Murine Breast Carcinoma Model Research Articles

Cerebral cavernous malformation proteins, CCM1, CCM2 and CCM3, are decreased in metastatic lesions in a murine breast carcinoma model

ABSTRACT Three genes are associated with cerebral cavernous malformations (CCMs): CCM1, CCM2 and CCM3. These genes participate in microvascular angiogenesis, cell-to-cell junctions, migration and apoptosis. We evaluated the expression in vivo of CCM genes in primary tumors and metastastases in a murine model of metastatic breast carcinoma. We used cell lines obtained from metastasis of 4T1, 4TLM and 4THM breast cancer to liver and heart. These cells were injected into the mammary ridge of Balb/C female mice. After 27 days, the primary tumors, liver and lung were removed and CCM proteins were assessed using immunohistochemistry and western blot analysis. CCM proteins were expressed in primary tumor tissues of all tumor-injected animals; however, no CCM protein was expressed in metastatic tumor cells that migrated into other tissues. CCM proteins still were observed in the lung and liver tissue cells. Our findings suggest that CCM proteins are present during primary tumor formation, but when these cells develop metastatic potential, they lose CCM protein expression. CCM protein expression was lost or reduced in metastatic tissues compared to the primary tumor, which indicates that CCM proteins might participate in tumorigenesis and metastasis.

In Situ Generated and Amplified Oxidative Stress with Metallo‐Nanodrug Assembly for Metastatic Cancer Therapy with High Specificity and Efficacy

AbstractCombination of anti‐metastasis antagonists with cytotoxic agents has shown clinical activity, yet is constrained with unconditional activation and low tumor selectivity. Here, a chemokine receptor‐targeted metallo‐nanodrug is reported for in situ generated and hyperthermia‐enhanced oxidative stress and metastatic pathway blockade with tumor specificity to treat breast cancer metastasis. The self‐assembled metallo‐nanodrugs trigger reactive oxygen species (ROS) generation in situ with tumor stimuli, self‐amplify ROS level upon intrinsic photothermal heating, and accurately block chemokine receptor CXCR4 to suppress tumor cell anti‐apoptosis, migration, and invasion. CXCR4‐targeted metallo‐nanodrugs plus laser irradiation induced complete inhibition of tumor growth in 80% of mice, and caused 92.8% reduction of the number of gross metastatic lung nodules in murine breast carcinoma models. The present work provides an intelligent and bioactivatable platform for precision nanomedicine of metastatic breast cancer.

Role of transient receptor potential ankyrin 1 (TRPA1) on nociception caused by a murine model of breast carcinoma

Breast carcinoma causes severe pain, which decreases the quality of life of patients. Current treatments produce adverse effects and have limited efficacy. Transient potential receptor ankyrin 1 (TRPA1) is related to the onset of cancer and neuropathic pain. The aim of this study was to evaluate the involvement of TRPA1 in a model of breast carcinoma. We injected 4T1 cells in the fourth caudal mammary fat pad of female BALB/c mice, and after 20 days we observed mechanical and cold allodynia and spontaneous nociception behavior (mouse grimace scale detection, MGS). TRPA1 selective antagonist (HC-030031 or A-967079) administration or intrathecal administration of TRPA1 antisense (AS) oligonucleotide was performed. The activity of NADPH oxidase, superoxide dismutase (SOD) and hydrogen peroxide (H2O2) levels were evaluated. The chemical hyperalgesia produced by a TRPA1 agonist (allyl isothiocyanate, AITC) was also detected. The administration of TRPA1 antagonists, TRPA1 AS, or antioxidant, transiently attenuated MGS, or mechanical and cold allodynia. Intraplantar injection of AITC also caused nociception. NADPH oxidase or SOD activity and H2O2 levels were increased in the sciatic nerve and hind paw skin samples. The 4T1 cells did not express TRPA1, and the use of HC-030031 or α-lipoic acid did not reduce the cytotoxic effect of a chemotherapeutic drug (paclitaxel). Thus, TRPA1 could be investigated as a target for breast carcinoma pain treatment.

