Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer.

Cells grown in three dimensions (3D) within natural extracellular matrices or synthetic scaffolds more closely recapitulate the phenotype of those cells within tissues in regard to normal developmental and pathobiological processes. This includes degradation of the surrounding stroma as the cells migrate and invade through the matrices. As 3D cultures of tumor cells predict efficacy of, and resistance to, a wide variety of cancer therapies, we employed tissue-engineering approaches to establish 3D pathomimetic avatars of human breast cancer cells alone and in the context of both their cellular and pathochemical microenvironments. We have shown that we can localize and quantify key parameters of malignant progression by live-cell imaging of the 3D avatars over time (4D). One surrogate for changes in malignant progression is matrix degradation, which can be localized and quantified by our live-cell proteolysis assay. This assay is predictive of changes in spatio-temporal and dynamic interactions among the co-cultured cells and changes in viability, proliferation, and malignant phenotype. Furthermore, our live-cell proteolysis assay measures the effect of small-molecule inhibitors of proteases and kinases, neutralizing or blocking antibodies to cytokines and photodynamic therapy on malignant progression. We suggest that 3D/4D pathomimetic avatars in combination with our live-cell proteolysis assays will be a useful preclinical screening platform for cancer therapies. Our ultimate goal is to develop 3D/4D avatars from an individual patient's cancer in which we can screen "personalized medicine" therapies using changes in proteolytic activity to quantify therapeutic efficacy.

Breast Cancer: Proteolysis and Migration.

Understanding breast cancer cell proteolysis and migration is crucial for developing novel therapies to prevent local and distant metastases. Human cancer cells utilize many biological functions comparable to those observed during embryogenesis conferring the cancer cells with survival advantages. One such advantage is the ability to secrete proteases into the tumor microenvironment in order to remodel the extracellular matrix to facilitate migration. These proteases degrade the extracellular matrix, which initially functions as a barrier to cancer cell escape from their site of origin. The extracellular matrix also functions as a reservoir for growth factors that can be released by the secreted proteases and thereby further aid tumor growth and progression. Other survival advantages of tumor cells include: the ability to utilize multiple modes of motility, thrive in acidic microenvironments, and the tumor cell's ability to hijack stromal and immune cells to foster their own migration and survival. In order to reduce metastasis, we must focus our efforts on addressing the survival advantages that tumor cells have acquired.

Il-6 signaling between ductal carcinoma in situ cells and carcinoma-associated fibroblasts mediates tumor cell growth and migration.

BACKGROUND: Ductal carcinoma in situ (DCIS) is a non-obligate precursor lesion of invasive breast cancer in which approximately half the patients will progress to invasive cancer. Gaining a better understanding of DCIS progression may reduce overtreatment of patients. Expression of the pro-inflammatory cytokine interleukin-6 increases with pathological stage and grade, and is associated with poorer prognosis in breast cancer patients. Carcinoma associated fibroblasts (CAFs), which are present in the stroma of DCIS patients are known to secrete pro-inflammatory cytokines and promote tumor progression. METHODS: We hypothesized that IL-6 paracrine signaling between DCIS cells and CAFs mediates DCIS proliferation and migration. To test this hypothesis, we utilized the mammary architecture and microenvironment engineering or MAME model to study the interactions between human breast CAFs and human DCIS cells in 3D over time. We specifically inhibited autocrine and paracrine IL-6 signaling to determine its contribution to early stage tumor progression. RESULTS: Here, DCIS cells formed multicellular structures that exhibited increased proliferation and migration when cultured with CAFs. Treatment with an IL-6 neutralizing antibody inhibited growth and migration of the multicellular structures. Moreover, selective knockdown of IL-6 in CAFs, but not in DCIS cells, abrogated the migratory phenotype. CONCLUSION: Our results suggest that paracrine IL-6 signaling between preinvasive DCIS cells and stromal CAFs represent an important factor in the initiation of DCIS progression to invasive breast carcinoma.

Metabotropic glutamate receptor-1 contributes to progression in triple negative breast cancer.

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.

Many Roles of CCL20: Emphasis on Breast Cancer.

CCL20 or MIP3α is a small ~8 kDa protein primarily expressed in the liver, colon, prostate, cervix, and skin. The cellular receptor for CCL20 is CCR6. CCl20 unlike many other cytokines only binds CCR6, making the CCL20/CCR6 pathway an attractive drug target. Since the initial discovery of CCL20 in the early 1990's, there has been an increase in the evidence implicating the chemokine and its receptor in a number of diseases, including rheumatoid arthritis and human immunodeficiency virus infection. CCL20 has also been linked to malignancies such as ovarian, colorectal and pancreatic cancers. CCL20 can also attract tumor-promoting immune-suppressive cells to the tumor microenvironment, which may contribute to the immune evasive potential of the tumor and tumor progression.

