and mesenchymal invasion. This three-dimensional (3D) culture assay utilizes the features of Matrigel and Collagen I to mimic the laminin-rich basement membrane and the stiff, fibrillar Collagen I tumor microenvironment allowing for spheroid invasion to be assessed at the interface between these two matrix components.

Definitive Endoderm from EpiSC Aggregates in Matrigel.

This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues.

Morphological and mechanical stability of bladder cancer cells in response to substrate rigidity

BackgroundMorphology of cells can be considered as an interplay between the accessibility of substrate anchoring sites, cytoskeleton properties and cellular deformability. To withstand tension induced by cell's environment, cells tend to spread out and, simultaneously, to remodel actin filament organization. MethodsIn this context, the use of polyacrylamide hydrogel substrates with a surface coated with laminin allows to trace remodeling of actin cytoskeleton during the interaction of cells with laminin-rich basement membrane. Reorganization of actin cortex can be quantified by a surface spreading area and deformability of single cells. ResultsIn our study, we demonstrated that morphological and mechanical alterations of bladder cancer cells in response to altered microenvironment stiffness are of biphasic nature. Threshold-dependent relations are induced by mechanical properties of cell microenvironment. Initially, fast alterations in cellular capability to spread and to deform are followed by slow-rate changes. A switch provided by cellular deformability threshold, in the case of non-malignant cells, triggers the formation of thick actin bundles accompanied by matured focal adhesions. For cancer cells, cell spreading and deformability thresholds switch between slow and fast rate of changes with weak reorganization of actin filaments and focal adhesions formation. ConclusionsThe presence of transition region enables the cells to achieve a morphological and mechanical stability, which together with altered expression of vinculin and integrins, can contribute to invasiveness of bladder cancers. General significanceOur findings show that morphological and mechanical stability is directly related to actin filament organization used by cancer cells to adapt to altered laminin-rich microenvironment.

Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices.

Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.

Dual role of pericyte α6β1-integrin in tumour blood vessels

ABSTRACTThe α6β1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6β1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRβ expression and AKT–mTOR signalling. Taken together, we show that pericyte α6β1-integrin regulates tumour blood vessels by both controlling PDGFRβ and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis.

The vitronectin RGD motif regulates TGF-β-induced alveolar epithelial cell apoptosis.

Transforming growth factor-β (TGF-β) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-β, which regulates changes in the cellular and matrix makeup of the lung during the repair and fibrosis phase. TGF-β can also initiate alveolar epithelial cell (AEC) apoptosis. Injury leads to destruction of the laminin-rich basement membrane, which is replaced by a provisional matrix composed of arginine-glycine-aspartate (RGD) motif-containing plasma matrix proteins, including vitronectin and fibronectin. To determine the role of specific matrix proteins on TGF-β-induced apoptosis, we studied primary AECs cultured on different matrix conditions and utilized mice with deletion of vitronectin (Vtn(-/-)) or mice in which the vitronectin RGD motif is mutated to nonintegrin-binding arginine-glycine-glutamate (RGE) (Vtn(RGE/RGE)). We found that AECs cultured on fibronectin and vitronectin or in wild-type mouse serum are resistant to TGF-β-induced apoptosis. In contrast, AECs cultured on laminin or in serum from Vtn(-/-) or Vtn(RGE/RGE) mice undergo robust TGF-β-induced apoptosis. Plasminogen activator inhibitor-1 (PAI-1) sensitizes AECs to greater apoptosis by disrupting AEC engagement to vitronectin. Inhibition of integrin-associated signaling proteins augments AEC apoptosis. Mice with transgenic deletion of PAI-1 have less apoptosis after bleomycin, but deletion of vitronectin or disruption of the vitronectin RGD motif reverses this protection, suggesting that the proapoptotic function of PAI-1 is mediated through vitronectin inhibition. Collectively, these data suggest that integrin-matrix signaling is an important regulator of TGF-β-mediated AEC apoptosis and that PAI-1 functions as a natural regulator of this interaction.

