The Th2 cytokines interleukin (IL)-4 and -13 are acknowledged regulators of lymphocyte proliferation and activation. They have also been well studied in the regulation of various myeloid-derived ...populations in tumor biology. It has become clear, however, that both cytokines can have direct effects on epithelial tumor cells expressing appropriate receptors. Changes in tumor proliferation, survival, and metastatic capability have all been ascribed to IL-4 and/or IL-13 action. Here, we evaluate the evidence to support direct tumor-promoting roles of these cytokines. We also identify the questions that should be addressed before proceeding with therapeutic approaches based on neutralization of IL-4 or IL-13 pathways.
Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis ...are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.
The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme.
AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascularized and contained more apoptotic cells compared to control and untreated tumors.
These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.
Catalytic oligonucleotides, known as DNAzymes, are a new class of nucleic acid-based gene therapy that have recently been used in preclinical animal studies to treat various cancers. In this study ...the systemic distribution, pharmacokinetics, and safety of intravenously administered anti-MMP (matrix metalloproteinase)-9 DNAzyme (AM9D) were determined in healthy FVB and in MMTV-polyoma virus middle T (PyMT) transgenic mice bearing mammary tumors. MMP-9 is known to be involved in tumor cell development, angiogenesis, invasion, and metastasis. Sulfur-35 ((35)S) labeled ((35)S-AM9D) administered intravenously, without the use of carrier molecules, to healthy and mammary tumor bearing MMTV-PyMT transgenic mice distributed to all major organs. The order of percentages of (35)S-AM9D accumulation in different organs of healthy and MMTV-PyMT mice were blood>liver>kidney>lung>spleen>heart and mammary tumor>blood≈liver>kidney>spleen>lung>heart, respectively. The amount of AM9D accumulated in mammary tumors 2 hours post injection was 0.6% and 0.2% higher than in either blood or liver, respectively, and its rate of initial clearance from mammary tissue was at least 50% slower than the other organs. Approximately 43% of the delivered dosage of (35)S-AM9D was cleared from the system via feces and urine over a period of 72 hours. No evidence of acute or chronic cytotoxicity, local or widespread, associated with AM9D treatment (up to 75 mg AM9D /kg of body weight) was observed in the organs examined. These data suggest that DNAzyme in general and AM9D in particular can be used systemically as a therapeutic agent to treat patients with breast cancer or other metastatic and surgically inaccessible tumors.
Abstract
The leading cause of cancer mortality is metastasis of a primary tumor to distant organs. It is thought that stromal micro-environmental changes occur at these secondary sites before the ...arrival of tumor cells, which facilitate future colonization and outgrowth. This occurrence is commonly termed as the formation of the “pre-metastatic niche.” Specific factors altered at pre-metastatic niches have already been described in literature (Nature. 2005 Dec 8;438(7069):820-7). These include VEGFR-1, VLA-4 and Fibronectin. Matrix Metalloproteinases (MMPs), known for their involvement in tumor invasion and metastasis, have also been implicated in niche formation. Our goals are to (1) determine the roles of MMPs in generation of the pre-metastatic niche and (2) develop a method of quantification for these niches in the lung utilizing a malignant breast cancer type.
In a distant murine LLC-implantation model, we developed immunofluorescent (IF) staining techniques to define pre-metastatic niches within the lung based on the molecules identified by Kaplan et al. Within this same model, qRT-PCR indicated a substantial increase in lung MMP9 (Gelatinase B) gene expression. This led us to reproduce the pre-metastatic niche within a breast cancer model using a time-coursed malignant murine 4T1-GFP implantation model within Balb/c mammary glands. Visualization was again achieved by IF staining of known factors obtained from Kaplan et al. We utilized these models and the clusters formed to create an algorithm for quantifying the pre-metastatic niche. To confirm this algorithm is specific for premetastatic niches in metastatic tumor models, we also implanted 4T1-GFP cells into MMP9-/- Balb/c mice. These mice result in unorganized protein expression and lack of cluster formation. In addition we utilized a non-metastatic mammary tumor model, 67NR also implanted in Balb/c mice to serve as a negative control for our algorithm. Thus, to confirm the pre-metastatic niche is specific to metastatic tumor models.
