Human Mammary Fibroblasts Research Articles

Paracrine Wnt signaling both promotes and inhibits human breast tumor growth

Wnt signaling in mouse mammary development and tumorigenesis has been heavily studied and characterized, but its role in human breast cancer remains elusive. Although Wnt inhibitors are in early clinical development, it is unclear whether they will be of therapeutic benefit to breast cancer patients, and subsequently, to which ones. To address this, we generated a panel of Wnt reporting human breast cancer cell lines and identified a previously unrecognized enrichment for the ability to respond to Wnt in the basal B or claudin-low subtype, which has a poor prognosis and no available targeted therapies. By co-injecting Wnt3A expressing human mammary fibroblasts with human breast cancer cell lines into mouse mammary fat pads, we showed that elevated paracrine Wnt signaling was correlated with accelerated tumor growth. Using this heterotypic system and a dual lentiviral reporter system that enables simultaneous real-time measurement of both Wnt-responsive cells and bulk tumor cells, we analyzed the outcome of elevated Wnt signaling in patient-derived xenograft (PDX) models. Interestingly, the PDX models exhibited responses not observed in the cell lines analyzed. Exogenous WNT3A promoted tumor growth in one human epidermal growth factor receptor 2-overexpressing PDX line but inhibited growth in a second PDX line obtained from a patient with triple-negative breast cancer. Tumor suppression was associated with squamous differentiation in the latter. Thus, our work suggests that paracrine Wnt signaling can either fuel or repress the growth of human breast cancers depending on yet to be determined aspects of the molecular pathways they express.

It takes two to tango in the microenvironment!

Using mortal non-tumorigenic human mammary epithelial cells and fibroblasts, Fordyce and colleagues show that an epithelial stress response promotes pro-tumorigenic changes in mammary fibroblasts. Fibroblast reprogramming was dependent on activin A or prostaglandin E2 produced by epithelial cells and, in turn, promoted enhanced migration of epithelial cells. These events in epithelial cells in vitro, including telomere loss, heightened DNA damage response, and activin A expression, are observed in breast ductal carcinoma in situ lesions surrounded by stroma bearing hallmarks of activated fibroblasts and immune and endothelial cell infiltration. Thus, reciprocal epithelial-stromal interactions facilitate progression to malignancy and occur even at the earliest stages of mammary tumorigenesis.

Abstract B17: A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts

Abstract Breast cancer progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer. This invasive transition of DCIS is defined by stromal invasion and is a life-threatening step accompanied by a dramatic drop in prognosis. In addition, the invasive transition of DCIS is largely driven by stromal alteration; thus it is critical to understand how DCIS alters the surrounding microenvironment, thus causing the cancer cells to become invasive. This study investigates the role of stromal fibroblasts in the vicinity of DCIS during the invasive transition using an innovative multidisciplinary approach that combines 3D cancer biology, microfluidics, and high-resolution imaging. We recently developed an efficient 3D microfluidic system that supports the transition from DCIS to IDC. The in vitro system employs microchannels with two inputs and one output enabling MCF10-DCIS.com cells (MCF-DCIS) and human mammary fibroblasts (HMF) to be loaded in two adjacent (side-by-side) compartments. This platform allows investigations of effects of spatial organization on the transition by independently analyzing their morphology and the modifications to the surrounding collagen architecture. Importantly, the compartmentalized platform enables monitoring of both MCF-DCIS and HMF independently including quantitative measures of the collagen architecture associated with each cell type. We observed that the HMF near MCF-DCIS became more protrusive versus HMF relatively far from MCF-DCIS. We have also begun to identify how the HMF become activated and protrusive when co-cultured with MCF-DCIS and to understand the biological function and impact of protrusive HMF during DCIS progression to IDC. We verified that the signaling based on Cathepsin D produced from MCF-DCIS and low-density lipoprotein receptor-related protein-1 (LRP1) from HMF is involved in regulation of the protrusive activity of HMF. Additionally, knocking down LRP1 in HMF inhibited the invasive transition of MCF-DCIS. This study demonstrates one possible route through which MCF-DCIS activate pre-existing fibroblasts and subsequently, leads to the modification of the ECM and the progression to IDC. Citation Format: Kyung Eun Sung, Carolyn Pehlke, Erwin Berthier, Kevin W. Eliceiri, Andreas Friedl, David J. Beebe. A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B17.