Three-dimensional culture model to distinguish normal from malignant human bronchial epithelial cells

In the present study, we have developed an in vitro three-dimensional model to differentiate normal lung cells from lung cancer cells in order to study the mechanisms resulting in lung cancer. Using a reconstituted laminin-rich basement membrane (Matrigel), we were able to culture normal human bronchial epithelial cells and a subset of malignant cells. The two cell types can be readily distinguished by the ability of normal cells to express a structurally and functionally differentiated phenotype within Matrigel. Human bronchial epithelial cells embedded in Matrigel as single cells were able to form multi-cellular spherical colonies with a final size close to that of true acini in situ. Sections of mature spheres revealed a central lumen surrounded by polarised epithelial cells. In contrast, none of malignant cells tested, cell lines and lung biopsies responded to basement membrane by lumen formation. These results demonstrated that this in vitro glandular tumour model can be useful for studies of bronchial oncogene. Indeed, these findings may provide the basis for a rapid assay to discriminate normal human bronchial epithelial cells from their malignant counterparts. In conclusion, the three-dimensional tumour bronchial epithelial acinar-like sphere represents a novel in vitro model to further investigate pathophysiological functions resulting in lung cancer.

Visualizing movement in the tumor microenvironment landscape

The local microenvironment influences tumor progression in several important ways. A recent study by Nguyen-Ngoc and colleagues used explants of primary human and mouse mammary tumors to examine how the composition of the extracellular matrix modulates tumor cell invasion. Culture in the presence of a three-dimensional laminin-rich basement membrane, similar to the mammary basement membrane in vivo, resulted in minimal invasion. In contrast, identical tumor fragments in a collagen I matrix which resembles the interstitial breast stroma exhibited a pronounced protrusive migration and local dissemination. These data emphasize the importance of the laminin-rich basement membrane in constraining tumor cell invasion.

Verteporfin-based photodynamic therapy overcomes gemcitabine insensitivity in a panel of pancreatic cancer cell lines

Pancreatic cancer is notoriously difficult to treat and resistant to virtually all therapeutics including gemcitabine, the standard front line agent for palliative chemotherapy. Early clinical studies point to a potential role for photodynamic therapy (PDT) in the management of this deadly disease. Here we examine PDT with verteporfin for treatment of cells that are nonresponsive to gemcitabine and identify intracellular and extracellular factors that govern sensitivity to each modality. Using MTS we assess cytotoxicity of verteporfin-PDT in gemcitabine-treated nonresponsive populations from a panel of five pancreatic cancer cell lines representing a range of tumor histopathology and origin. We conduct Western blots for pro-/anti-apoptotic proteins bax and Bcl-XL to identify factors relevant to PDT and gemcitabine sensitivity. To examine the role of extracellular matrix influences we compare response to each modality in traditional cell culture conditions and cells grown on a laminin-rich basement membrane. All cell lines have gemcitabine nonresponsive populations (17-33%) at doses up to 1 mM while moderate total verteporfin PDT doses (1-6 µM J/cm2) produce nearly complete killing. Our data shows that cells that are nonresponsive to sustained gemcitabine incubation are sensitive to verteporfin PDT indicating that the latter is agnostic to gemcitabine sensitivity. Verteporfin-based PDT decreases Bcl-XL and increases the bax/Bcl-XL ratio toward a pro-apoptotic balance. Insensitivity to gemcitabine is increased in cells that are adherent to basement membrane relative to traditional tissue culture conditions. Collectively these results indicate the ability of verteporfin-based PDT to bypass intracellular and extracellular cues leading to gemcitabine resistance and point to the emerging role of this therapy for treatment of pancreatic cancer.

Abstract 555: 3D tumor models for the assessment of molecular targeted therapies

Abstract It is becoming increasingly apparent that the interaction between the tumor and the surrounding stromal cells is critical to tumor growth, invasion and metastasis. These paracrine interactions can profoundly alter EMT profiles and modulate key growth factor signaling cascades which can lead to altered responsiveness to molecular targeted therapies such as EGF-R TKI inhibitors. We have developed a novel 3D tumor growth assay, where the paracrine interaction between tumor derived fibroblasts and epithelial cells are restored. Utilizing luminescent and fluorescent labeled cell populations we are able to monitor tumor-microenvironment driven tumor growth in the presence of targeted therapies in comparison to standard platinum dublet therapies. Primary tumor associated fibroblasts and mesenchymal stem cells drive growth and proliferation of non-small cell lung cancer cell lines of adenocarcinoma (A549, NCI-H358, NCI-H460) and squamous subtypes (SK-MES1, H226) as well as early stage disease (NCI-H322M). The paracrine interaction promotes 3D structure formation and invasion into laminin rich basement membrane extract. Cells grown in 3D assays show a slowed proliferation and lower sensitivity to anti-mitotic agents such as paclitaxel. As proof of concept we show that responsiveness to EGF-R TKI is significantly decreased in the presence of HGF secreting stromal cells irrespective of EGF-R and KRAS mutational status. The 3D growth assay provides a more tumor microenvironment relevant model for pre-clinical testing of molecular targeted therapies. 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 555. doi:10.1158/1538-7445.AM2011-555

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.

