Rat Model Of Breast Cancer Research Articles

Abstract 193: Effect of omega-3 fatty acid and dietary energy restriction on mammary carcinogenesis, circulating biomarkers and molecular regulators in rats.

Abstract Available evidence indicates that diets enriched in the ratio of n-3 fatty acids or dietary energy restriction (DER) have a broad range of health-related benefit including prevention of breast cancer. However, the number of studies in the combined effects of these intervention strategies have been investigated is limited. The objective of this investigation was to determine the effect of the combination of dietary n-3 and DER on mammary carcinogenesis, on plasma levels of cancer associated growth factors and cytokines and on molecular regulators in mammary carcinomas using a pre-clinical rat model for breast cancer. Female Sprague Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter randomized to four groups (n=30/group), 1) control (dietary n-6/n-3=1:1); 2) high n-3 (dietary n-6/n-3=1:10); 3) 20%DER (dietary n-6/n-3=1:1); 4) high n-3 + 20%DER (dietary n-6/n-3=1:10). Rats were fed a modification of the AIN-93G diet formulation that contained 30% of dietary calories from fat (currently recommended in the US Dietary guidelines). It was observed that mammary cancer incidence, cancer multiplicity and tumor burden were reduced and cancer latency was elongated for the rats in groups 2-4 compared to the rats in group 1, and the significant differences were observed between group 1 (n-6/n-3=1:1) and 4 (n-6/n-3=1:10 + 20%DER). The protective dietary combination of high n-3 and DER was associated with significant reductions in plasma IGF-1, insulin, and leptin and increased adiponectin compared to rats fed the control diet (n-6/n-3=1:1, p<0.01). Carcinomas induced in the protected animals were smaller in mass with altered levels of proteins in the PPAR and mTOR pathways on which plasma insulin had the most impact. This work was supported by grant KG081632 from the Susan G. Komen Foundation. Citation Format: Weiqin Jiang, Zongjian Zhu, John N. McGinley, Andrea Manni, Henry J. Thompson. Effect of omega-3 fatty acid and dietary energy restriction on mammary carcinogenesis, circulating biomarkers and molecular regulators in rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 193. doi:10.1158/1538-7445.AM2013-193

Abstract 4873: Genistein and soy isoflavone isolate decreased tumor incidence and multiplicity in a Sprague-Dawley ER-positive breast cancer model.

Abstract This study evaluated the chemopreventive efficacy of the pure soy isoflavone genistein and of a soy isoflavone mixture (PTI G-4660) containing roughly 90% genistein in an ER-positive breast cancer model in rats. Female Sprague Dawley rats were injected i.v. with N-nitroso-N -methylurea (MNU) at 50 days of age. In a model examining the effects on cancer initiation, promotion, and progression, rats were fed either a standard Teklad diet or a diet supplemented with genistein (200 or 2000 mg/kg diet) beginning at 43 d of age. Supplemented or standard diets were administered continuously thereafter until the end of the study. In a separate study testing the effects on progression, rats were administered standard Teklad diet or diet supplemented with PTI G-4660 (2000 mg/kg diet) beginning at 55 d of age. All rats were weighed weekly, palpated for mammary tumors twice per week, and checked daily for signs of toxicity. The studies were terminated 126 d after MNU treatment and tumors were weighed. After 126 days, genistein at 200 and 2000 mg/kg diet decreased tumor incidence in a dose-dependent fashion by 15 and 20% (p<0.05), respectively, and significantly decreased tumor multiplicity by 49.6 and 60.3% (p<0.05), respectively. PTI non-significantly decreased tumor incidence by 14% and tumor multiplicity by 24%. In addition, gene expression profiling of livers from rats treated with standard Teklad diet or genistein revealed pathways modified by genistein which may be useful as biomarkers. Citation Format: Holly L. Nicastro, Ronald A. Lubet, Barbara K. Dunn, Clinton J. Grubbs. Genistein and soy isoflavone isolate decreased tumor incidence and multiplicity in a Sprague-Dawley ER-positive breast cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4873. doi:10.1158/1538-7445.AM2013-4873