Characterization of Cancer-Induced Nociception in a Murine Model of Breast Carcinoma.

Severe and poorly treated pain often accompanies breast cancer. Thus, novel mechanisms involved in breast cancer-induced pain should be investigated. Then, it is necessary to characterize animal models that are reliable with the symptoms and progression of the disease as observed in humans. Explaining cancer-induced nociception in a murine model of breast carcinoma was the aim of this study. 4T1 (104) lineage cells were inoculated in the right fourth mammary fat pad of female BALB/c mice; after this, mechanical and cold allodynia, or mouse grimace scale (MGS) were observed for 30 days. To determine the presence of bone metastasis, we performed the metastatic clonogenic test and measure calcium serum levels. At 20 days after tumor induction, the antinociceptive effect of analgesics used to relieve pain in cancer patients (acetaminophen, naproxen, codeine or morphine) or a cannabinoid agonist (WIN 55,212-2) was tested. Mice inoculated with 4T1 cells developed mechanical and cold allodynia and increased MGS. Bone metastasis was confirmed using the clonogenic assay, and hypercalcemia was observed 20 days after cells inoculation. All analgesic drugs reduced the mechanical and cold allodynia, while the MGS was decreased only by the administration of naproxen, codeine, or morphine. Also, WIN 55,212-2 improved all nociceptive measures. This pain model could be a reliable form to observe the mechanisms of breast cancer-induced pain or to observe the efficacy of novel analgesic compounds.

ID:2038 THE ROLE OF THE CASPASE FAMILY in PF4-INDUCED APOPTOSIS IN BREAST CANCER IN VIVO STUDY

Background: Apoptosis is critical to the development and homeostasis of metazoans. Apoptotic dysregulation leads to various human pathologies including cancer, autoimmune and neurodegenerative disorders. Caspases, sub-members of cysteine proteases, are the central components of apoptotic response. The ELR-negative chemokine platelet factor 4 (PF4) was initially identified as an antiangiogenic agent. It inhibits endothelial cell proliferation, migration and angiogenesis in in vitro and in vivo and was further proving in the in vivo models of human colon carcinoma and murine melanoma xenograft mouse. However, there are no studies (either in-vivo or ex-vivo) which have conclusively demonstrated and characterized the PF4 effects on inducing apoptosis in breast cancer setting and therefore, this becomes the study’s central objective.
 
 Aim: This study aimed to investigate PF4's effects on the apoptosis regulation mediated by caspase -3, -6, -7, -8 and -9 in 1-methyl-1-nitrosourea-induced (MNU) invasive murine breast carcinoma model.
 Methods: Breast carcinoma were induced in 60, 21-day-old female Sprague Dawley Rat (SDR) using 1-methyl-1-nitrosourea until its size reached 14.5±0.5 mm. The tumors were divided into two groups: Group 1 (control pre-treated, n=20 and post treated, n=20), and Group 2 (PF4, n=20). PF4 were administered through focal intralesional injections at 20 µg/lesion dosage. After 5 days of treatment, the SDRs were sacrificed. Subsequently the tumor specimens were prepared for haematoxylin and eosin staining. The expression of caspases family members (caspase-3,-6,-7,-8 and-9) were detected using immunohistochemistry. The specimens were morphologically examined and the cell counts with positive caspases expressions were recorded.
 Results: There was a significant reduction of tumor size after PF4 treatment compared to the controls. PF4 demonstrated differential effects on downstream effector caspases; both upregulated caspase-3 and -6, and downregulated caspase-7 expression was observed compared to controls. PF4 exhibited significant effects on the three caspases-3, -6 and -7 (p <0.001, p=0.004 and p=0.024, respectively). PF4 had no effect on caspase-8 (p=0.067) and caspase-9 (p=0.061) expressions.
 Conclusion: This study found that PF4 significantly reduced the tumor sizes in a short time (less than 3 days) and acts differentially on the caspase-mediated apoptosis pathways and on the downstream levels of the apoptotic machinery.

