Mouse Model Of Liver Metastasis Research Articles

Marked Antitumor Effect of NK012, a SN-38-incorporating Micelle formulation, in a Newly Developed Mouse Model of Liver Metastasis Resulting From Gastric Cancer

Gastric cancer with liver metastasis (LM) is associated with poor prognosis due to rapid progression. It is, therefore, important to develop a quantitative and highly reproducible animal model of LM using human gastric cancer cells. Cells of a human gastric cancer cell line, HSC-57, were injected into the portal vein to produce LMs. Cells from some of these metastatic foci were expanded in vitro and subsequently implanted into the portal veins of mice. This procedure was repeated nine times. The antitumor effects of CPT-11 and NK012 were compared using the LM model. The potent metastatic clone 57L9 was obtained. NK012 exerted a stronger antitumor effect than CPT-11 against 57L9 cells integrated with the luciferase gene (57L9Luc). The survival rates on day 131 in the 57L9Luc mouse model were 100% and 0% for the NK012 and CPT-11 groups, respectively. This 57L9Luc LM model was found to be useful for monitoring the responses to NK012 and CPT-11.

Comparison of Fenestra LC, ExiTron nano 6000, and ExiTron nano 12000 for Micro-CT Imaging of Liver and Spleen in Mice

The purpose of this study was to compare different contrast agents for longitudinal liver and spleen imaging in a mouse model of liver metastasis. Mice developing liver metastases underwent longitudinal micro-computed tomography imaging after injection of Fenestra LC, ExiTron nano 6000, or ExiTron nano 12000. Elimination times and contrast enhancement of liver and spleen were compared. For all contrast agents, liver contrast peaked at approximately 4 hours and spleen contrast at 48 hours postinjection. A single dose of 100 μL of ExiTron nano 6000 or 12000 resulted in longstanding enhancement of liver and spleen tissue for longer than 3 weeks, whereas repeated injections of 400 μL of Fenestra LC were required to retain contrast at acceptable levels and allowed imaging of the liver/spleen for up to 2 and 9 days, respectively. Both ExiTron nano agents provide longer and stronger contrast enhancement of liver and spleen compared to Fenestra LC, and they do so at a 75% lower injection volume in mice.

Role of endothelin a receptor in colon cancer metastasis: In vitro and in vivo evidence

The endothelin (ET)-1/endothelin A receptor (ETAR) axis is reportedly involved in tumor cell invasion, survival, and metastasis. However, the role of ETAR in colon cancer metastasis and the underlying mechanisms have not been defined. In the present study, we assessed the role of ETAR in colon cancer metastasis in vitro and in vivo. Overexpression and knockdown of ETAR were respectively performed in SW480 and SW620 human colon cancer cells. Overexpression of ETAR in SW480 cells significantly increased cell survival against cisplatin, cell invasion, and matrix metalloproteinase (MMP)-2 expression, which was strengthened by exogenous ET-1 and abolished by selective ETAR antagonist BQ123 and phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Knockdown of ETAR in SW620 cells markedly decreased cell survival against cisplatin, cell invasion, and MMP-2 expression, which was strengthened by BQ123 and LY294002, and partially rescued by exogenous ET-1. In a colon cancer liver metastasis mouse model, while ETAR overexpression promoted colon cancer liver metastases, ETAR knockdown markedly decreased liver metastases. In conclusion, our in vitro data demonstrate that ETAR mediates the promoting effects of ET-1 on colon cancer cell survival, invasion and MMP-2 expression by a PI3K-mediated mechanism. Our in vivo data indicate that ETAR markedly promotes colon cancer liver metastasis. This study provides direct evidence for a critical role of ETAR in colon cancer metastasis, which suggests that ETAR antagonism could benefit patients with metastatic colon cancer.

