Overexpression of monocarboxylate anion transporter 1 and 4 in T24-induced cancer-associated fibroblasts regulates the progression of bladder cancer cells in a 3D microfluidic device

Stromal fibroblasts are essential for tumor proliferation and invasion. Here we presented a 3-dimensional (3D) microfluidic co-culture device to reconstruct an in vivo-like tumor microenvironment for investigation of the interactions of cancer-associated fibroblasts (CAFs) and bladder cancer cells. With this device, we verified that the cytokines secreted by bladder cancer cells T24 effectively transform the fibroblasts into CAFs. Compared to fibroblasts, the CAFs, which undergo the aerobic glycolysis, showed higher ability to produce lactate and provide energy for bladder cancer cell proliferation and invasion. We also demonstrated that this kind of tumor-promoting effect was associated with the upregulation of monocarboxylate anion transporter 1 (MCT1) and MCT4 expression in CAFs. We concluded that MCT1 and MCT4 are involved in bladder cancer cell proliferation and invasiveness. Moreover, this 3D microfluidic co-culture device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.     

Cadherin-23 mediates heterotypic cell-cell adhesion between breast cancer epithelial cells and fibroblasts

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis.     

Interactions with fibroblasts are distinct in Basal-like and luminal breast cancers

Basal-like breast cancers have several well-characterized distinguishing molecular features, but most of these are features of the cancer cells themselves. The unique stromal-epithelial interactions, and more generally, microenvironmental features of basal-like breast cancers have not been well characterized. To identify characteristic microenvironment features of basal-like breast cancer, we performed cocultures of several basal-like breast cancer cell lines with fibroblasts and compared these with cocultures of luminal breast cancer cell lines with fibroblasts. Interactions between basal-like cancer cells and fibroblasts induced expression of numerous interleukins and chemokines, including IL-6, IL-8, CXCL1, CXCL3, and TGFβ. Under the influence of fibroblasts, basal-like breast cancer cell lines also showed increased migration in vitro. Migration was less pronounced for luminal lines; but, these lines were more likely to have altered proliferation. These differences were relevant to tumor biology in vivo, as the gene set that distinguished luminal and basal-like stromal interactions in coculture also distinguishes basal-like from luminal tumors with 98% accuracy in 10-fold cross-validation and 100% accuracy in an independent test set. However, comparisons between cocultures where cells were in direct contact and cocultures where interaction was solely through soluble factors suggest that there is an important impact of direct cell-to-cell contact. The phenotypes and gene expression changes invoked by cancer cell interactions with fibroblasts support the microenvironment and cell-cell interactions as intrinsic features of breast cancer subtypes.     

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model

In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME.     

Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation

Tumors arise from cells that have sustained genetic mutations resulting in deregulation of several of their normal growth-controlling mechanisms. Much of the research concerning the origins of cancer has focused on the genetic mutations within tumor cells, treating tumorigenesis as a cell-autonomous process governed by the genes carried by the tumor cells themselves. However, it is increasingly apparent that the stromal microenvironment in which the tumor cells develop profoundly influences many steps of tumor progression. In various experimental tumor models, the microenvironment affects the efficiency of tumor formation, the rate of tumor growth, the extent of invasiveness, and the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by paracrine signaling between epithelial tumor cells and neighboring stromal fibroblasts. In this review, we summarize recent advances in understanding the paracrine signaling interactions between epithelial cancer cells and associated fibroblasts and examine the effects of these bidirectional interactions on various aspects of carcinoma formation. We note, however, that paracrine signaling between other cell types within the carcinomas, such as endothelial cells and inflammatory cells, may play equally important roles in tumor formation and we will refer to these heterotypic interactions where relevant.     

Functional Investigation of NCI-H460-Inducible Myofibroblasts on the Chemoresistance to VP-16 with a Microfluidic 3D Co-Culture Device

Fibroblasts, the major cell type in tumor stroma, are essential for tumor growth and survival, and represent an important therapeutic target for cancers. Here we presented a microfluidic co-culture device in which the three-dimensional (3D) matrix was employed to reconstruct an in vivo-like fibroblast-tumor cell microenvironment for investigation of the role of myofibroblasts induced by lung cancer cells in the chemoresistance to VP-16. Composed of a double-layer chip and an injection pump, the device houses fibroblasts and lung cancer cells co-cultured in 3D matrix and 2D mode to induce fibroblasts to become myofibroblasts with the supplement of the medium continuously. With this device, we verified that the cytokines secreted by lung cancer cells could effectively transform the fibroblasts into myofibroblasts. Moreover, compared to fibroblasts, the myofibroblasts showed higher resistance to anticancer drug VP-16. We also demonstrated that this kind of acquired resistance in myofibroblasts was associated with the expression of Glucose-regulated protein 78 (GP78). We concluded that this device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.     