Abstract A20: Investigating gene expression alterations in the tumor microenvironment based on receptor status in breast cancer using a microfluidic multiculture platform

Abstract Numerous cellular influences regulate the behavior of cancer cells, contributing to an orchestration of responses to stimuli within the tumor microenvironment. Development of novel therapies and drug targets specific to tumor receptor status in breast cancer necessitate the understanding of tumor microenvironment interactions. The current four tumor subgroups based on receptor status differ in tumor grade, recurrence and metastasis making these tumor types unique to one another in their microenvironments. Of particular interest, the HER2+ subgroup, is associated with higher recurrence, metastasis and poor prognosis. Microfluidic multi-culture techniques provide the opportunity to manipulate multiple cell types in a highly-controlled fashion, permitting evaluation of the tumor microenvironment that is not possible in traditional culture techniques. Utilizing a microfluidic multi-culture platform, where we have the ability to culture more than two cell types in compartmentalized culture chambers separated by diffusion ports to allow for soluble factor exchange, the HER2+ breast carcinoma cell lines SKBR3 (HER2+/ER-/PR-), and BT474 (HER2+/ER+/PR+) were cultured in co- and tri-culture combinations with bone marrow stroma, human mammary fibroblast, and the human M2 monocyte lines to examine signaling interactions between cancer, fibroblast and immune cell types. Gene expression changes of tumor progression markers were then evaluated using RT-qPCR for a constellation of growth factors, receptors and cytokines. Importantly, this new device allows isolation and gene expression analysis of individual cell types subsequent to culture without the need for downstream cell separation techniques. Significant changes in gene expression of the two breast cancer cell lines were detected in multi-culture that was dependent on the tumor microenvironment. Differences in gene expression between the cancer cells based on receptor status in multi-culture were also shown. These findings suggest that both the tumor microenvironment and receptor status may be interrelated and together influence tumor progression. The use of a micro multi-culture platform allows one to easily recapitulate individual complex tumor microenvironments. Even in as simple a model as two or three cell types, shapes tumor responses, which can now be rapidly dissected using this model system. Micro multi-culture techniques have the potential to provide insight on essential tumor microenvironment interactions that would otherwise be difficult to detect in traditional culture methods, paving the way for the design of new and personalized therapies. Citation Format: Lindsey J. Maccoux, Emma M. Weinberger, Scott M. Berry, Elaine T. Alarid, David J. Beebe. Investigating gene expression alterations in the tumor microenvironment based on receptor status in breast cancer using a microfluidic multiculture platform. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A20.

Cytotoxic activity of a plant extract on cancer cells

Chemoprevention by natural products may be considered a promising approach to cancer control and management [1]. Many studies have demonstrated antiproliferative, cytostatic and cytotoxic activities of phytochemicals against cancer cells [2]. In this study, a plant extract from Arctium lappa, Berberis vulgaris and Eschscholtia californica was tested as potential anticancer agent. The antitumoral activity of this plant extract was tested on four human cancer cell lines: MCF-7 (breast carcinoma cells), Huh-7 (hepatic carcinoma cells), HTB-43 (oropharyngeal carcinoma cells) and ECV- 304 (urinary bladder carcinoma cells). The efficacy of the extract was compared to the common chemotherapeutic agent cyclophosphamide. Three plant extract concentrations were tested: 800, 650 and 450 ng/ml; for cyclophosphamide, three concentrations were assayed, according to literature data: 1300, 1000 and 850 ng/ml [3]. In addition, plant extract and cyclophosphamide were tested on two primary cell lines as controls, human gingival fibroblasts and human mammary fibroblasts. Cell viability was evaluated by the MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma] colorimetric assay and the new xCELLigence system (Roche) for real-time monitoring of cell viability. All concentrations of plant extract exhibited a high level of cytotoxicity on MCF-7, Huh-7, HTB-43 and ECV-304 cancer cells, similar to cyclophosphamide, though they slightly reduced viability of human gingival and mammary fibroblasts. Conversely, the conventional chemotherapeutic drug showed a marked cytotoxicity on control cells. The potential of the plant extract has been demonstrated in vitro on various types of cancers, suggesting a possible use of this natural product as a promising anticancer agent. Further studies are needed to ascertain its efficacy in vivo and to elucidate its mechanism(s) of action at molecular and biochemical levels.