Changes of T Cells and Cytokines TGF-β1 and IL-10 in Mice During Liver Metastasis of Colon Carcinoma: Implications for Liver Anti-tumor Immunity

BackgroundThe local and systemic regulation of the immune system may play important roles in the process of liver metastasis of colorectal carcinoma. The aim of this study was to establish a reproducible experimental liver metastasis model, to identify changes in T cells and cytokines TGF-β1 and IL-10, and to explore a possible mechanism of liver metastasis of colon carcinoma. MethodsWe used a colon carcinoma liver metastasis model, in which different numbers of CT-26 murine colon carcinoma cells (1 × 103, 5 × 103, 1 × 104, 5 × 104, and 1 × 105) were injected into the spleen. The liver and spleen tissues were examined for T cell markers using flow cytometry. Liver tissues were analyzed for IL-10 and transforming growth factor beta 1 (TGF-β1) expression using immunohistochemistry. ResultsSpleen injection of colon carcinoma cells is a reproducible animal model for liver metastases, which resulted in quantity-dependent metastatic growth. We provided a snapshot of the hepatic immune microenvironment in the mouse liver metastasis model. Injection of A large number of tumor cells (5 × 104 and 1 × 105) decreased anti-tumor cell counts, such as CD4+ and CD8+ T cells, and increased immune-suppressive cell counts (CD4+CD25+ Treg cells). In addition, the expression levels of immunosuppressive cytokines IL-10 and TGF-β1 were also increased with the number of tumor cells. ConclusionsChanges in the systemic and local immunological environment contribute to immunological escape mechanisms during liver metastasis of colon carcinoma, and therapies aiming at immune microenvironment may prove a useful strategy in the treatment of metastatic disease in the future.

Abstract 3888: Host-tumor interactions that promote liver metastasis of colorectal cancer.

Abstract Colorectal cancer (CRC) is the third most frequent cancer in the United States. Metastases is the major cause of death, reducing the five-year survival to less than 12%, with liver as the frequent target organ. Metastasis occurs due to productive collaborations between tumor cells and host-derived cells in the tumor microenvironment. Molecular signals from the primary tumor mediate long distance communication with the hematopoietic compartment and stimulate immune cell mobilization to the primary tumor and target organ environments. These interactions promote invasiveness of tumor cells in the primary lesion and the establishment of a pre-metastatic niche in the distant organ where cancer cells can attach and develop into a metastatic lesion. Therefore, understanding these interactions can provide us with valuable opportunities for early diagnosis of metastasis or blocking its progression by disengaging the cancer cells from their metastasis promoting networks. To understand these interactions and identify the molecular regulators, particularly at the early stages, we established an orthotopic mouse model of liver metastasis of CRC that can recapitulate all stages of tumor growth and metastasis. We used in vivo selection to isolate a highly metastatic cell line, CT26-FL3, from CT26 colon adenocarcinoma cells. Tumors derived from CT26-FL3 were more proficient in inducing a metastasis-friendly host environment compared to CT26 cells. These suggest that transition from CT26 to CT26-FL3 might be due to genetic alterations resulting from interactions between tumor cells and the host environment. We hypothesized that molecules secreted by the primary tumor can direct genetic changes in the liver microenvironment creating a fertile niche for invading cancer cells. Identifying these alterations in both the tumor cells and the host microenvironment, will allow us to understand the mechanisms by which host-tumor interactions promote liver metastasis of CRC. We carried out microarray analyses to determine the genetic signatures of the parental CT26 and highly metastatic CT26-FL3 cells, and to determine the genetic changes in the liver microenvironment in mice bearing CT26-FL3 tumors prior to metastasis. The results identified genes in signaling, cancer, metabolic, and various pathways that were differentially expressed between CT26 and CT26-FL3, and between liver from tumor-bearing and non-tumor bearing mice. More importantly, several genes in the chemokine and the cytokine-cytokine receptor pathways were expressed up to 50-fold or higher in CT26-FL3 cells or in the liver microenvironment of tumor-bearing mice. The gene products are likely mediators of the complex cross-talk between the primary tumor and the liver microenvironment. Using our orthotopic mouse model, we will assess their potential alone or in combination as viable markers for early diagnosis or as therapeutic targets to alleviate metastatic disease. Citation Format: Yu Zhang, Celestia Davis, Maria Marjorette Pena. Host-tumor interactions that promote liver metastasis of colorectal cancer. [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 3888. doi:10.1158/1538-7445.AM2013-3888

Abstract 1564: Establishment and characterization of an orthotopic mouse model for human uveal melanoma hepatic metastasis.