Systemic siRNA Delivery with a Dual pH-Responsive and Tumor-targeted Nanovector for Inhibiting Tumor Growth and Spontaneous Metastasis in Orthotopic Murine Model of Breast Carcinoma.

Phenylboronic acid (PBA)-mediated tumor targeting nanovector is an attractive strategy for enhancing siRNA delivery and treatment of metastatic cancers. However, its nonspecific binding with various biological membranes containing cis-diol moieties restricts its potential application by systematic administration. Herein, we constructed a novel pH-activated “sheddable” PEG-coated nanoparticle for effective treatment of primary tumors and metastases, which was based on the conjugation of catechol group modified poly(ethylene glycol) (PEG-Cat) and PBA-terminated polyethylenimine (PEI-PBA) via the borate ester formed between PBA and Cat. By virtue of the pH-dependent stability of borate ester in an aqueous medium, the PEG-shell could “shield” the PBA ligand in systemic circulation to reduce its “off-target effect”, while PEG was detached at tumor extracellular pH (~6.5) to expose intact PBA moiety. Simultaneously, the PBA ligand could bind with overexpressed sialic acid residues on cancer cells, giving rise to enhanced cellular internalization. In addition, the PBA moieties could also couple with each 3'-end ribose of double-stranded siRNA. siRNAs were used as both a payload and a pH-responsive intermolecular cross-linker, and thereby acquired sufficient stability during circulating in blood and a rapidly triggered release in response to acidic endosomal/lysosomal pH-stimuli. As a result, this dual pH-sensitive nanoparticle showed enhanced siRNA uptake, gene silencing efficacy and anti-metastatic effects in vitro. Furthermore, in vivo studies demonstrated that PBA-based nanoparticles effectively accumulated in tumor and inhibited tumor growth and metastasis in 4T1 orthotopic mammary tumor model after intravenous administration.

Abstract LB-151: A novel TGFβ 1/2 specific antibody suppresses 4T1 tumor growth by enhancing NK cell recruitment and function

Abstract TGFβ can play an important role in tumor progression. Whether expressed by the tumor, or the tumor microenvironment, it has a profound impact on not only the tumor itself but also the host immune system. There are three isoforms of TGFβ: TGFβ1, TGFβ2 and TGFβ3. The functions of TGFβ1 and 2 have been widely studied. However, little is known about how TGFβ3 affects both tumor growth and the host. Here we used a novel human antibody that neutralizes only TGFβ1 and 2, while leaving TGFβ3 activity untouched. By specifically blocking TGFβ1 and 2 functions, we were able to investigate the role of TGFβ3 in modulating the immune compartment, and its ability to combat tumor growth. We used the 4T1 murine breast carcinoma model, which secretes all three isoforms of TGFβ. Tumor-bearing mice were treated with 10mg/kg the novel anti-TGFβ1/2 antibody, which inhibited 25% of 4T1 tumor growth after 20 days. We evaluated the immune cells from tumors and found there was a significant increase (14%) in the NK cells that infiltrated the tumor site in the antibody-treated group. To explore the mechanism underlying this phenomenon, we evaluated the effect of tumor-secreted TGFβ isoforms on NK cells in vitro in a co-culture system. When 4T1 tumor cells were co-cultured with enriched NK cells, the TGFβ1/2 antibody elevated the levels of IFNγ, GzmB and perforin. However, the expression levels of NKG2D, CD69, Ly49A and C were unchanged. We also detected a significant upregulation of FasL on NK cells treated with the TGFβ1/2 antibody. Moreover, NK cells treated with the TGFβ1/2 antibody in the co-culture system showed improved killing activity towards Yac-1 cells, which was confirmed to be mediated by GzmB. In summary, a novel therapeutic antibody candidate that specifically neutralizes TGFβ1 and 2 inhibits 4T1 tumor growth by (i) changing the tumor microenvironment to attract NK cells to tumor sites; and (ii) by increasing NK cell function via upregulation of FasL and GzmB. Citation Format: Ou Li, Shireen Khan, Nerissa Mendoza, Sandra Vanegas, Cat Tran, Amer Mirza. A novel TGFβ 1/2 specific antibody suppresses 4T1 tumor growth by enhancing NK cell recruitment and function. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2014-LB-151

Significance of interleukin-33 and its related cytokines in patients with breast cancers.