Protective effect of melatonin against Adriamycin-induced cardiotoxicity

The aim of this in vivo study was to explore the protective properties of melatonin against Adriamycin-induced myocardial toxicity. A rat model of breast cancer was established and the rats were randomly divided into the blank group (Blank), the solvent group [Diss; dehydrated alcohol: physiological saline (1:9)], the Adriamycin group (ADM), the melatonin group (MLT) and the melatonin + Adriamycin group (M+A). The concentrations of lipid peroxide (LPO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in myocardial tissues were detected, the changes in myocardial tissues were observed using light microscopy and electron microscopy, and the 1-month survival rates of each group of rats were compared. Breast cancer was established in 116 rats. In the ADM group, the concentration of LPO was higher and the concentrations of SOD and GSH-Px were significantly lower than those in the blank group. In the M+A group, compared with the ADM group, the concentration of LPO was lower (P<0.05) and the concentrations of SOD and GSH-Px were higher (P<0.05). It was observed using light and electron microscopy that the myocardial injuries to the M+A group were significantly alleviated in comparison with those in the ADM group; the 1-month survival rate in the M+A group was higher than that in the ADM group. Melatonin may have a protective role in the myocardium by reducing Adriamycin-induced myocardial oxidative damage.

Identification of a Molecular Signature Underlying Inhibition of Mammary Carcinoma Growth by Dietary N-3 Fatty Acids

An increased ratio of dietary n-3 relative to n-6 fatty acids has been shown to inhibit the development of mammary cancer in animal models. However, the molecular mechanisms by which n-3 fatty acids affect tumor growth remain unknown. Here, we investigated the effects of varying dietary ratios of n-3:n-6 fatty acids on cell signaling in a rat model of chemically induced mammary carcinoma. Cell proliferation was reduced by 60% in carcinomas from the high n-3:n-6 treatment group compared with the low n-3:n-6 treatment group. These changes were associated with decreased cyclin-D1 and phospho-retinoblastoma protein expression and increased levels of cyclin-dependent kinase inhibitors, CIP1 (p21) and KIP1 (p27). In addition, the apoptotic index was increased in carcinomas from the high n-3:n-6 group and was associated with elevated apoptotic protease-activating factor 1 and a higher ratio of Bax/Bcl-2. Interestingly, changes in protein expression were consistent with reduced inflammation and suppressed mTOR activity, and the molecular signature associated with high n-3:n-6 treatment revealed changes in PPARγ activation and suppression of lipid synthesis. Together, our findings indicate that the molecular effects of high dietary n-3 to n-6 ratios are heterogeneous in nature but point to consistent changes in lipid metabolism pathways, which may serve as potential therapeutic targets for cancer prevention and control. This study identifies the pathways modulated by dietary fatty acid ratios in a rat model of breast cancer, with implications for cancer prevention.

Oxidative Stress in the in vivo DMBA Rat Model of Breast Cancer: Suppression by a Voltage‐gated Sodium Channel Inhibitor (RS100642)

Breast cancer (BCa) was induced in vivo in female rats with 7,12-dimethylbenz(a)anthracene (DMBA). Two main questions were addressed. Firstly, would the carcinogenesis be accompanied by oxidative stress as signalled by superoxide dismutase, glutathione peroxidase, malondialdehyde and total nitrate? Secondly, would treating the rats additionally with a blocker of voltage-gated sodium channel (VGSC) activity, shown previously to promote BCa progression, affect the oxidative responses? The DMBA-induced increases in the antioxidant systems were completely blocked by the VGSC inhibitor RS100642, which also significantly prolonged the lifespan. We conclude that VGSC inhibition in vivo can significantly protect against oxidative stress and improve survival from tumour burden.

Abstract SY31-02: Behavioral consequences of peripheral tumors: Neuroimmune mechanisms

Abstract SY31-02: Behavioral consequences of peripheral tumors: Neuroimmune mechanisms

Abstract 3743: NVP-BYL719, a selective inhibitor of the class Ia PI3K isoform alpha impairs angiogenesis and microvascular permeability.