Abstract Background: The survival of patients with metastatic uveal melanoma remains poor due to lack of effective treatment. Uveal melanoma patients preferentially develop hepatic metastasis, which is the major cause of death. Therefore, a suitable animal model of hepatic metastasis with human uveal melanoma is critical for the investigation of its biological behavior and designing an effective therapy. Methods: Human hepatic metastatic uveal melanoma cell line (UM001), established in our laboratory, and NOD scid gamma mice (NSG mice) were used for orthotopic hepatic metastasis model. The UM001 cell line has typical characters of human melanoma, including a GNAQ mutation and expression of IGF-1 receptors. 1.00 -1.25 x 106 of UM001 cells in 15-20μl suspension of RPMI1640 medium were implanted using 27G needles under the capsule of the liver through a 1.5 cm abdominal incision. After the development of liver tumors, the NSG mice were sacrificed and investigated the behavior of the tumor cells in the liver both macro- and microscopically. Immunohistochemical analysis was performed to confirm the expression of human melanoma markers (HMB45 and Melan-A), IGF-1R. Tumor specimens were also stained for mouse CD31 (constituent of the endothelial intercellular junction of the vessels) and human laminin (major basement component of the vessels) to investigate neovascularization and vasculogenic mimicry patterns in the tumor. In addition, we performed Western blot analysis and immunohistochemistry to investigate the activation of PI3K-Akt and MAPK signaling pathways. Results: We achieved 91% (10/11cases) success rate of tumor formation using the method described in the present report. UM001 cells formed localized macroscopic nodules in the mouse liver within 4 weeks and then developed intrahepatic and peritoneal metastases within 12 weeks after implantation. Immunohistochemical analysis showed the expression of melanoma markers, IGF-1R, neovascularization positive for mouse CD31, vasculogenic mimicry patterns composed of human laminin in the hepatic tumors. In addition, activation of phosphorylated Akt and Erk1/2 in the hepatic tumor specimens was also noted. Conclusion: This orthotopic mouse model of human uveal melanoma liver metastasis showed similar histological findings as those observed in human liver. Moreover, PI3K-Akt and MAPK signaling pathway are activated in the hepatic tumor tissue. Taken together, this model is a useful tool to investigate the biological behavior of metastatic uveal melanoma in vivo, and to test novel therapies for the treatment of human uveal melanoma metastasizing to liver. Citation Format: Shinji Ozaki, Makoto Yoshida, Mizue Terai, Senthamil Selvan, Michael J. Mastrangelo, Takami Sato. Establishment and characterization of an orthotopic mouse model for human uveal melanoma hepatic metastasis. [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 1564. doi:10.1158/1538-7445.AM2013-1564

Abstract A36: Expression profiling reveals characteristics of host-tumor interactions that promote liver metastasis of colorectal cancer

Abstract Colorectal cancer (CRC) is the third most frequent cancer and the third leading cause of cancer deaths in the United States. Metastasis is the major cause of death. When diagnosed at the localized stage, the five-year survival rate is approximately 90%, however, after metastasis has occurred, the survival rate drops to less than 12%. The most frequent target organ is the liver. Successful metastasis depends on productive collaborations between tumor cells and host-derived cells in the tumor microenvironment as well as long distance communication mediated by molecular signals from the primary tumor that stimulate immune cell mobilization between the primary tumor and target organ environments and the hematopoietic compartment. Prior to the arrival of metastatic cells in the secondary organ, signals from the primary tumor direct organ specific mobilization of bone marrow derived cells to prepare a niche that promotes the attachment of cancer cells and their subsequent development into a metastatic lesion. These complex processes occur asymptomatically, thus despite its devastating impact on the patient, metastasis is often diagnosed at the final stage when very little can be done to alleviate its effects. To understand the mechanisms underlying these processes and identify the molecular regulators, particularly at the early stages, we established an orthotopic mouse model of liver metastasis of CRC that can recapitulate all stages of tumor growth and metastasis. We used in vivo selection to isolate a highly metastatic mouse carcinoma cell line, CT26-FL3, from CT26 colon adenocarcinoma cells. Tumors derived from CT26-FL3 were more proficient in inducing a metastasis-friendly host environment as compared to the parental cell line. We hypothesized that the transition from CT26 to CT26-FL3 was driven by genetic alterations resulting from interactions between tumor cells and host environment, thereby contributing to a higher incidence of liver metastasis from tumors derived from CT26-FL3. We further hypothesized that molecules secreted by the primary tumor can direct genetic changes in the liver microenvironment that promote the creation of a fertile niche for invading cancer cells. By identifying these alterations in both the tumor cells and the host microenvironment, we can begin to understand the mechanisms by which host-tumor interactions promote liver metastasis of CRC. We therefore carried out microarray analyses to: 1) determine the genetic signatures of the parental CT26 and highly metastatic CT26-FL3 cells, and 2) determine the genetic changes in the liver microenvironment in mice bearing CT26-FL3 tumors prior to metastasis. The results identified a number of genes in signaling, cancer, metabolic, and various signaling pathways that were differentially expressed between CT26 and CT26-FL3, and between liver from tumor-bearing mice and non-tumor bearing mice. More importantly, the results showed that several genes in the chemokine and the cytokine-cytokine receptor pathways were expressed by up to 50-fold or higher in CT26-FL3 cells or in the liver microenvironment of tumor-bearing mice. The products of these genes are likely mediators of the complex cross-talk between the primary tumor and the liver microenvironment. Using our orthotopic mouse model, we examine the kinetics of the changes in gene expression during metastatic progression to assess the potential of these gene products alone or in combination as viable markers in blood serum for early diagnosis of metastasis, or as targets to alleviate metastatic disease. Citation Format: Yu Zhang, Celestia Davis, Maria Marjorette O. Peña. Expression profiling reveals characteristics of host-tumor interactions that promote liver metastasis of colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A36.