Interleukin-33 (IL-33) is a recently identified cytokine, an important member of the interleukin-1 family. IL-33 binds to its receptor ST2 to induce type 2 cytokines and exert both pro-inflammatory and protective functions in host defense and disease. Murine breast carcinoma models suggest disruption of ST2 signaling may enhance the anti-tumor immune response, suggesting IL-33 impedes anti-tumor immunity. However, the role of IL-33 in patients with breast cancers (BC) is not elucidated. We detected the expression of IL-33 in tumor tissue, and IL-33 and its related cytokines in serum from BC patients. Using Luminex and immunohistochemistry methods, we found that serum levels of IL-33 were nearly twofold higher in patients with BC, compared to patients with benign breast diseases. In cancer tissues, expression of IL-33 was higher than matched normal breast tissues from the same patients, and was also associated with a well-differentiated phenotype, HER2 overexpression, more lymph nodes involvement, and a family history of malignant carcinoma. These results suggest that IL-33 may play an important role in the progress of BC and may be a useful biomarker for predicting the progress and metastasis of BC.

A novel probe for the non-invasive detection of tumor-associated inflammation

A novel dual-mode contrast agent was formulated through the addition of an optical near infrared (NIR) probe to a perfluorocarbon (PFC)-based 19F magnetic resonance imaging (MRI) agent, which labels inflammatory cells in situ. A single PFC-NIR imaging agent enables both a qualitative, rapid optical monitoring of an inflammatory state and a quantitative, detailed and tissue-depth independent magnetic resonance imaging (MRI). The feasibility of in vivo optical imaging of the inflammatory response was demonstrated in a subcutaneous murine breast carcinoma model. Ex vivo optical imaging was used to quantify the PFC-NIR signal in the tumor and organs, and results correlated well with quantitative 19F NMR analyses of intact tissues. 19F MRI was employed to construct a three-dimensional image of the cellular microenvironment at the tumor site. Flow cytometry of isolated tumor cells was used to identify the cellular localization of the PFC-NIR probe within the tumor microenvironment. Contrast is achieved through the labeling of host cells involved in the immune response, but not tumor cells. The major cellular reservoir of the imaging agent were tumor-infiltrating CD11b+ F4/80low Gr-1low cells, a cell subset sharing immunophenotypic features with myeloid-derived suppressor cells (MDSCs). These cells are recruited to sites of inflammation and are implicated in immune evasion and tumor progression. This PFC-NIR contrast agent coupled to non-invasive, quantitative imaging techniques could serve as a valuable tool for evaluating novel anticancer agents.

Using the allergic immune system to target cancer: activity of IgE antibodies specific for human CD20 and MUC1

Monoclonal antibodies are widely used in the treatment of many B cell lymphomas and certain solid tumors. All currently approved therapeutic monoclonal antibodies are of the immunoglobulin G (IgG) isotype. We hypothesized that tumor-specific monoclonal antibodies of the IgE isotype may serve as effective cancer therapeutics. To test this hypothesis, we produced mouse-human chimeric IgE antibodies specific for the human B cell antigen CD20 and the epithelial antigen MUC1. We demonstrate here that anti-hCD20 IgE antibodies have in vitro cytotoxic activity when used with purified allergic effector cells derived from umbilical cord blood. At an effector-tumor ratio of 2:1, mast cells and tumor-specific IgE induced a 2.5-fold increase in tumor cell death, as compared to control IgE. Similar results were observed when eosinophils were used as effector cells. In an in vivo murine model of breast carcinoma, administration of anti-hMUC1 IgE reduced the growth of MUC1(+) tumors by 25-30% in hFcεRI transgenic mice. In contrast, local production of IgE and cytokines chemotactic for macrophages, eosinophils and mast cells led to complete tumor eradication. These results suggest that allergic effector cells activated by IgE and cell surface antigens have the capacity to induce tumor cell death in vitro and in vivo. The use of chimeric antibodies and hFcεRI transgenic mice will greatly enhance investigations in the nascent field of allergo-oncology.