Lysophosphatidic acid cooperates with EGF in inducing branching morphogenesis of embryonic mouse salivary epithelium

Epithelial morphogenesis is supported by diffusible growth factors and by nondiffusible cell substrata, such as laminin and fibronectin. When embedded in a laminin-rich basement-membrane substratum, embryonic mouse submandibular epithelium undergoes cell proliferation and branching morphogenesis in response to epidermal growth factor (EGF) in mesenchyme-free culture but not in serum-free medium. In this study, we sought to identify the biologically active factor in serum. As this factor was heat-stable and trypsin-resistant, the lipid fraction was analyzed. Horse serum was fractionated by ethanol extraction, Folch partition with chloroform-methanol-water, and high-performance liquid chromatography, and we tested the branch-inducing activity of each fraction. We also analyzed the partially purified fraction with a mass spectrometer, indicating that the active fraction largely consisted of lysophosphatidyl-hexose. Finally we identified the molecule as lysophosphatidic acid (LPA), because, whereas lysophosphatidyl-inositol had only a slight branch-inducing activity, its relevant LPA fully substituted for serum and induced branching morphogenesis in cooperation with EGF. LPA receptor genes were expressed in submandibular epithelial cells. DNA-synthesizing cells were abundant only when cultured in the presence of both EGF and LPA, but not either singly.

Endothelial Extracellular Matrix

The extracellular matrix (ECM) is critical for all aspects of vascular biology. In concert with supporting cells, endothelial cells (ECs) assemble a laminin-rich basement membrane matrix that provides structural and organizational stability. During the onset of angiogenesis, this basement membrane matrix is degraded by proteinases, among which membrane-type matrix metalloproteinases (MT-MMPs) are particularly significant. As angiogenesis proceeds, ECM serves essential functions in supporting key signaling events involved in regulating EC migration, invasion, proliferation, and survival. Moreover, the provisional ECM serves as a pliable scaffold wherein mechanical guidance forces are established among distal ECs, thereby providing organizational cues in the absence of cell-cell contact. Finally, through specific integrin-dependent signal transduction pathways, ECM controls the EC cytoskeleton to orchestrate the complex process of vascular morphogenesis by which proliferating ECs organize into multicellular tubes with functional lumens. Thus, the composition of ECM and therefore the regulation of ECM degradation and remodeling serves pivotally in the control of lumen and tube formation and, finally, neovessel stability and maturation.

Homeobox D10 Induces Phenotypic Reversion of Breast Tumor Cells in a Three-Dimensional Culture Model

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. We previously reported that HoxD10 helps to maintain a quiescent, differentiated phenotype in endothelial cells by suppressing expression of genes involved in remodeling the extracellular matrix and cell migration. Here we investigated whether HoxD10 could also promote or maintain a differentiated phenotype in epithelial cells. We observed that HoxD10 expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HoxD10 in the malignant breast tumor cells MDA-MB-231 significantly impaired migration, and when these cells were cultured in a three-dimensional laminin-rich basement membrane (3DlrBM) model, they formed polarized, acinar structures. This phenotypic reversion was accompanied by decreased alpha3 integrin expression and reduced proliferation. Importantly, expression of HoxD10 in the MDA-MB-231 cells inhibited their ability to form tumors in mouse xenografts. Taken together, our results suggest that HoxD10 has tumor-suppressive functions for mammary epithelial cells.