Immunotherapy for the liver metastases prevention with a vaccine strain of an attenuated Salmonella Typhi

Event Abstract Back to Event Immunotherapy for the liver metastases prevention with a vaccine strain of an attenuated Salmonella Typhi Alejandrina Vendrell1*, A Tesone1, J De Toro1, L Herschlik1, M J. Gravisaco2, J C. Goin1, G Larotonda3, Claudia Mongini1 and Claudia Waldner1 1 CEFYBO-CONICET/UBA, Argentina 2 Instituto de Biotecnología, CICVyA, Argentina 3 LAVET Lab Argentina, Argentina In previous studies we demonstrated that an attenuated Salmonella Typhi vaccine strain (CVD 915) induces tumor-specific immune and therapeutic responses, following intratumoral and subcutaneous inoculations, in mice bearing a subcutaneous mammary adenocarcinoma (LM3). The aim of this work was to study whether orogastric (og) administration of CVD 915 can prevent tumor cell implantation in liver, in a mouse model of liver metastases. Each BALB/c mouse received og 5x109UFC of CVD 915 or PBS (control group). After 24 hours, mice were subjected to surgery and 50,000 LM3 cells were injected into their spleens. Then, a splenectomy was performed. After 24 h of bacterial treatment, CVD 915-treated mice exhibited elevated IFN-γserum levels. Seven days later, Salmonella treatment stimulated the proinflammatory Th1 cytokines production (enhanced IFN-γ level in serum, p<0.001, and detectable serum TNF- α and IL-12 levels). The prophylactic treatment with Salmonella significantly decreased the occurrence of liver metastases as well as their average volume (1.9 vs. 6.2 mm3) 21 days after surgery, as compared with control mice (p<0.05). Moreover, at that time, an increased frequency of CD4+lymphocytes but a decreased proportion of B cells (B220+) in both liver and peripheral blood were observed in CVD 915-treated animals (p<0.05). These results indicate that a CVD 915-based immunotherapy used preventively via og route induces an antitumor immunity and prevents the implantation of metastasis in a vital organ. The use of this vaccine as an adjunct therapy to conventional cancer treatments, such as surgery, should be assessed in the future. Keywords: Cancer, Immunotherapy, liver metastasis, Salmonella typhi, mamary adenocarcinoma Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Vendrell A, Tesone A, De Toro J, Herschlik L, Gravisaco MJ, Goin JC, Larotonda G, Mongini C and Waldner C (2013). Immunotherapy for the liver metastases prevention with a vaccine strain of an attenuated Salmonella Typhi. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01202 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Sep 2013; Published Online: 11 Sep 2013. * Correspondence: Dr. Alejandrina Vendrell, CEFYBO-CONICET/UBA, Buenos Aires, Argentina, alejandrinavendrell@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alejandrina Vendrell A Tesone J De Toro L Herschlik M J Gravisaco J C Goin G Larotonda Claudia Mongini Claudia Waldner Google Alejandrina Vendrell A Tesone J De Toro L Herschlik M J Gravisaco J C Goin G Larotonda Claudia Mongini Claudia Waldner Google Scholar Alejandrina Vendrell A Tesone J De Toro L Herschlik M J Gravisaco J C Goin G Larotonda Claudia Mongini Claudia Waldner PubMed Alejandrina Vendrell A Tesone J De Toro L Herschlik M J Gravisaco J C Goin G Larotonda Claudia Mongini Claudia Waldner Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Nanoamplifiers synthesized from gadolinium and gold nanocomposites for magnetic resonance imaging