Abstract 3349: Nanog-expressing breast cancer tumor initiating cells (TICs) are highly metastatic and are resistant to the effects of proteasome inhibition

Abstract Functional heterogeneity of transformed cells exists within a single tumor mass and between distinct tumor nodules. Many tumors have also been demonstrated to contain a subpopulation of tumor initiating cells (TICs) whose epigenetic modifications convey resistance to conventional therapies. It has been hypothesized that TICs represent the terminal product of epithelial-mesenchymal transition which is linked to metastatic potential. Therefore we examined the metastatic potential of TICs in the 4T1 murine breast carcinoma model, and evaluated Nanog expression as a biomarker for highly metastatic 4T1 sub-populations. Traditionally TICs have been identified and isolated through expression of selected cell surface markers and functional assays, however cell surface phenotype alone fails to identify CSC in all tumors from the same organ or in all patients. We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency (Oct-4, Sox-2 and Nanog) could be superior to cell surface phenotype for identification of TICs. Thus we adapted a commercial promoter reporter system where the Nanog promoter drives copepod GFP which has been destabilized to better reflect rapid changes in transcriptional activity. I ntroduction of a Nanog promoter reporter construct into 4T1.2 cells resulted in stable expression of GFP in about 0.5% of the cells. In vivo tumor transfer studies confirmed that Nanog promoter activity (4T1.2-GFPpos cells) was associated with a significantly enhanced ability to form experimental lung metastases. In addition, in vitro TIC surrogate assays showed that 4T1.2 GFPpos cells produced increased numbers of spheroids in both soft agar and under non-adherent growth conditions. Finally Nanog-GFPpos cells were more resistant to killing by Paclitaxel than Nanog-GFPneg cells, but were more sensitive to Salinomycin. Together these findings suggest that Nanog promoter activity is a robust marker of metastatic 4T1.2 TICs. We have previously observed that administration of the proteasome inhibitor Bortezomib (Bzb) has moderate anti-4T1.2 tumor activity in vivo therefore we examined the sensitivity of the various 4T1.2 populations to Bzb treatment in vitro. 4T1.2 Nanog-GFPpos cells were resistant to the cytostatic effects of Bzb while 4T1.2 Nanog-GFPneg cells were sensitive. The molecular basis of this difference is under investigation, but is not due to differences in accumulation of ubiquitinated proteins. Taken together, these findings show that Nanog promoter activity is a marker of highly metastatic TICs, and suggest that eradication of TICs may be required for curative anti-cancer therapies. 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 3349. doi:10.1158/1538-7445.AM2011-3349

Multidonor bone marrow transplantation improves donor engraftment and increases the graft versus tumor effect while decreasing graft-versus-host disease

In partially matched donor transplantation, mandatory T-cell depletion (TCD) increases the risks of rejection/graft failure, relapse, and post-transplant infections. A multi-donor approach was offered to resolve some of these drawbacks. This hypothesis was previously tested in a TCD fully mismatched murine model. However, the effect of multi-donor transplantation (MDT) on graft-versus-host disease (GVHD) and graft versus tumor (GVT) effect were never tested. To assess the safety and efficacy of MDT, we used it in non-TCD transplantation and murine breast carcinoma model. We found that when transplanting non-TCD MDT composed by C57Bl/6 and C3H cells into BALB/c, a consistent trichimerism is established, dominated by C57Bl/6 cells. Following MDT the study animals experienced reduced GVHD compare with those transplanted from C57Bl/6 alone, while the GVT effect was superior. We conclude that MDT may serve as a technique that suppresses GVHD while maintaining the GVT effect.