Enabling screening in 3D microenvironments: probing matrix and stromal effects on the morphology and proliferation of T47D breast carcinoma cells

During breast carcinoma progression, the three-dimensional (3D) microenvironment is continuously remodeled, and changes in the composition of the extracellular matrix (ECM) occur. High throughput screening platforms have been used to decipher the complexity of the microenvironment and to identify ECM components responsible for cancer progression. However, traditional screening platforms are typically limited to two-dimensional (2D) cultures, and often exclude the influence of ECM and stromal components. In this work, a system that integrates 3-dimensional cell culture techniques with an automated microfluidic platform was used to create a new ECM screening platform that cultures cells in more physiologically relevant 3D in vitro microenvironments containing stromal cells and different ECM molecules. This new ECM screening platform was used to culture T47D breast carcinoma cells in mono- and co-culture with human mammary fibroblasts (HMF) with seven combinations of three different ECM proteins (collagen, fibronectin, laminin). Differences in the morphology of T47D clusters, and the proliferation of T47D cells were found in ECM compositions rich in fibronectin or laminin. In addition, an MMP enzyme activity inhibition screening showed the capabilities of the platform for small molecule screening. The platform presented in this work enables screening for the effects of matrix and stromal compositions and show promises for providing new insights in the identification of key ECM components involved in breast cancer.

Cell-Type Specific DNA Methylation Patterns Define Human Breast Cellular Identity

DNA methylation plays a role in a variety of biological processes including embryonic development, imprinting, X-chromosome inactivation, and stem cell differentiation. Tissue specific differential methylation has also been well characterized. We sought to extend these studies to create a map of differential DNA methylation between different cell types derived from a single tissue. Using three pairs of isogenic human mammary epithelial and fibroblast cells, promoter region DNA methylation was characterized using MeDIP coupled to microarray analysis. Comparison of DNA methylation between these cell types revealed nearly three thousand cell-type specific differentially methylated regions (ctDMRs). MassARRAY was performed upon 87 ctDMRs to confirm and quantify differential DNA methylation. Each of the examined regions exhibited statistically significant differences ranging from 10–70%. Gene ontology analysis revealed the overrepresentation of many transcription factors involved in developmental processes. Additionally, we have shown that ctDMRs are associated with histone related epigenetic marks and are often aberrantly methylated in breast cancer. Overall, our data suggest that there are thousands of ctDMRs which consistently exhibit differential DNA methylation and may underlie cell type specificity in human breast tissue. In addition, we describe the pathways affected by these differences and provide insight into the molecular mechanisms and physiological overlap between normal cellular differentiation and breast carcinogenesis.

Cell-extrinsic consequences of epithelial stress: activation of protumorigenic tissue phenotypes

IntroductionTumors are characterized by alterations in the epithelial and stromal compartments, which both contribute to tumor promotion. However, where, when, and how the tumor stroma develops is still poorly understood. We previously demonstrated that DNA damage or telomere malfunction induces an activin A-dependent epithelial stress response that activates cell-intrinsic and cell-extrinsic consequences in mortal, nontumorigenic human mammary epithelial cells (HMECs and vHMECs). Here we show that this epithelial stress response also induces protumorigenic phenotypes in neighboring primary fibroblasts, recapitulating many of the characteristics associated with formation of the tumor stroma (for example, desmoplasia).MethodsThe contribution of extrinsic and intrinsic DNA damage to acquisition of desmoplastic phenotypes was investigated in primary human mammary fibroblasts (HMFs) co-cultured with vHMECs with telomere malfunction (TRF2-vHMEC) or in HMFs directly treated with DNA-damaging agents, respectively. Fibroblast reprogramming was assessed by monitoring increases in levels of selected protumorigenic molecules with quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunocytochemistry. Dependence of the induced phenotypes on activin A was evaluated by addition of exogenous activin A or activin A silencing. In vitro findings were validated in vivo, in preinvasive ductal carcinoma in situ (DCIS) lesions by using immunohistochemistry and telomere-specific fluorescent in situ hybridization.ResultsHMFs either cocultured with TRF2-vHMEC or directly exposed to exogenous activin A or PGE2 show increased expression of cytokines and growth factors, deposition of extracellular matrix (ECM) proteins, and a shift toward aerobic glycolysis. In turn, these "activated" fibroblasts secrete factors that promote epithelial cell motility. Interestingly, cell-intrinsic DNA damage in HMFs induces some, but not all, of the molecules induced as a consequence of cell-extrinsic DNA damage. The response to cell-extrinsic DNA damage characterized in vitro is recapitulated in vivo in DCIS lesions, which exhibit telomere loss, heightened DNA damage response, and increased activin A and cyclooxygenase-2 expression. These lesions are surrounded by a stroma characterized by increased expression of α smooth muscle actin and endothelial and immune cell infiltration.ConclusionsThus, synergy between stromal and epithelial interactions, even at the initiating stages of carcinogenesis, appears necessary for the acquisition of malignancy and provides novel insights into where, when, and how the tumor stroma develops, allowing new therapeutic strategies.