We have synthesized an efficient and highly sensitive nanoamplifier composed of gadolinium-doped silica nanoparticles and gold nanoparticles (AuNPs). Magnetic resonance imaging (MRI) in vitro and in vivo assays revealed enhancement of signal sensitivity, which may be explained by electron transfer between water and gadolinium-doped nanoparticles, apparent in the presence of gold. In vitro and in vivo evaluation demonstrated nanoamplifier incurred minimal cytotoxicity and immunotoxicity, increased stability, and gradual excretion patterns. Tumor targeted properties were preliminarily determined when the nanoamplifier was injected into mouse models of colon cancer liver metastasis. Furthermore, although AuNPs departed from the nanoamplifiers in specific mice tissues, optical and magnetic resonance imaging was efficient, especially in metastatic tumors. These assays validate our nanoamplifier as an effective MRI signal enhancer with sensitive cancer diagnosis potential.

Extracellular signal-regulated kinase and Akt activation play a critical role in the process of hepatocyte growth factor-induced epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) has recently been studied to elucidate mechanisms of the liver metastatic process. We investigated EMT in the process of liver metastasis and the effects of chemotherapy on EMT cells as therapeutic strategy for colorectal liver metastasis. We used the CT26 murine colorectal carcinoma cell line to create an in vivo mouse liver metastasis model. Liver tumors were stained immuno-histochemically. Expression of proteins associated with TGF-β/Smad and hepatocyte growth factor (HGF)/c-Met pathways were investigated by western blotting. Cells with c-Met mRNA knockdown by siRNA techniques showed clearly reduced liver metastases compared with regular cells at 21 days. TGF-β and HGF induced EMT expression, but signal transduction was quite different. TGF-β induced ERK, but not Akt phosphory-lation. HGF mediated both ERK and Akt phosphorylation. Akt inhibitor blocked Akt phosphorylation but did not affect TGF-β-induced activation of ERK, Snail and Slug. U-0126 did not reduce Snail activity by TGF-β at a concentration to block ERK phosphorylation. However, Akt inhibitor and U-0126 completely inhibited HGF-induced Slug activation. 5-FU mediated cell death in the EMT process induced by TGF-β more effectively than HGF. ERK/Akt signaling, but not the Smad pathway, may be one of the main processes in HGF-induced EMT, despite the Smad pathway, but not ERK/Akt, being critical for TGF-β-induced EMT. The MAPK/Akt pathway is indispensable in HGF/c-Met signaling. The ERK/Akt pathway particularly may be critical in the HGF-induced EMT process. However, long-term use of chemotherapeutic agents may induce drug resistance and distant metastases through EMT-related signaling pathway activation.

Combined effect of dehydroxymethylepoxyquinomicin and gemcitabine in a mouse model of liver metastasis of pancreatic cancer

Activation of nuclear factor-κB (NF-κB) has been implicated in metastasis of pancreatic cancer. We investigated the effects of the novel NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) on the inhibition of liver metastasis of pancreatic cancer in a mouse model of clinical liver metastasis. Nude mice were xenografted by intra-portal-vein injection with the human pancreatic adenocarcinomas cell line AsPC-1 via small laparotomy. Mice were treated with DHMEQ and gemcitabine (GEM), alone or in combination. The combination of GEM+DHMEQ showed a stronger antitumor effect than either monotherapy. Apoptosis induction in the metastatic foci was greatest in the DHMEQ+GEM group. Significant reductions in the numbers of neovessels were also seen in the DHMEQ and/or GEM groups. Cell growth inhibition assays revealed no synergistic effect of combination therapy, although each monotherapy had an individual cytotoxic effect. Combination therapy produced the greatest inhibition of tumor cell invasiveness in chemoinvasion assay. In addition, combination therapy significantly down-regulated the expression level of matrix metalloproteinase (MMP)-9 mRNA in AsPC-1 cells. DHMEQ also markedly down-regulated interleukin-8 and MMP-9, while GEM caused moderate down-regulation of vascular endothelial growth factor in metastatic foci, demonstrated by quantitative reverse transcription-polymerase chain reaction. These results demonstrate that DHMEQ can exert anti-tumor effects by inhibiting angiogenesis and tumor cell invasion, and by inducing apoptosis. Combination therapy with DHMEQ and GEM also showed potential efficacy. DHMEQ is a promising drug for the treatment of advanced pancreatic cancer.