Abstract 2406: Increased efficacy of combination of IL-13 receptor-targeted cytotoxin and DNA vaccine in murine tumor models

Abstract Recent studies show that interleukin-13 receptor α2 chain (IL-13Rα2) is a tumor-associated antigen that is a promising potential target for immunotherapy. High levels of IL-13Rα2 are expressed on a variety of human tumors, but its precise role in tumor immunology is not known. IL13-PE, a targeted cytotoxin composed of IL-13 fused to mutated form of Pseudomonas exotoxin, induces specific killing of tumor cells expressing abundant levels of IL-13Rα2. In this study, we sought to determine the effect of IL13-PE in combination with DNA vaccination in murine MCA304 sarcoma and 4T1 breast carcinoma, which naturally over-express IL-13Rα2 antigen. Three intramuscular prophylactic IL-13Rα2 DNA immunizations in syngeneic mice (n=6) on −8, −4, and −2 weeks resulted in partial protection (382 mm3) against subcutaneous MCA304 tumor challenge compared to mock vector vaccine (1498 mm3) on day 21. Three additional intratumoral (i.t.) treatments of IL13-PE on day 4, 6 and 8 post-tumor challenge further decreased tumor burden (85 mm3) by 78%, measured on the same day (day 21) compared to DNA vaccination alone. In an established MCA304 tumor model, mice receiving three i.t. injections of IL13-PE (on day 7, 9, and 11) and four subsequent DNA vaccinations (on day 13, 18, 23 and 28) reduced tumor size to 44 mm3 (94% less) compared to DNA vaccination alone (693 mm3) on day 24. For comparison, the untreated control tumors had a size of 1579 mm3 on day 24. Interestingly, the combination therapy with IL13-PE and DNA vaccination produced 50% complete responders; however, tumors in remaining 50% partial responders grew slowly. Combination therapy prolonged the mean survival to 60 days compared to control (24 days) and DNA only vaccinated groups (36 days). Similar results were also observed in the 4T1 murine breast carcinoma model. Combination therapy group showed higher CTL activity and IFN-γ release from splenocytes compared to the single therapy groups in both tumor models. Immunohistochemical analysis showed the infiltration of CD4 and CD8 positive T cells and expression of IFN-γ-induced chemokines (CXCL9 and CXCL10) in tumor tissues of immunized mice. These results suggest that combination therapy with IL13-PE and IL-13Rα2 DNA vaccination inhibited tumor growth in a T cell dependent manner. In addition to the combination therapy strategy, elimination of immunosuppressive elements such as regulatory T cells and myeloid-derived suppressor cells may further enhance tumor immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2406.

(-)-Epigallocatechin gallate sensitizes breast cancer cells to paclitaxel in a murine model of breast carcinoma

IntroductionPaclitaxel (Taxol®) is a microtubule-targeted agent that is widely used for cancer treatment. However, resistance to paclitaxel is frequently encountered in the clinic. There is increasing interest in identifying compounds that may increase the sensitivity to conventional chemotherapeutic agents. In this study, we investigated whether green tea polyphenol (-)-epigallocatechin gallate (EGCG) could sensitize breast carcinoma to paclitaxel in vivo.MethodsBreast cancer cells were treated with or without EGCG and paclitaxel followed by detection of cell survival and apoptosis. c-Jun NH2-terminal kinase (JNK) phosphorylation and glucose-regulated protein 78 (GRP78) expression were detected by Western blotting. For in vivo study, 4T1 breast cancer cells were inoculated into Balb/c mice to establish a transplantation model. The tumor-bearing mice were treated with or without EGCG (30 mg/kg, i.p.) and paclitaxel (10 mg/kg, i.p.). Tumor growth was monitored. Apoptosis in tumor tissues was detected. Cell lysates from tumors were subjected to Western blot analysis of GRP78 expression and JNK phosphorylation.ResultsEGCG synergistically sensitized breast cancer cells to paclitaxel in vitro and in vivo. EGCG in combination with paclitaxel significantly induced 4T1 cells apoptosis compared with each single treatment. When tumor-bearing mice were treated with paclitaxel in combination with EGCG, tumor growth was significantly inhibited, whereas the single-agent activity for paclitaxel or EGCG was poor. EGCG overcame paclitaxel-induced GRP78 expression and potentiated paclitaxel-induced JNK phosphorylation in 4T1 cells both in vitro and in vivo.ConclusionsEGCG may be used as a sensitizer to enhance the cytotoxicity of paclitaxel.