Controlling Wettability of Boron Nitride Nanotube Films and Improved Cell Proliferation

Desired wettability has been achieved on highly hydrophobic boron nitride nanotube (BNNT) films using nitrogen/hydrogen (N2/H2) gas plasma treatments under controlled input energies and modes. Both hydrophilicity (contact angle (CA) ∼60°) and superhydrophilicity (CA < 5°) are demonstrated on BNNT films with little change of the surface morphology or the structure of individual BNNTs. The combination of continuous wave and pulse mode (CW+P) plasma shows more effective wettability modification and introduces more amine functional groups than the continuous wave (CW) plasma alone at a given input energy. (Super)hydrophilic/hydrophobic patterns have been created on BNNT films using masked plasma methods. The cell response to BNNT films is investigated for the first time. The proliferation of human primary mammary fibroblasts and a transformed mammary cell line (TXP RFP3) shows that the untreated hydrophobic BNNT films can support the growth of both cell lines, but the plasma treatments greatly enhance (up to ...

Abstract 4053: A label-free spectroscopic signature associated with hormone sensitivity in 3D co-culture models of breast cancer

Abstract Treatment of estrogen-receptor positive breast cancers relies on endocrine therapies; however, up to 30% of patients become resistant to this treatment. It is beneficial to identify patients who will remain sensitive to endocrine therapy using markers inherent in tissue at the time of diagnosis. Using a three-dimensional (3D) co-culture, we developed a method to determine the chemical signature of hormone sensitivity based on Fourier Transform Infrared (FT-IR) spectroscopic imaging, a highly sensitive label-free method that can detect chemical changes in tissue samples. We used a 3D co-culture model comprised of MCF7 breast cancer cells and MCF7 Tam-resistant cells grown as spheroids on Matrigel™ and different densities of human mammary fibroblasts (HMFs) embedded within collagen. Upon treatment with 17β-estradiol (E2) or tamoxifen (Tam), samples were paraffin embedded for imaging and, in parallel, RNA was isolated to determine hormone sensitivity. MCF7 grown in 3D showed comparable E2 and Tam sensitivity compared to 2D culture as determined through expression of estrogen-responsive genes. Co-culture led to a decrease in the ability of E2 and Tam to regulate known response genes, and this was dependent on fibroblast density. Furthermore, EMT markers were up-regulated after co-culture, showing that this model more closely mimics an early, more invasive tumor stage. FT-IR detection of spectroscopic features identified changes occurring after treatment with Tam in MCF7 grown alone or in co-culture. A significant decrease in a peak associated with nucleic acids (1080 cm-1) was seen compared to untreated or Tam-resistant cells, emphasizing the inhibitory effects of Tam on proliferation of MCF7. Moreover, Tam induced a significant decrease in peaks associated with fatty acid synthesis (2930 cm-1, 2850 cm-1) that have been related to apoptosis. When cells were exposed to E2 in the presence of HMFs, there was a distinct increase in the peak associated with glycosylation (1030 cm-1), suggesting a synergistic metabolic effect between E2 and the surrounding microenvironment. Based on the application of FT-IR, we identified a novel spectroscopic signature that can distinguish between hormone-responsive and non-responsive tumor cell lines. Although there is already considerable insight into molecular signatures of endocrine response in cell culture and in vivo models of breast cancer, FT-IR spectroscopic imaging provides a label-free and global chemical signature that could be used in addition to immunohistochemical analysis of the initial biopsy as a way to predict endocrine sensitivity. (Supported by grants from The Breast Cancer Research Foundation to BSK, DOD postdoctoral fellowship (W81XWH-09-1-0398) to AB, NIH NCI Alliance for Nanotechnology in Cancer ‘Midwest Cancer Nanotechnology Training Center’ (R25 CA154015A) to SEH, and National Institutes of Health (RO1CA138882) to RB.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4053. doi:1538-7445.AM2012-4053