Analysis of pairwise cell interactions using an integrated dielectrophoretic‐microfluidic system

Blood vessel formation, during either normal vascular reconstruction or pathogenic tumour formation, relies upon highly organized cell–cell interactions. Isolating the function of any particular component of this cell–cell communication is often difficult, given the vast complexity of communication networks in multicellular systems. One way to address this problem is to analyse cell–cell communication on the most elementary scale—cell pairs. Here, we describe an integrated dielectrophoretic (DEP)‐microfluidic device allowing for such analysis. Single cancer and endothelial cells (ECs) and cell pairs were patterned using DEP force and cultured within a minimally stressful microfluidic channel network. Controlling both the initial cell positions and extracellular environment, we investigated cell motility in homo‐ and heterotypic cell pairs under diverse conditions. We found that secreted collagen IV and soluble vascular endothelial growth factor have considerable guidance effect on ECs at the level of two interacting cells. Cell interaction rules extracted from the experiments of cell pairs were used to mathematically predict branching patterns characteristic of developing multicellular blood vessels. This integrative analysis method can be extended to other systems involving complex multicellular interactions.     

Catalytically inactive human cathepsin D triggers fibroblast invasive growth

The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial-fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D-deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras-MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.     

Quantitative analysis of epithelial cell aggregation in the simple metazoan Hydra reveals a switch from homotypic to heterotypic cell interactions

Hydra, a member of the diploblastic phylum Cnidaria, exhibits the most basic type of organized metazoan tissues. Two unicellular sheets of polarized epithelial cells - ectoderm and endoderm - form a double layer throughout the body column. The double layer can be reestablished from single-cell suspensions by tissue-specific cell-sorting processes. However, the underlying pattern of interactions between ectodermal and endodermal epithelial cells responsible for double-layer formation is unclear. By analyzing cell interactions in a quantitative adhesion assay using mechanically dissociated Hydra epithelial cells, we show that aggregation proceeds in two steps. First, homotypic interactions within ectodermal epithelial cells (ecto-ecto) and within endodermal epithelial cells (endo-endo) form homotypic cell clusters. Second, at an aggregate size of about ten epithelial cells/cluster, ectodermal and endodermal clusters start to form heterotypic aggregates. Homotypic ecto-ecto interactions are inhibited by a polyclonal anti-Hydra membrane antiserum, and under these conditions homotypic endo-endo interactions do not proceed beyond a size of about ten epithelial cells/cluster. These data suggest that homotypic cell clusters reduce their initial homotypic affinity and acquire a new heterotypic affinity. A link between cell adhesion and cell signaling in early Hydra aggregates is discussed.     

Heterotypic cell interactions on a dually patterned surface

It is worth investigating heterotypic cell-cell interactions by mimicking their in vivo structures and environment. In the present study, physiological cellular response and behavior of hepatocytes and endothelial cells were investigated by controlling their contact periphery in a new co-culture system. Rat primary hepatocytes and bovine endothelial cells were co-cultured on a dually patterned surface. Hepatic physiological functions such as albumin secretion and ammonium metabolism were enhanced by increasing heterotypic cell-cell interactions in a patterned co-culture. Furthermore, enhanced hepatic functions through heterotypic interactions are effective within a limited area apart from endothelial cells as evidenced by immunofluorescence staining of hepatic intracellular albumin, indicating that heterotypic interactions act in a paracrine manner. Thus, heterotypic cell communications that play indispensable roles in increasing hepatic physiological functions should be obtained with an increasing periphery of two-cell domains. These findings are important for the reconstruction of complex tissues such as liver and pancreas.     

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 μm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.     

System-Wide Analysis Reveals a Complex Network of Tumor-Fibroblast Interactions Involved in Tumorigenicity

Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five) played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8) or minimally (STC1) significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti-stromal therapeutic strategies will need to be multi-targeted.     