Abstract Tumor blood vessels are distinctly abnormal in structure and function. When compared to normal vessels, they are enlarged, tortuous, leaky, poorly covered by pericytes and with a defective basement membrane. VEGF is a prominent cytokine responsible for the hyperpermeable state of tumor microvasculature to plasma macromolecules. The phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway functions downstream of VEGF and can activate the production of the free radical gas nitric oxide (NO) through the enzyme endothelial NO synthase (eNOS), a key player for the in vivo biological activity of VEGF. In the present study, we investigated the effect of NVP-BYL719, a novel, synthetic low molecular mass compound, which potently and selectively inhibits class I PI3K isoform alpha activity, on vascular leakage in tumors and its impact on tumor progression in an orthotopic breast cancer model (BN472) in syngeneic rats. Effects on the vasculature were investigated in normal and tumor tissues via tumor interstitial fluid pressure (IFP) measurements in conscious animals via radio-telemetry and a modified Miles assay. The antiangiogenic effect of NVP-BYL719 and its impact on tumor progression was also assessed in an U87MG glioblastoma subcutaneous xenograft tumor model. Pathway inhibition was confirmed using tumor histology and Reverse Phase Protein Array (RPPA) approaches. NVP-BYL719 inhibits VEGF driven tissue formation as well as neo-vascularization of the newly formed tissue. Moreover, the compound strongly inhibited microvessel permeability in normal tissue as determined in the modified miles assay. Similarly, tumor interstitial fluid pressure (IFP), a phenomenon that is directly dependent on tumor vessel permeability, was significantly reduced (up to 43%) by NVP-BYL719 in a dose-dependent manner, upon oral administration in BN472 mammary carcinoma grown orthotopically in syngeneic rats. Concomitantly, tumor growth was significantly inhibited in BN472 mammary carcinoma and U87MG xenografts. The compound was well tolerated with no significant body weight loss at any tested dose. In conclusion, our data suggest that NVP-BYL719, a Class I PI3K selective inhibitor of p110 alpha, interferes with the tumor vasculature system. Thus, the anti-angiogenic proprieties of NVP-BYL719 might participate to the antitumor activity (or efficacy) of the compound. 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 3743. doi:1538-7445.AM2012-3743

Doxorubicin-Conjugated PEGylated Dendrimers Show Similar Tumoricidal Activity but Lower Systemic Toxicity When Compared to PEGylated Liposome and Solution Formulations in Mouse and Rat Tumor Models

PEGylated polylysine dendrimers show promise as novel drug delivery systems with the potential to direct site specific deposition patterns and to reduce toxicity at nontarget sites. Here the activity and toxicity profiles of a generation 5 polylysine dendrimer with 50% surface conjugation of PEG1100 and 50% surface conjugation of doxorubicin (via an acid labile 4-hydrazinosulfonyl benzoic acid linker) have been compared in a Walker 256 rat tumor model and a human MDA-MB231 xenograft in mice. A direct comparison was also made to a PEGylated liposomal formulation of doxorubicin and a doxorubicin solution. In both rat and mouse breast cancer models, the dendrimer formulation gave equivalent antitumor efficacy when compared to the liposomal or solution doxorubicin formulations and administration of all three doxorubicin formulations resulted in a significant reduction (>75%) in tumor growth in both models at doses ranging from 2 to 10 mg/kg doxorubicin equivalents. The dendrimer formulation, however, was better tolerated by both rats and mice, and approximately 2-fold higher doses were required to induce similar levels of toxicity (as assessed by organ weight, peripheral white cell counts, body weight and survival curves) when compared to administration of the doxorubicin solution or PEGylated liposomal doxorubicin. In rats the appearance of palmar plantar erythematosis (PPE), or hand foot syndrome, was also less evident after administration of dendrimer doxorubicin when compared to the liposome. Finally, even after administration to mice at 2-fold higher doses, dendrimer-doxorubicin resulted in a reduced incidence of cardiotoxicity when compared with a simple solution formulation of doxorubicin. The data suggest that dendrimer-based doxorubicin formulations may provide advantage over solution and liposomal formulations of doxorubicin via a reduction in systemic toxicity.