Abstract 468: 70kDa fibronectin splice variant induced human mammary fibroblast migration

Abstract A soluble factor hypothesized to play an important role in metastasis is migration stimulating factor (MSF). MSF is a truncated splice variant of fibronectin, with a structure nearly identical to the long-studied 70 kDa N-terminal catheptic-D fragment. MSF is secreted by fetal fibroblasts, some tumor cells, tumor-associated macrophages (TAM), cancer associated fibroblasts (CAF), and tumor-associated vascular endothelial cells. MSF is a potent bioactivator of normal adult fibroblast, and endothelial cell migration, but previous studies have failed to regulate and characterize the MSF gradient and the gradient effect on cell migration. In the current study, we compared the motogenic activity of the 70 kDa fibronectin N-terminal fragment (70kDA-FN) at different concentrations to baseline media containing 2% fetal calf serum (FCS) using microfluidic channels with well-characterized chemoattractant gradients. Microfluidic channels with 5um x 1um x 500 um dimensions were developed from PDMS. They comprise of two half-Y-shaped channels connected by a 1.35 um long gradient controlling migration channel. In this design, cells were introduced into the right half channel. To distinguish gradient dependent migration from random migration, MSF was introduced into the non-cell-containing half channel for chemotaxis, or placed directly into the cell containing half channel for chemokinesis. GFP-labeled human mammary fibroblast (HMF; originally termed RMF/EG) cells were provided by Dr. Kuperwasser, and maintained in high glucose DMEM supplemented with 10% FCS. Prior to the experiments, HMFs were cultured overnight in DMEM supplemented with 2% FCS. Microfluidic channels were imaged after 36 hours by an Olympus IX71 microscope, and migrating cells were quantified using the custom image analysis program JeX. Prior to the migration experiments, time-lapse fluorescent imaging of FITC-labeled 70kDa-FN was used to characterize and verify the presence of the chemoattractant gradient for 36 hours. The numbers of cells present in different experimental conditions were compared using two-sided Mann-Whitney Wilcoxon test. There was a statistically significant difference between the chemotactic experimental conditions containing 70kDa-FN concentrations of 14pMol/mL (p&amp;lt;0.01) and 1.4pMol/mL (p&amp;lt;0.05). Normalizing to migrating cells present in the control channel, the 14pMol/mL and 1.4pMol/mL 70kDa-FN chemotaxis channels on average contained 70% and 53% more cells, respectively. Furthermore, the experimental setups testing chemokinesis on average contained more cells compared to the control, supporting a possible role for 70kDa-FN in HMF chemokinesis. MSF and 70kDa-FN are potent activators of fibroblast migration. In the current experiment we utilized microfluidic channels to test and confirm that 70kDa-FN induced fibroblast migration is both chemokinetic and chemotactic, and dose dependent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 468. doi:1538-7445.AM2012-468

Abstract 1500: Bone morphogenetic proteins stimulate mammary fibroblasts to promote mammary tumorigenesis

Abstract Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor ≤ (TGFβ) superfamily. BMPs have recently been shown to be overexpressed in human breast cancers, however loss of BMP signaling in mammary carcinomas has also been shown to accelerate metastases. Stimulation of dermal fibroblasts with BMP4 can enhance pro-tumorigenic factors uniquely from the epidermal response. Additionally, treatment of carcinoma-associated fibroblasts (CAFs) derived from a mouse prostate carcinoma with BMP have been reported to stimulate tumor-associated angiogenesis. We sought to determine the effect of BMP treatment on mammary fibroblasts and determine their response during mammary tumor progression. We first identified secreted pro-inflammatory cytokines and matrix-metalloproteinases (MMPs) upregulated in response to BMP4 treatment. We found that fibroblasts stimulated with BMP4 could enhance tumor invasion, which was inhibited by a BMP receptor kinase antagonist. We next demonstrated that BMP signaling was specifically absent in the stroma of human ductal and lobular carcinoma in situ (DCIS &amp; LCIS). Yet after progression to invasion, breast tumors of many distinct subtypes contained a stroma active for BMP signaling. We further extended these observations with human normal mammary fibroblasts and compared them to human fibroblasts from breast cancer and found that BMP was capable of inducing MMP3 and IL-6 secretion in normal fibroblasts. However carcinoma associated fibroblasts had constitutively elevated levels of MMP3 and IL-6 that corresponded to enhanced BMP signaling in addition to loss of secreted BMP antagonists. These experiments demonstrate that BMP may play important tumor promoting functions within the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1500. doi:1538-7445.AM2012-1500