Physiological and genomic consequences of adrenergic deficiency during embryonic/fetal development in mice: impact on retinoic acid metabolism.

Adrenergic hormones are essential for early heart development. To gain insight into understanding how these hormones influence heart development, we evaluated genomic expression changes in embryonic hearts from adrenergic-deficient and wild-type control mice. To perform this study, we used a mouse model with targeted disruption of the Dopamine ß-hydroxylase (Dbh) gene, whose product is responsible for enzymatic conversion of dopamine into norepinephrine. Embryos homozygous for the null allele (Dbh(-/-)) die from heart failure beginning as early as embryonic day 10.5 (E10.5). To assess underlying causes of heart failure, we isolated hearts from Dbh(-/-) and Dbh(+/+) embryos prior to manifestation of the phenotype and examined gene expression changes using genomic Affymetrix 430A 2.0 arrays, which enabled simultaneous evaluation of >22,000 genes. We found that only 22 expressed genes showed a significant twofold or greater change, representing ~0.1% of the total genes analyzed. More than half of these genes are associated with either metabolism (31%) or signal transduction (22%). Remarkably, several of the altered genes encode for proteins that are directly involved in retinoic acid (RA) biosynthesis and transport. Subsequent evaluation showed that RA concentrations were significantly elevated by an average of ~3-fold in adrenergic-deficient (Dbh(-/-)) embryos compared with controls, thereby suggesting that RA may be an important downstream mediator of adrenergic action during embryonic heart development.

Adrenergic deficiency leads to impaired electrical conduction and increased arrhythmic potential in the embryonic mouse heart.

To determine if adrenergic hormones play a critical role in the functional development of the cardiac pacemaking and conduction system, we employed a mouse model where adrenergic hormone production was blocked due to targeted disruption of the dopamine ß-hydroxylase (Dbh) gene. Immunofluorescent histochemical evaluation of the major gap junction protein, connexin 43, revealed that its expression was substantially decreased in adrenergic-deficient (Dbh-/-) relative to adrenergic-competent (Dbh+/+ and Dbh+/-) mouse hearts at embryonic day 10.5 (E10.5), whereas pacemaker and structural protein staining appeared similar. To evaluate cardiac electrical conduction in these hearts, we cultured them on microelectrode arrays (8×8, 200 µm apart). Our results show a significant slowing of atrioventricular conduction in adrenergic-deficient hearts compared to controls (31.4±6.4 vs. 15.4±1.7 ms, respectively, p<0.05). To determine if the absence of adrenergic hormones affected heart rate and rhythm, mouse hearts from adrenergic-competent and deficient embryos were cultured ex vivo at E10.5, and heart rates were measured before and after challenge with the ß-adrenergic receptor agonist, isoproterenol (0.5 µM). On average, all hearts showed increased heart rate responses following isoproterenol challenge, but a significant (p<0.05) 225% increase in the arrhythmic index (AI) was observed only in adrenergic-deficient hearts. These results show that adrenergic hormones may influence heart development by stimulating connexin 43 expression, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm during a critical phase of embryonic development.

Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart.

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the ß-galactosidase (ßGAL) reporter gene in cells of an adrenergic lineage. ßGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

Generation of novel reporter stem cells and their application for molecular imaging of cardiac-differentiated stem cells in vivo.

Stem cell therapies offer the potential for repair and regeneration of cardiac tissue. To facilitate evaluation of stem cell activity in vivo, we created novel dual-reporter mouse embryonic stem (mES) cell lines that express the firefly luciferase (LUC) reporter gene under the control of the cardiac sodium-calcium exchanger-1 (Ncx-1) promoter in the background of the 7AC5-EYFP mES cell line that constitutively expresses the enhanced yellow fluorescent protein (EYFP). We compared the ability of recombinant clonal cell lines to express LUC before and after induction of cardiac differentiation in vitro. In particular, one of the clonal cell lines (Ncx-1-43LUC mES cells) showed markedly enhanced LUC expression (45-fold increase) upon induction of cardiac differentiation in vitro. Further, cardiac differentiation in these cells was perpetuated over a period of 2-4 weeks after transplantation in a neonatal mouse heart model, as monitored by noninvasive bioluminescence imaging (BLI) and confirmed via postmortem immunofluorescence and histological assessments. In contrast, transplantation of undifferentiated pluripotent Ncx-1-43LUC mES cells in neonatal hearts did not result in detectable levels of cardiac differentiation in these cells in vivo. These results suggest that prior induction of cardiac differentiation in vitro enhances development and maintenance of a cardiomyocyte-like phenotype for mES cells following transplantation into neonatal mouse hearts in vivo. We conclude that the Ncx-1-43LUC mES cell line is a novel tool for monitoring early cardiac differentiation in vivo using noninvasive BLI.