Local Delivery of Chitosan/VEGF siRNA Nanoplexes Reduces Angiogenesis and Growth of Breast CancerIn Vivo

Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.

P2-09-05: Molecular Imaging of Hedgehog Induced Epithelial-Stromal Interactions.

Abstract Introduction: Epithelial-stromal Hedghehog (HH) pathway signaling, originating from Sonic Hedgehog (SHH) ligand secreted by tumor cells, promotes breast cancer growth and metastasis. Furthermore, high tumor SHH expression is associated with poor prognosis in invasive ductal carcinoma. Studies have also shown high expression of Patched (PTCH) mRNA in SHH expressing tumors as well as cancer stem-like cells. This is in part due to a feedback loop that results in increased PTCH gene expression upon HH signaling. We sought to exploit this feedback loop by using radiolabeled derivative of SHH to identify tumors that have a high level of hedgehog signaling. These agents can potentially be used for molecular imaging of HH induced changes in PTCH expression originating from tumor expressed SHH. Methods: Recombinant N-terminal SHH derivatives were radioiodinated with iodine-131 using the idogen method. Iodinated proteins were obtained with radiolabeling yields of 50–60 % and specific activity of 9.3E10Ci/mol. Receptor binding studies were performed on breast cancer cell lines with varying SHH expression. Studies were also conducted on cells cultured in stem cell promoting conditions (mammospheres), after exposure to ionizing radiation or treatment with the hedgehog inhibitor cyclopamine. Scatchard plots were generated using Graph pad software. Pilot studies were conducted with 131I-SHH to test the potential of imaging PTCH receptor expression in a rat model of breast cancer. Fisher rats bearing breast cancer xenografts were injected with approximately 250 microCi of 131I-SHH. Planar scinitigraphy was conducted at 2, 4 and 24 hours. Studies were performed with iodinated BSA as a control for extracellular perfusion and retention. Tissue biodistribution studies were also performed using this model. Results: Receptor binding of 131I-SHH was significantly increased in cell lines with high endogenous HH pathway activation. Receptor binding was also increased after exposure to ionizing radiation and significantly decreased following treatment with hedgehog inhibitors. Scatchard analysis revealed up to a 14 fold increase in PTCH receptor sites in mammospheres compared to the entire breast cancer population. A 10-fold increase in PTCH receptor binding sites was also observed in cells after exposure to ionizing radiation. In vivo imaging and biodistribution studies revealed significant accumulation of 131I-SHH within tumor tissue as compared to normal organs. Tumor:muscle ratios were approximately 8:1 at 4 hours, while tumor to blood and tumor to bone were 2:1 and 5:1 respectively. Significant uptake was also observed in liver tissue, as a result of protein degradation and excretion and endogenous PTCH expression. Conclusions: These studies show that expression of the PTCH receptor is increased in cells with active HH signaling and in breast cancer mammospheres. Our findings also show that PTCH receptor expression is decreased upon treatment with HH signaling inhibitors. Preliminary imaging studies show that 131I-SHH is capable of in vivo detection of breast tumors with high hedgehog signaling. Our data suggests that radiolabeled SHH derivatives may provide a method to follow epithelial-stromal HH interactions and determine response to SHH targeted therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-09-05.

Fingolimod potentiates the effects of sunitinib malate in a rat breast cancer model