EBAG9 inducing hyporesponsiveness of T cells promotes tumor growth and metastasis in 4T1 murine mammary carcinoma

The estrogen receptor-binding fragment-associated gene 9 (EBAG9) has been identified as an estrogen-responsive gene and was recently identified as a tumor-promoting and prognostic factor for renal cell carcinoma. We investigated whether EBAG9 expression was correlated with primary tumor growth and distant tumor metastasis in a murine breast carcinoma model. Knockdown expression of EBAG9 by small interfering RNA significantly suppressed tumor growth and metastasis in vivo in a highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model. 4T1 cells stably overexpressing EBAG9 developed larger and faster tumor growth and lung metastasis compared with parental 4T1 or 4T1 expressing vector alone. Strong specific cytotoxic T lymphocyte activity and enhanced gamma-interferon and interleukin-2 productions were induced in mice that received EBAG9 small interfering RNA therapy. Gene silencing of EBAG9 prolonged the survival of tumor-bearing mice and induced more intensive infiltration of CD8+ T cells in tumor mass. EBAG9 induced apoptosis of T cells, enhanced glycogen synthase kinase 3beta phosphorylation and inhibited gamma-interferon production of T cells when T lymphocytes were cocultured with 4T1 cells overexpressing EBAG9. Furthermore, overexpression of EBAG9 in 4T1 cells was accompanied with enhanced expression of chemokine (C-X-C motif) receptor 4, which might be involved in tumor metastasis. Taken together, our results suggested that EBAG9 promoted primary 4T1 mammary carcinoma growth and distant metastasis, and EBAG9 small interfering RNA exerted overt regression of tumor growth and metastasis. These findings might provide insights into the mechanism through which tumors evade immunosurveillance and provide a strategy for therapeutic intervention of cancer metastases.

Novel Cytosine Deaminase Fusion Gene Enhances the Effect of Radiation on Breast Cancer in Bone by Reducing Tumor Burden, Osteolysis, and Skeletal Fracture

Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression. The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CD(y)). CD-expressing tumor cells (4TCD(y)) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCD(y), but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCD(y) intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).

Antitumor effect of valorphin in vitro and in vivo: Combined action with cytostatic drugs

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative £]-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 µM valorphin and 1 µM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 µM (but not 1 µM) epirubicin added 24 h prior to 1 µM valorphin; 1 µM valorphin added 48 h prior to 0.1 µM epirubicin, or 0.1 µM vincristine, or 0.05 µM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg/m2 epirubicin and 1 mg/kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.

IRF-1 suppresses tumor formation and protects against tumor rechallenge in a murine model of breast carcinoma: The role of tumor specific immunity

Introduction. IRF-1 is involved in multiple stages of the immune response. We previously demonstrated that IRF-1 causes apoptosis in breast cancer; however, the role of immune up regulation by IRF-1 remains to be elucidated. We hypothesize that IRF-1 expression results in enhanced tumor immunogenicity of murine breast cancer in vivo. Methods. Murine breast carcinoma C3L5 cells were mock, Ad-Psi5 (empty vector), or Ad-IRF-1 (recombinant adenovirus expressing IRF-1) infected and then injected s.c. into syngeneic C3H/HeJ and nude mice at 500,000 cells/mouse. In subsequent experiment, mock, Ad-Psi5 or Ad-IRF-1-infected cells were injected into C3H/HeJ mice, and at 3 weeks, tumor, if present, was excised, and mice were rechallenged with 500,000 uninfected, parental tumor cells into contralateral flank. Results. Ex vivo transduction of C3L5 cells with Ad-IRF-1 prevented tumor formation in 80% of syngeneic mice, whereas 0% of nude mice were protected. Mock and Ad-Psi5 animals all developed tumor. “Vaccination” with Ad-IRF-1-treated cells protected 60% syngeneic mice from tumor formation after rechallenge with parental tumor cells. Vaccination with mock and Ad-Psi5-treated cells did not provide any protection. Median tumor-free survival was 70 days for Ad-IRF-1 (the length of the experiment), 27 days for Ad-Psi5, and 29 days for mock ( P < 0.05 by log rank test). Conclusions. Tumor suppression by Ad-IRF-1 is immune-mediated to a significant extent, involving T cells. An adaptive immune response to parental tumor cell rechallenge was achieved, reflecting tumor specific immunity. These data support the use of agents that increase IRF-1 in breast cancer.