MiR-493 induction during carcinogenesis blocks metastatic settlement of colon cancer cells in liver

Liver metastasis is a major lethal complication associated with colon cancer, and post-intravasation steps of the metastasis are important for its clinical intervention. In order to identify inhibitory microRNAs (miRNAs) for these steps, we performed 'dropout' screens of a miRNA library in a mouse model of liver metastasis. Functional analyses showed that miR-493 and to a lesser extent miR-493(*) were capable of inhibiting liver metastasis. miR-493 inhibited retention of metastasized cells in liver parenchyma and induced their cell death. IGF1R was identified as a direct target of miR-493, and its inhibition partially phenocopied the anti-metastatic effects. High levels of miR-493 and miR-493(*), but not pri-miR-493, in primary colon cancer were inversely related to the presence of liver metastasis, and attributed to an increase of miR-493 expression during carcinogenesis. We propose that, in a subset of colon cancer, upregulation of miR-493 during carcinogenesis prevents liver metastasis via the induction of cell death of metastasized cells.

Multiple roles of angiotensin in colorectal cancer

Colorectal cancer (CRC) cells express renin and chymase through which they can activate angiotensin. Renin expression is induced by hyperglycemic conditions. As angiotensinogen is produced in the liver, CRC cells that can activate angiotensin have an enhanced ability to metastasize to this organ. In human CRC cases, patients with diabetes have higher activities of rennin and angiotensin-II in primary tumors, and on average, have a more progressed disease stage, especially with respect to liver metastasis. These patients exhibit a stronger association with Hemoglobin A1c levels and metastasis compared to patients without diabetes. In a combined diabetes/CRC liver metastasis mouse model, concurrent treatment with anti-angiotensin and hypoglycemic agents shows a synergic effect in terms of reduced liver metastasis and improved survival. The effect of anti-angiotensin treatment and blood sugar control as a baseline management for colon cancer patients with diabetes needs to be examined in clinical trials to establish whether it can prevent liver metastasis.

Intratumoral injection of interleukin‐2 augments the local and abscopal effects of radiotherapy in murine rectal cancer

Recent studies have suggested that tumor shrinkage in response to radiotherapy (RT) is greatly dependent on the host immune response. A Balb/c mouse model of simultaneous subcutaneous tumor and liver metastasis of Colon26 was prepared and, after irradiation of the subcutaneous tumor (2 Gy × 5 day × 2 cycles), interleukin-2 (IL-2) (2 × 10(4) U) was injected intra-tumorally, and the fate of both the subcutaneous tumor and liver metastatic lesions was evaluated. Intratumoral injection of IL-2 greatly enhanced the anti-tumor effects of RT and completely eradicated the established subcutaneous tumor. Interestingly, although RT was given locally to the subcutaneous tumor, liver metastasis formation was also inhibited in mice receiving only local RT. More impressively, the combination of RT + IL-2 completely inhibited liver metastasis formation. Splenocytes in mice receiving RT + IL-2 contained a higher percentage of CD4(+) T cells, but lower percentages of CD4(+)CD25(+) regulatory T cells and CD11b(+) Gr-1(+) myeloid-derived suppressor cells. Immunohistochemical investigation of human rectal cancer revealed that the density of CD8(+) cells infiltrating into irradiated rectal tumor was positively associated with a lower frequency of distant metastasis as well as histological response grade. Local administration of IL-2 not only enhances shrinkage of the irradiated tumor itself, but can also suppress the development of distant metastasis located outside the RT field, possibly though the induction of a systemic T cell response. Augmentation of T-cell-mediated antitumor immunity during RT might be critical for improvement of the clinical efficacy of neoadjuvant RT for the treatment of advanced rectal cancer.