Most of the antiangiogenic strategies used in oncology principally target endothelial cells through the vascular endothelial growth factor (VEGF) pathway. Multiple kinase inhibitors can secondarily reduce mural cell stabilization of the vessels by blocking platelet-derived growth factor receptor (PDGFR) activity. However, sphingosine-1-phosphate (S1P), which is also implicated in mural cell recruitment, has yet to be targeted in clinical practice. We therefore investigated the potential of a simultaneous blockade of the PDGF and S1P pathways on the chemotactic responses of vascular smooth muscle cells (VSMCs) and the resulting effects of this blockade on breast tumor growth. Due to crosstalk between the S1P and PDGF pathways, we used AG1296 and/or VPC-23019 to inhibit PDGFR-β and S1PR1/S1PR3 receptors, respectively. We showed that S1PR1 and S1PR3 are the principal receptors that mediate the S1P chemotactic signal on rat VSMCs and that they act synergistically with PDGFR-β during PDGF-B signaling. We also showed that simultaneous blockade of the PDGFR-β and S1PR1/S1PR3 signals had a synergistic effect, decreasing VSMC migration velocity toward endothelial cell and breast carcinoma cell-secreted cytokines by 65-90%. This blockade also strongly decreased the ability of VSMCs to form a three-dimensional cell network. Similar results were obtained with the combination of sunitinib malate (a VEGFR/PDGFR kinase inhibitor) and fingolimod (an S1P analog). Sunitinib malate is a clinically approved cancer treatment, whereas fingolimod is currently indicated only for treatment of multiple sclerosis. Orally administered, the combination of these drugs greatly decreased rat breast tumor growth in a syngeneic cancer model (Walker 256). This bi-therapy did not exert cumulative toxicity and histological analysis of the tumors revealed normalization of the tumor vasculature. The simultaneous blockade of these signaling pathways with sunitinib malate and fingolimod may provide an effective means of reducing tumor angiogenesis, and may improve the delivery of other chemotherapies.

Lignan transformation by gut bacteria lowers tumor burden in a gnotobiotic rat model of breast cancer

High dietary lignan exposure is implicated in a reduced breast cancer risk in women. The bacterial transformation of plant lignans to enterolignans is thought to be essential for this effect. To provide evidence for this assumption, gnotobiotic rats were colonized with the lignan-converting bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis (LCC rats). Germ-free rats were used as the control. All animals were fed a lignan-rich flaxseed diet and breast cancer was induced with 7,12-dimethylbenz(a)anthracene. The lignan secoisolariciresinol diglucoside was converted into the enterolignans enterodiol and enterolactone in the LCC but not in the germ-free rats. This transformation did not influence cancer incidence at the end of the 13 weeks experimental period but significantly decreased tumor numbers per tumor-bearing rat, tumor size, tumor cell proliferation and increased tumor cell apoptosis in LCC rats. No differences between LCC and control rats were observed in the expression of the genes encoding the estrogen receptors (ERs) α, ERβ and G-coupled protein 30. The same was true for IGF-1 and EGFR involved in tumor growth. The activity of selected enzymes involved in the degradation of oxidants in plasma and liver was significantly increased in the LCC rats. However, plasma and liver concentrations of reduced glutathione and malondialdehyde, considered as oxidative stress markers, did not differ between the groups. In conclusion, our results show that the bacterial conversion of plant lignans to enterolignans beneficially influences their anticancer effects.

Cell signaling pathways associated with a reduction in mammary cancer burden by dietary common bean ( Phaseolus vulgaris L.)

Emerging evidence indicates that common bean (Phaseolus vulgaris L.) is associated with reduced cancer risk in human populations and rodent carcinogenesis models. This study sought to identify cancer-associated molecular targets that mediate the effects of bean on cancer burden in a chemically induced rat model for breast cancer. Initial experiments were conducted using a high dietary concentration of bean (60% wt/wt) where carcinoma burden in bean-fed rats was reduced 62.2% (P < 0.001) and histological and western blot analyses revealed that the dominant cellular process associated with reduced burden was induction of apoptosis. Further analysis of mammary carcinomas revealed changes in the phosphorylation states of mammalian target of rapamycin (mTOR) substrates (4E-binding protein 1 and p70S6 kinase) and mTOR regulators adenosine monophosphate-activated protein kinase and protein kinase B (Akt) (P < 0.001). Effects on mTOR signaling in carcinomas were also found at lower dietary concentrations of bean (7.5-30% wt/wt). Liquid chromatography-time of flight-mass spectrometry analysis of plasma provided evidence of altered lipid metabolism consistent with reduced mTOR network activity in the liver (P < 0.001). Plasma concentrations of insulin and insulin-like growth factor-1 were reduced by 36.3 and 38.9%, respectively, (P < 0.001), identifying a link to Akt regulation. Plasma C-reactive protein, a prognostic marker for long-term survival in breast cancer patients, was reduced by 23% (P < 0.001) in bean-fed rats. Identification of a role for the mTOR signaling network in the reduction of cancer burden by dietary bean is highly relevant given that this pathway is deregulated in the majority of human breast cancers.