Abstract 1502: Microtubule stabilization alters tumor secretome and fibroblast activation

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs that target MTs, like the taxanes, often fail in the metastatic setting. Therefore it is of utmost importance to fully understand the roles of MTs and MT-targeting agents in tumor biology. An important process for tumor development and metastasis involves the remodeling of the 3D microenvironment surrounding tumor cells. In order to successfully proliferate and metastasize, tumor cells need to dynamically respond to, manipulate, and remodel their surrounding microenvironment. We hypothesized that MTs, as a dynamic cytoskeletal component, regulates the cellular signaling process that controls tumor-mediated remodeling of the microenvironment. To test this hypothesis, we assessed the MT-dependence of metastatic MDA-MB-231 (231) breast cancer cell activation of HMF3S human mammary fibroblasts. We collected media conditioned by vehicle- or paclitaxel (PTX)-pretreated 231 cells and then applied this conditioned media to HMF3S fibroblasts. Application of 231-derived conditioned media activated HMF3S fibroblasts as evidenced by their enhanced directional 3D motility towards a serum gradient. However, application of conditioned media from PTX-pretreated 231 cells to HMF3S fibroblasts inhibited their 3D directional motility. Importantly, we demonstrated that this effect was due to PTX-induced MT stabilization as conditioned media from PTX-treated 231 cells resistant to PTX did not inhibit fibroblasts’ 3D motility. Together, these results suggest that the composition of the tumor secretome changes upon MT stabilization by PTX, which in turn affects the ability of tumor cells to communicate with fibroblasts. In order to identify the factors responsible for this MT-dependent cell-cell communication we performed a differential proteomic analysis of the conditioned media from vehicle- and PTX-pretreated 231 cells. Using splitless nanoflow chromatography coupled with quadrupole time-of-flight mass spec, we identified a total of 2124 proteins within these tumor secretomes. Using a 2-fold change threshold, we identified 78 under-represented and 31 over-represented proteins in the secretome of PTX- versus vehicle-pretreated cells. We focused our analysis on under-represented proteins based on the hypothesis that MT stabilization would inhibit their secretion. From this analysis we have identified proteins involved in cell motility, cell-cell communication, or ECM remodeling. TGFβ, CTGF, cMet, fibronectin, and lysyl oxidase 2 were key down-regulated proteins identified in the tumor secretome upon PTX-induced MT stabilization. Currently we are investigating the mechanisms by which stabilization of the MT cytoskeleton results in the down-regulation of these factors. These findings point to a novel role of interphase MTs in tumor biology and can possibly enhance our understanding of taxane clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1502. doi:1538-7445.AM2012-1502

Scavenging of CXCL12 by CXCR7 promotes tumor growth and metastasis of CXCR4-positive breast cancer cells

Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7 dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈ 30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small molecule inhibitor of CXCR7 chemokine scavenging limited growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes (1-3). The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression(4,5). These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues (6). The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients. Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis (7-10). CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model. In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance (11). We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Epigenetic regulation of normal human mammary cell type–specific miRNAs

Epigenetic mechanisms are important regulators of cell type-specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type-specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type-specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type-specific miRNA expression.

Automation of three-dimensional cell culture in arrayed microfluidic devices.

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions.