Eventually, pre-metastatic MMP activity may indicate potential roles and markers for metastatic spread. Further, this algorithm will provide a new quantitative standard for the field. Thus, this algorithm has translational potential as a test for specific drug efficacy.
Citation Format: Miranda A. Hallett, E. Ashley Dozier, Joseph T. Roland, Barbara Fingleton. Quantifying the contribution of matrix metalloproteinases to the development of the pre-metastatic niche in the lung and liver microenvironments. abstract. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B57. doi:10.1158/1538-7445.CHTME14-B57
The Th2 cytokines interleukins-4 and -13 (IL4; IL13) are acknowledged regulators of lymphocyte proliferation and activation. They have also been well-studied in the regulation of various ...myeloid-derived populations in tumor biology. It has become clear however, that both that cytokines can have direct effects on epithelial tumor cells expressing appropriate receptors. Changes in tumor proliferation, survival and metastatic capability have all been ascribed to IL4 and/or IL13 action. Here, we evaluate the evidence to support direct tumor-promoting roles of these cytokines. We also identify the questions that should be addressed before proceeding with therapeutic approaches based on neutralization of IL4 or IL13 pathways.
BACKGROUND: Tumor cell proliferation, invasion and metastasis are known to be mediated, at least in part, through degradation of basement membrane by neutral metalloproteinases (MMP) produced by ...tumor and stromal cells. MMP−9 is known to play a significant role in breast tumor cell invasion and metastasis via degradation of ECM components and activation of cytokines and chemokines. MMP−9 is known to cleave type IV collagen, one of the most abundant collagens of the extracellular matrix. Our lab has found that the fully processed (68 kDa) form of MMP−9 generated in tumor and stromal cells is also capable of cleaving type I collagen molecule with similar characteristics of MMP−1. This observation raises questions regarding the significance of each MMP in tumor cell invasion and metastases. Thus, in combating tumor growth and invasion, understanding the mechanism of substrate specificity and design of a novel therapeutic compound that is capable of specifically down regulating MMP−9 expression are of interest. METHOD: DNAzymes are catalytic oligonucleotides that bind to and cleave specific mRNA, resulting in a decreased protein expression. The safety and efficacy of anti−MMP−9 DNAzyme (AM9D) in vivo was determined by injecting 35S−labeled AM9D (35S−AM9D) into the tail vein of healthy and MMTV−PyMT transgenic mice and the amount of 35S−AM9D accumulated in different tissues was measured as a function of time. The effect of AM9D treatment on breast tumor progression was tested by four once−weekly intratumoral injections of two concentrations of AM9D into mammary tumors of MMTV−PyMT transgenic mice at early stages of tumor development. The tumor sizes were measured at the end of the experiments. The effect of AM9D treatment on early stage experimental metastasis and survival was evaluated by injecting FVB female mice with 1x106 luciferase labeled mammary tumor epithelial cells (luc−MTEC) isolated from MMTV−PyMT mice and treating the animals with AM9D, control DNAzyme, or PBS for 3 weeks or up to 10 weeks, respectively. The mechanism of substrate specificity of 68 kDa MMP−9 was determined by generating a series of truncated and site directed mutant forms of MMP−9. Specific amino acid residue in sequence 444PRPEPEPRPPTTT 456 in the hinge region responsible for substrate selectivity of the enzyme was then identified by alanine−scanning site directed mutagenesis. The charged amino acid residues (Arg445, Glu447, Glu449, and Arg451) were individually replaced by Ala and the proteolytic activity of the mutant enzymes toward Mca−PLGL(Dpa)AR−NH2, gelatin, and type I collagen was determined. RESULTS: Treatment of MDA−MB−231 breast cancer cell line with AM9D in vitro resulted in decreased invasion potential of the cells and intratumoral treatment of MMTV−PyMT mice in vivo resulted in delayed rate of tumor growth and retarded final tumor volume by up to 51%. This decrease in tumor growth was correlated with decreased MMP−9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascular compared to control and untreated tumors. Furthermore, DNAzyme administered IV is distributed to major organs including lung, without showing any organ toxicity. Intravenous administration of AM9D and control DNAzyme in animals bearing luc−MTECs were able to