Differential microstructure and physiology of brain and bone metastases in a rat breast cancer model by diffusion and dynamic contrast enhanced MRI

Pharmacological approaches to treat breast cancer metastases in the brain have been met with limited success. In part, the impermeability of the blood brain barrier (BBB) has hindered delivery of chemotherapeutic agents to metastatic tumors in the brain. BBB-permeable chemotherapeutic drugs are being developed, and noninvasively assessing the efficacy of these agents will be important in both preclinical and clinical settings. In this regard, dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) are magnetic resonance imaging (MRI) techniques to monitor tumor vascular permeability and cellularity, respectively. In a rat model of metastatic breast cancer, we demonstrate that brain and bone metastases develop with distinct physiological characteristics as measured with MRI. Specifically, brain metastases have limited permeability of the BBB as assessed with DCE and an increased apparent diffusion coefficient (ADC) measured with DWI compared to the surrounding brain. Microscopically, brain metastases were highly infiltrative, grew through vessel co-option, and caused extensive edema and injury to the surrounding neurons and their dendrites. By comparison, metastases situated in the leptomenengies or in the bone had high vascular permeability and significantly lower ADC values suggestive of hypercellularity. On histological examination, tumors in the bone and leptomenengies were solid masses with distinct tumor margins. The different characteristics of these tissue sites highlight the influence of the microenvironment on metastatic tumor growth. In light of these results, the suitability of DWI and DCE to evaluate the response of chemotherapeutic and anti-angiogenic agents used to treat co-opted brain metastases, respectively, remains a formidable challenge.

High-Resolution Computed Tomography of Single Breast Cancer Microcalcifications in Vivo

Microcalcification is a hallmark of breast cancer and a key diagnostic feature for mammography. We recently described the first robust animal model of breast cancer microcalcification. In this study, we hypothesized that high-resolution computed tomography (CT) could potentially detect the genesis of a single microcalcification in vivo and quantify its growth over time. Using a commercial CT scanner, we systematically optimized acquisition and reconstruction parameters. Two ray-tracing image reconstruction algorithms were tested: a voxel-driven "fast" cone beam algorithm (FCBA) and a detector-driven "exact" cone beam algorithm (ECBA). By optimizing acquisition and reconstruction parameters, we were able to achieve a resolution of 104 μm full width at half-maximum (FWHM). At an optimal detector sampling frequency, the ECBA provided a 28 μm (21%) FWHM improvement in resolution over the FCBA. In vitro, we were able to image a single 300 μm × 100 μm hydroxyapatite crystal. In a syngeneic rat model of breast cancer, we were able to detect the genesis of a single microcalcification in vivo and follow its growth longitudinally over weeks. Taken together, this study provides an in vivo "gold standard" for the development of calcification-specific contrast agents and a model system for studying the mechanism of breast cancer microcalcification.

Abstract 3696: Effect of dietary omega-3/omega-6 ratio on mammary carcinogenesis, circulating biomarkers and molecular regulators in rats