The advantages of co-culture over mono cell culture in simulating in vivo environment

Breast cancer tissue consists of both carcinoma cells and stromal cells, and intratumoral stroma is composed of various cell types such as fibroblasts, adipocytes, inflammatory including lymphocytes and macrophage and lymphatic and blood capillaries including pericytes and endothelial cells. Recently, cell-cell communications or interactions among these cells have been considered to play an important role to cancer initiation, promotion, and progression. In particular, intratumoral fibroblasts are well known as cancer-associated fibroblast (CAF). CAF is considered to be different from normal fibroblasts in terms of promoting cancer progression through the cytokine signals. Carcinoma cell lines have contributed to the advancement of our understanding of cancer cell biology. Numerous researches have employed these carcinoma cell lines as a single- or mono-culture. However, it is also true that this mono-culture system cannot evaluate interactions between carcinoma and intratumoral stromal cells. Co-culture compositions of two different cell type of cancer tissues i.e., carcinoma cell lines and fibroblasts, were established in order to evaluate cell-cell interactions in these cancer microenvironment. This co-culture condition has the advantage of evaluating cell-cell interactions of cancer microenvironment. Therefore, in this review, we focused upon co-culture system and its application to understanding of various biological phenomenon as an ex vivo evaluation method of cancer microenvironment in breast cancer.     

Developing therapeutic proteins by engineering ligand-receptor interactions

Ligand-receptor interactions govern myriad cell signaling pathways that regulate homeostasis and ensure that cells respond properly to stimuli. Growth factors, cytokines and other regulatory elements use these interactions to mediate cell responses, including proliferation, migration, angiogenesis, immune responses and cell death. Proteins that inhibit these processes have potential as therapeutics for cancer and autoimmune disorders, whereas proteins that stimulate these processes offer promise in regenerative medicine. Although much of the focus in this area over the past decade has been on monoclonal antibodies, recently there has been increased interest in the use of non-antibody proteins as therapeutic agents. Here, we review recent advances and accomplishments in the use of rational and combinatorial protein engineering approaches to developing ligands and receptors as agonists and antagonists against clinically important targets.     

Senescence,Wound Healing,and Cancer: the PAI-1 Connection

Prolonged propagation of primary diploid fibroblasts in culture activates an ageing process known as replicative senescence, which is considered to provide a barrier against oncogenic transformation. Remarkably, both cell autonomous tumor-suppressive and cell non-autonomous tumor-promoting effects of senescent cells have been reported. Recently, we described that the p53 target gene plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of replicative senescence. PAI-1 antagonizes the protease urokinase-type plasminogen activator (uPA). Both are secreted factors and involved in heterotypic signaling processes such as wound healing, angiogenesis and metastasis. Both uPA and PAI-1 are expressed in senescent cells and their relative abundance controls proliferation downstream of p53. Here, we present data that the effects of PAI- 1 and uPA in the senescence response are not strictly cell autonomous. We discuss these findings in the context of the emerging roles of PAI-1 and uPA in heterotypic cellular signaling in senescence, wound healing and metastasis.     

The Role of the Microenvironment in Mammary Gland Development and Cancer

The mammary gland is composed of a diverse array of cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the importance of cellular interactions and microenvironmental signals in mammary gland development and cancer.The mammary gland is composed of a combination of multiple cell types that together form complex interaction networks required for the proper development and functioning of the organ. The branching milk ducts are formed by an outer myoepithelial cell layer producing the basement membrane (BM) and an inner luminal epithelial cell layer producing milk during lactation. The ducts are surrounded by the microenvironment composed of extracellular matrix (ECM) and various stromal cell types (e.g., endothelial cells, fibroblasts, myofibroblasts, and leukocytes). Large amount of data suggest that cell-cell and cell-microenvironment interactions modify the proliferation, survival, polarity, differentiation, and invasive capacity of mammary epithelial cells. However, the molecular mechanisms underlying these effects are poorly understood. The purification and comprehensive characterization of each cell type comprising normal and neoplastic human breast tissue combined with hypothesis testing in cell culture and animal models are likely to improve our understanding of the role these cells play in the normal functioning of the mammary gland and in breast tumorigenesis. In this article, we overview cellular and microenvironmental interactions that play important roles in the normal functioning of the mammary gland and their abnormalities in breast cancer.     

Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation.

Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.     

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.