decrease the number of gross lung macro metastasis. In addition, AM9D treatment increased progression−free survival but did not have an effect on overall survival of animals inoculated with luc−MTECs compared to control DNAzyme and PBS treatment. These data indicate that AM9D can be used individually or as an adjuvant to current chemotherapy for breast cancer. To further illuminate the role of MMP−9 in tumor growth and metastases the mechanism of substrate specificity of the enzyme was studied. Site directed and deletion mutagenesis revealed that interaction of Glu415 in the active site with Glu447 and/or Arg451 in the hinge region makes the active site rigid, preventing full length MMP-9 from cleaving type I collagen. Disruption of this interaction, on the other hand, provides the flexibility necessary for the enzyme's active site to change conformation and be able to bind and cleave type I collagen substrate. CONCLUSION: Given that MMP-9 is capable of cleaving type I collagen and its down regulation hinders the development of breast cancer in an animal model, AM9D could prove useful as an adjuvant therapy against breast carcinoma.
Death threatens migrants physically during perilous border crossings between Central and North America, but many also experience legal, social, and economic mortality. Rooted in histories of ...colonialism and conquest, exclusionary policies and practices deliberately take aim at racialized, dispossessed people in transit. Once in the new land, migrants endure a web of systems across every facet of their world—work, home, healthcare, culture, justice—that strips them of their personhood, denies them resources, and creates additional obstacles that deprive them of their ability to live fully.As laws and policies create ripe conditions for the further extraction of money, resources, and labor power from the dispossessed, the contributors to this vibrant anthology, Migration and Mortality, examine restrictive immigration policies and the broader capitalist systems of exploitation and inequality while highlighting the power of migrants' collective resistance and resilience. The case studies in this timely collection explore border deaths, detention economies, asylum seeking, as well as the public health and mental health of migrants. Ultimately, these examples of oppression and survival contribute to understanding broader movements for life and justice in the Americas.
We investigated the effect of electrode area and inter-electrode distance on the spatial distribution of the current density in transcranial direct current stimulation (tDCS). For this purpose, we ...used the finite element method to compute the distribution of the current density in a four-layered spherical head model using various electrode montages, corresponding to a range of electrode sizes and inter-electrode distances. We found that smaller electrodes required slightly less current to achieve a constant value of the current density at a reference point on the brain surface located directly under the electrode center. Under these conditions, smaller electrodes also produced a more focal current density distribution in the brain, i.e. the magnitude of the current density fell more rapidly with distance from the reference point. The combination of two electrodes with different areas produced an asymmetric current distribution that could lead to more effective and localized neural modulation under the smaller electrode than under the larger one. Focality improved rapidly with decreasing electrode size when the larger electrode sizes were considered but the improvement was less marked for the smaller electrode sizes. Also, focality was not affected significantly by inter-electrode distance unless two large electrodes were placed close together. Increasing the inter-electrode distance resulted in decreased shunting of the current through the scalp and the cerebrospinal fluid, and decreasing electrode area resulted in increased current density on the scalp under the edges of the electrode. Our calculations suggest that when working with conventional electrodes (25-35 cm(2)), one of the electrodes should be placed just 'behind' the target relative to the other electrode, for maximum current density on the target. Also electrodes with areas in the range 3.5-12 cm(2) may provide a better compromise between focality and current density in the scalp than the traditional electrodes. Finally, the use of multiple small return electrodes may be more efficient than the use of a single large return electrode.
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