Abstract Available evidence indicates that diets enriched in the ratio of n-3 to n-6 fatty acids have a broad range of health-related benefit; however, the epidemiological and preclinical data for breast cancer risk are mixed. The majority of animal carcinogenesis experiments have been done using diets that were high in the percent of dietary calories provided from fat (40 to 50%) and few studies have investigated the impact of the n-3/n-6 ratio despite the fact that considerable emphasis has been placed on this ratio in hypothesized mechanisms of action. The objective of this investigation was to determine the effect of a wide range of dietary n-3/n-6 ratios on mammary carcinogenesis, on plasma levels of cancer associated growth factors and cytokines and on molecular regulators in mammary carcinomas using a pre-clinical rat model for breast cancer. Female Sprague Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter randomized to groups (n=30/group) in which the n-3/n-6 ratio varied widely and diets were fed in the presence or absence of tamoxifen treatment. Rats were fed a modification of the AIN-93G diet formulation that contained 30% of dietary calories from fat (currently recommended in the US Dietary guidelines). It was observed that mammary cancer incidence, cancer multiplicity and tumor burden were reduced when the dietary ratio of n-3/n-6 was &amp;gt; 5:1. Cancer inhibition was observed in both the presence and absence of tamoxifen. The protective dietary n-3/n-6 ratio was associated with significant reductions in plasma IGF-1, insulin, and leptin and increased adiponectin compared to rats fed the control diet (n-3/n-6=1:1, p&amp;lt;0.05), effects that were not modified by tamoxifen treatment. Carcinomas induced in the protected animals were smaller in mass with altered levels of proteins in the PPAR-NFkB pathway. This work was supported by grant KG081632 from the Susan G. Komen Foundation. 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 3696. doi:10.1158/1538-7445.AM2011-3696

The manner in which calories are restricted impacts mammary tumor cancer prevention

Although treatments for breast cancer have improved and long-term survival after diagnosis is now common, prevention of the disease is the ultimate goal. Weight loss or weight maintenance is one approach that has been recommended to reduce the risk of breast cancer, particularly for peri/postmenopausal women. This approach is supported by decades of data indicating that calorie restriction prevents spontaneous and chemically induced mammary tumor development in rodents. In most cases, calorie restriction was implemented by a consistent daily reduction of calories, i.e. chronic calorie restriction (CCR). There have also been several studies where periods of reduced caloric intake were followed by periods of refeeding, i.e. intermittent calorie restriction (ICR), resulting in the prevention of spontaneous mammary tumorigenesis. In most of the early studies, there were no direct comparisons of CCR to ICR. One study using moderate calorie restriction in a chemically induced breast cancer rat model found a slight increase in mammary tumor incidence compared with ad libitum fed and CCR rats. However, recently, it has been demonstrated in several transgenic mouse models of breast cancer that ICR consistently provided a greater degree of protection than CCR. This review will provide a detailed comparison of ICR and CCR for breast cancer prevention. It will also examine potential mechanisms of action that may include periods of reduced IGF-I and leptin as well as an increase in the adiponectin:leptin ratio. Application of this approach to at-risk women may provide an approach to lower the risk of breast cancer in overweight/obese women.

Abstract P4-05-04: Lack of the Effect of Mesenchymal Stem Cells on Breast Cancer Initiation and Growth in a Somatic Mouse Breast Cancer Model