JNK in Fibronectin Splicing

The basement membrane communicates with the adjacent cells through cell surface proteins and can alter gene expression and transcript splicing. Pelisch et al. now provide evidence for the involvement of the c-Jun N-terminal kinase (JNK) pathway and the phosphatidyinositol 3-kinase (PI3K) pathway in the alternative splicing of fibronectin transcripts in a mammary epithelial cell line (EpH4) in response to laminin-rich basement membrane (lr-BM). Twelve isoforms of fibronectin are generated by alternative splicing of three discrete regions: EDII (extra domain II), EDI (extra domain I), and IIICS (type III connecting segment). EpH4 cells plated on lr-BM showed decreased inclusion of the EDI exon, no change in the inclusion of the EDII exon, and decreased inclusion of the IIICS exon that contains the integrin-binding motif LDV. Cells plated with lr-BM showed activation of JNK and extracellular signal-regulated kinase (ERK) but not of p38 or PI3K. Inhibition of JNK pharmacologically with silencing RNA (siRNA) or by expression of a dominant-negative blocked the effect of lr-BM on fibronectin splicing. Although PI3K inhibition or p38 inhibition did decrease the ratio of EDI + :EDI − and LDV + :LDV − transcripts in untreated cells, exposure to lr-BM further decreased these ratios, indicating that these two kinases are not involved in the lr-BM regulation of fibronectin splicing. Exposure of cells to lr-BM produced a sustained activation of JNK, but a transient activation of ERK was followed by dephosphorylation below basal levels. Pharmacological inhibition of ERK in untreated cells suggested that ERK (like p38 and PI3K) is involved in the basal inclusion of EDI- and LDV-containing IIICS exons. Overexpression of active JNK was not sufficient to alter splicing in the absence of lr-BM, which suggests that the interplay between JNK and ERK signaling may be important for the changes in splicing of fibronectin. F. Pelisch, M. Blaustein, A. R. Kornblihtt, A. Srebrow, Cross-talk between signaling pathways regulates alternative splicing: A novel role for JNK. J. Biol. Chem. 280 , 25461-25469 (2005). [Abstract] [Full Text]

Cross-talk between Signaling Pathways Regulates AlternativeSplicing: A NOVEL ROLE FORJNK

The regulation of alternative splicing by extracellular signals represents a key event in the control of gene expression. There is increasing evidence showing that many extracellular cues regulate alternative splicing. Nevertheless, the broad picture regarding the role of different signaling pathways and their interaction remains incomplete. Using the fibronectin gene as a model, we show that a laminin-rich basement membrane regulates the alternative splicing of two out of three regions of the transcript (extra domain I and type III connecting segment) in mammary epithelial cells, through a non-stress c-Jun N-terminal kinase (JNK) signaling pathway. We propose that dephosphorylation of the extracellular signal-regulated kinase is involved in this regulatory process. Furthermore, the laminin-rich basement membrane blocks the effect of a mammary mesenchymal cell-conditioned medium, which stimulates the inclusion of extra domain I and type III connecting segment through a phosphatidylinositol3-kinase-dependent cascade, indicating that JNK signaling can inhibit the phosphatidylinositol 3-kinase-mediated splicing regulation. These results implicate JNK in the regulation of alternative splicing and provide new evidence on how extracellular stimuli are converted into changes in splicing patterns, strengthening the view that the control of alternative splicing is as complex and relevant as transcriptional control, together accounting for the spatiotemporal requirements of gene expression.

A breast cancer progression model: the importance of three-dimensional tissue architecture and metalloproteinases

Previous studies from our laboratory have shown that non-malignant and malignant cells can be distinguished easily and rapidly by their morphology and growth rate when cultured in three-dimensional (3D) laminin-rich basement membrane but not when cultured on traditional tissue culture plastic (two-dimensional [2D]) [1,2]. In addition, we have shown that cellular responses to signaling inhibitors and apoptotic agents differ in cells cultured in 2D versus 3D [3,4]. This applies also to our finding with reverted tumor cell lines [3-8]. In this presentation, I will address two inter-related topics. First, we asked how the 3D morphology and gene expression profiles for a panel of 60 breast cancer cell lines for which the Gray laboratory has obtained 2D expression as well as CGH profiles may differ, and whether any of the surrogate genes or phenotypes could track with response to therapy. The cell lines examined so far fell into four distinct morphologies of 'round', 'mass', 'grape-like' and 'stellate'. An ANOVA analysis of Affymetrix gene expression profiles for each of these cell lines was used to identify genes, the expression profiles of which could distinguish the other known parameters of the cultured cells. Of the 22,283 genes on the Affymetrix 133A chip, ~5800 genes were identified where expression patterns differed between different cell lines both in 2D and 3D, and ~2000 genes were identified where expression differed between the non-malignant and malignant cell lines. About 700 genes differed between 2D and 3D, and ~800 correlated with the morphological differences seen in 3D. These genes fall into a number of functional classes, which we are currently analyzing to identify common signaling themes and/or morphological regulators that will be tested by manipulation of expression and correlated with therapeutic response of these cell lines in 2D and 3D to Herceptin and other chemotherapeutic drugs. Second, we have also shown previously that loss of basement membrane in both cultured mammary mouse cells [9] and in transgenic animals led to epithelial to mesenchymal transition (EMT) and mammary tumors [10]. We have now determined the molecular pathways induced by MMP-3 to lead to EMT and genomic instability via production of reactive oxygen species [11]. These mechanisms will be discussed.