Abstract 423: Decipher the molecular mechanisms by which syndecan-1 in breast cancer stroma fibroblasts regulates extracellular matrix fiber organization and carcinoma cell motility

Abstract The cell surface proteoglycan syndecan-1 (Sdc1) is frequently induced in the stromal fibroblasts of invasive breast carcinomas. We have recently identified a striking correlation between stromal Sdc1 expression and extracellular matrix (ECM) fiber architecture, both In vivo (in human breast carcinoma samples) and in vitro. Bioactive, cell-free 3D ECMs generated from cultures of Sdc1-positive human mammary fibroblasts (HMF), termed ECM-Sdc1, showed an organized, aligned fiber architecture, which contrasted markedly with the random fiber arrangement seen in the ECM of Sdc1-negative HMFs (ECM-mock). We further demonstrated that aligned ECM fiber architecture was responsible for the enhanced directional migration and invasion of the breast carcinoma cells when seeded into the fibroblast-free ECM-Sdc1. To decipher the molecular mechanisms responsible for the formation of an invasion-permissive ECM, a series of Sdc1 mutants was introduced into HMF. These include a heparan sulfate (HS) -deficient, a cytoplasmic domain (CD) deletion and a transmembrane domain (TMD) substitution mutant. Early experiments suggest that HS are required for the activity of Sdc1 in regulating ECM alignment, while TDM is dispensable. Integrins play a key role in the assembly of the provisional fibronectin-rich ECM and Sdc1 has been shown to be an important regulator of integrin activity. Therefore, we have begun to investigate the the role of integrins in Sdc1-mediated ECM fiber alignment. HMF expresses various integrins, as determined by Western blotting and flow cytometry. Using siRNA oligos, we are targeting different integrin subunits including α4, α5, αV and β3 to determine, which integrins are responsible for Sdc1-dependent ECM fiber alignment and consequently the invasion-permissive properties of the ECM. A better understanding of the mechanisms governing ECM organization may lead to the development of novel stroma-targeted therapy for breast cancer. 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 423. doi:10.1158/1538-7445.AM2011-423

Abstract 435: MT regulation of tumor-dependent microenvironment remodeling

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs that target MTs, like the taxanes, often fail in the metastatic setting. Most studies are performed in a two-dimensional monoculture thus, overlook the complexity of the tumor microenvironment. Therefore, to gain a better understanding of tumor MT biology within a physiological context we studied the role of MT dynamics in 3D co-cultures closer mimicking the tumor microenvironment. Since tumor cells need to remodel their ECM in order to metastasize we hypothesized that the interphase MT cytoskeleton, through the coordination of cellular signaling regulates this process. To test this hypothesis we first assessed the MT-dependence of tumor-mediated ECM remodeling, using a floating collagen gel contraction assay. In this assay, breast cancer cells were embedded into a floating collagen gel and monitored for their ability to contract the gel as a result of cell generated traction force. We observed that the MDA-MB-231 metastatic breast cancer cells exhibited greater contractility as compared with the MCF-7 non-metastatic breast cancer cells or the non-tumorigenic human breast epithelial cell line MCF10A. However upon disruption of MTs with paclitaxel (PTX), tumor cell contractility was severely inhibited at drug concentrations that did not affect cell number, suggesting that dynamic MTs are essential for cell traction force generation. In addition, we observed that human mammary fibroblasts when co-cultured with breast cancer cells in 3D exhibited increased motility as compared to 3D fibroblast monocultures. Interestingly, fibroblast activation was inhibited in co-cultures with breast cancer cells previously treated with a taxane, suggesting that tumor MT stabilization negatively affects fibroblast activation. To investigate the factors that mediated the tumor cell communication with surrounding fibroblasts we monitored fibroblast motility in 3D monocultures following incubation with conditioned medium collected from either untreated or Taxol pre-treated breast cancer cells. In agreement with our 3D co-culture results, fibroblast 3D motility was inhibited following exposure to conditioned medium from the Taxol-pretreated cells. These results suggest that the composition of the tumor secretome changes upon MT stabilization, which in turn affects the ability of tumor cells to communicate with stromal fibroblasts. Currently, we are performing proteomic analysis of the conditioned media to identify soluble factors whose secretion is dependent on dynamic microtubules. In addition, to determine whether the effects of taxane treatment is solely due to microtubule stabilization we are performing the 3D co-culture experiments using a Taxol-resistant β-tubulin mutant breast cancer cell line. These findings point to a novel role of interphase MTs in tumor biology and can possibly lead to new avenues for taxane clinical efficacy. 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 435. doi:10.1158/1538-7445.AM2011-435