Combined effect of para-aminobenzoic acid (PABA) and ionizing radiation in a syngeneic murine model of breast carcinoma

3189 Background: PABA or p-amino benzoic acid is a water-soluble natural compound and a component of Vitamin B-12, thought to play a role in nucleotide base synthesis. It is widely available as a dietary supplement and is a common component of sunscreens. Previously, we observed that PABA enhanced the effects of radiation in B16F10 and G361 melanoma and Lewis Lung carcinoma in vitro, as well as in 4T1 breast carcinoma and B16F10 melanoma in a chick CAM in vivo model (ACR-NCI-EORTC, Nov. 2003). To further explore the role of PABA pre-clinically, we studied its effects as a radiosensitizer in the 4T1 syngeneic murine model of mammary carcinoma (Fred Miller, The Michigan Cancer Center). Methods: 4T1 tumor cells (5 x 104) were injected subcutaneously into the flank of BALB/C mice. Three days later, 28 mice were randomly assigned to PABA, administered daily for the duration of the experiment (1 mg/day), or no PABA treatment. At 14 days post-implantation, a randomization to radiation treatment (RT) occurred in both the PABA and non-PABA treated groups. A 60Co source was used to deliver a single fraction of 10 Gy to the tumor. Tumors were measured and volumes were calculated weekly for 28 days ((L2xW)/2). Results: At day 28, PABA injection alone had minimal effect on tumor growth (P=0.14); 10 Gy of ionizing radiation therapy significantly inhibited tumor growth by 47% (P=0.019); and PABA+10 Gy RT significantly inhibited tumor growth by 83% (P=0.000217) as compared to untreated controls. In addition, PABA+10 Gy RT resulted in a 68% inhibition of tumor growth as compared to radiation alone (P=0.00036). Conclusions: The most effective inhibition of tumor growth was achieved by PABA+10 Gy RT, suggesting that PABA is a radiosensitizer in this model. Given the high tolerability of PABA, these data may have significant translational potential. No significant financial relationships to disclose.

Combined effect of para-aminobenzoic acid (PABA) and ionizing radiation in a syngeneic murine model of breast carcinoma

3189 Background: PABA or p-amino benzoic acid is a water-soluble natural compound and a component of Vitamin B-12, thought to play a role in nucleotide base synthesis. It is widely available as a dietary supplement and is a common component of sunscreens. Previously, we observed that PABA enhanced the effects of radiation in B16F10 and G361 melanoma and Lewis Lung carcinoma in vitro, as well as in 4T1 breast carcinoma and B16F10 melanoma in a chick CAM in vivo model (ACR-NCI-EORTC, Nov. 2003). To further explore the role of PABA pre-clinically, we studied its effects as a radiosensitizer in the 4T1 syngeneic murine model of mammary carcinoma (Fred Miller, The Michigan Cancer Center). Methods: 4T1 tumor cells (5 x 104) were injected subcutaneously into the flank of BALB/C mice. Three days later, 28 mice were randomly assigned to PABA, administered daily for the duration of the experiment (1 mg/day), or no PABA treatment. At 14 days post-implantation, a randomization to radiation treatment (RT) occurred in ...