Abstract Background: The impact of mesenchymal stem cells (MSCs) on breast cancer (BC) progression and growth remains controversial. Some studies using xenograft BC models in nude mice suggest that MSCs stimulate BC metastases, whereas other studies in syngeneic rat BC models suggest that MSCs suppress BC development and growth. In addition, whether MSCs have a role in BC initiation has not been tested. The objective of this study was to determine if bone marrow-derived MSCs affect BC initiation and progression in vitro and in clinically relevant somatic and synegeneic BC mouse models. Materials and Methods: MSCs were isolated from bone marrows of FVB wild-type mice, cultured, and characterized for their potential to differentiate into adipocytes and express flow-cytometric cell surface markers. MSC-conditioned medium (CM) was used to culture RCAS-Neu and RCAS-PyMT BC cell lines, derived from TVA-transgenic mice infected with an avian retroviral vector encoding Neu or polyoma middle T antigen (PyMT). Cell proliferation was analyzed with CellTiter 96 proliferation assay and 5-bromo-2-deoxyuridine labeling. The effect of RCAS-Neu and RCAS-PyMT cells and their CM on MSC migration was determined with Boyden chamber migration assay. In a somatic BC model, TVA-transgenic mice expressing the receptor for an avian retrovirus vector, RCAS, were infected with RCAS-PyMT vector by intraductal injection and treated with MSC (2x106cells/mouse) by i.v. injection. Mice were observed for BC development by palpation. In a syngeneic BC model, RCAS-Neu BC cells were co-implanted with MSCs (5x105/gland) at the ratios 1:0, 1:0.2, or 1:1 into the fat pad of FVB female mice. BC growth was monitored for 9 weeks. Results: CM from MSC did not significantly affect the proliferation of RCAS-Neu and RCAS-PyMT cells. However, RCAS-Neu and RCAS-PyMT cells and their CM induced morphologic changes in MSC and dramatically increased their motility. In somatic model, MSCs had no effect on BC initiation or growth with the mean tumor latency 27.5±7.5 days in MSC-treated mice and 29±5.5 days in control mice treated with PBS. In syngeneic BC model, there was no significant difference in the growth of RCAS-Neu alone or RCAS-Neu cells co-implanted with MSCs. Since BC in these models does not metastasize to distant organs, it could not be determined whether MSCs affect BC metastases. Discussion: Our results demonstrated that BC cell lines and their CM are able to induce MSCs migration and possibly differentiation. However, MSCs had no effect on BC initiation in a somatic BC model and on tumor growth in a syngeneic BC model. It is possible that the number of MSCs and schedule of MSC injection were not optimized or strong PyMT oncogene rapidly induced BC and overcame the effect of MSCs on BC formation. Experiments are ongoing to determine if multiple administrations of MSCs affect BC development induced by PyMT and Neu oncogenes. Lack of effect of MSCs in BC initiation and progression, if confirmed, will suggest that MSCs are safe for delivering novel antitumor agents for BC treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-04.

Molecular Histopathology by Spectrally Reconstructed Nonlinear Interferometric Vibrational Imaging

Sensitive assays for rapid quantitative analysis of histologic sections, resected tissue specimens, or in situ tissue are highly desired for early disease diagnosis. Stained histopathology is the gold standard but remains a subjective practice on processed tissue taking from hours to days. We describe a microscopy technique that obtains a sensitive and accurate color-coded image from intrinsic molecular markers. Spectrally reconstructed nonlinear interferometric vibrational imaging can differentiate cancer versus normal tissue sections with greater than 99% confidence interval in a preclinical rat breast cancer model and define cancer boundaries to ± 100 μm with greater than 99% confidence interval, using fresh unstained tissue sections imaged in less than 5 minutes. By optimizing optical sources and beam delivery, this technique can potentially enable real-time point-of-care optical molecular imaging and diagnosis.

Wheel Running–Induced Changes in Plasma Biomarkers and Carcinogenic Response in the 1-Methyl-1-Nitrosourea–Induced Rat Model for Breast Cancer

The purpose of this investigation was to identify pathways by which physical activity, implemented as running in an activity wheel, inhibits carcinogenesis. The focus of this analysis was on 20 plasma biomarkers for glucose homeostasis, inflammation, cytokine function, and endocrine activity, known to be affected by a physically active lifestyle. Plasma for analysis was obtained from previously reported carcinogenesis experiments in which the effects on mammary carcinogenesis, induced by i.p. injection of 1-methyl-1-nitrosurea, of running on a motorized activity wheel or a nonmotorized free wheel were compared with sedentary controls. Wheel running reduced cancer incidence (P = 0.0004) and the number of cancers per animal (P = 0.005). Principal components analysis was used to reduce the 20 plasma biomarkers to a concise index that was significantly different by treatment group assignment (P < 0.0001). Statistical analyses provided evidence that supported the hypothesis of a mediational role of these molecules in accounting for the protective effect of wheel running on the carcinogenic process. In addition, the plasma biomarker index derived from principal components analysis was a good discriminator of treatment group assignment (only 4.5% of animals were misclassified). These findings suggest that the plasma biomarkers evaluated have utility in assessing the breast cancer response to a physical activity intervention. Identification of such biomarkers is critical in clinical studies for which evaluating the effects of physical activity on cancer outcomes (diagnosis, recurrence, or mortality) is not possible.