Abstract 3533: DNA binding molecules as a new tool to regulate cancer cell fate

Abstract Pyrrole-Imidazole (PI) polyamide molecule was originally designed from structures of natural DNA binding molecule, such as Distamicine and Diocarbamicine and has been discovered as a synthetic molecule which recognizes the minor groove of Watson-Click base pair of double-stranded DNA in a sequence-dependent manner. We have recently developed a semi-automatic synthesis system for PI polyamide, which are able to regulate specific target gene-expression under specific transcription factor binding inhibition for biological functional studies and perhaps patients’ therapy. PI polyamide immediately penetrated the nucleus in vitro and in vivo without any vehicle. After intra venous injection It rapidly reduce the serum concentration, delivered to most of tissue cells, excreted to urine or bile juice and did not metabolize in animals. The PI polyamides, designed for anti-Tgfb1 and anti-MMP9 activity, were well tolerated, reduced target gene expression and showed therapeutic effects in animal models of human diseases. For instance, when performed two studies using I.V. treatment by either of two independent anti-MMP9 polyamides, tumor metastasis was significantly suppressed in both studies using the same mouse model of human liver metastasis of colon cancer. This new auto-synthetic chemicals can be designed for many transcriptional regulation of transcripts and applied to prove many biological hypothesis of transcriptional regulation for cancer research, and may be used for cancer therapy in the future. 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 3533. doi:10.1158/1538-7445.AM2011-3533

Efficient in vivo microRNA targeting of liver metastasis

Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors.

Clinical significance of aggressive hepatectomy for colorectal liver metastasis, evaluated from the HGF/c-Met pathway

Liver metastasis is one of the most critical factors in deciding the prognosis of patients with colorectal cancer (CRC). Hepatectomy is the most curative treatment for liver metastasis of CRC. The high amount of hepatocyte growth factor (HGF) is produced to promote liver regeneration by hepatectomy. Theoretically, HGF produced after hepatectomy stimulates the progression of CRC cells with c-Met in residual liver. This study was aimed to evaluate the value of hepatectomy towards liver metastasis of CRC in relation to the HGF/c-Met pathway. Ninety-four patients with CRC (including 24 liver metastasis cases) were operated at Gifu University Hospital (2002-2004). For these cases, the expression of c-Met in the primary and liver metastatic sites was evaluated by immunohistochemistry and Western blot. Experiments were also conducted on CT26 murine CRC cell line and a mouse liver metastasis model. In clinical study, the c-Met expression in liver metastatic sites was lower than in the primary sites in 87% of 24 cases. In basic study, the expression of c-Met protein in the liver tumor was significantly lower than in culture cells according to Western blot (p=0.033). The growth of residual liver tumors was not significantly different between 30% hepatectomy group and no operation group. The over-expression of c-Met was closely associated with CRC liver metastases. On the other hand, in liver metastatic lesions, the c-Met expression was reduced in comparison to primary lesions. Therefore, even if serum HGF levels increased due to liver resection during the regeneration period, residual liver metastases of CRC was not promoted in its progression. Aggressive hepatectomy would still be acceptable and favorable as a curative therapy.

Preclinical analysis of targeting miR-182 against melanoma liver metastasis.

8554 Background: Malignant melanoma is a devastating cancer and the overall survival is extremely poor for stage IV disease. The paucity of effective treatments clearly illustrates the dire need of alternative therapies for metastatic melanoma. Targeting oncogenic microRNAs has the potential to become an important strategy for targeted therapy. Studies from our lab have demonstrated a prometastatic role for miR-182 in melanoma. Moreover, we proved that miR-182 repression in vitro impairs the invasive capacity of melanoma cells and triggers an antiproliferative response. Thus, we hypothesized that the silencing of miR-182 can be of therapeutic benefit in melanoma. Methods: We have tested the effect of anti-miR- 182 oligonucleotides synthesized with a phosphorothioate backbone in a mouse model of liver metastasis after intrasplenic injection of melanoma cells. 1E04 A375 cells stably transduced with the luciferase cDNA were injected into the spleen of NOD/Scid/IL2gR-/- (NOG) mice. Thirty mice received intrasplenic injections of A375 cells, and then were distributed into two groups to either receive anti-miR-182 or a control anti-miR (25 mg/Kg), administered by intraperitoneal injection twice weekly. Mice were monitored using in vivo fluorescence imaging for tumor cell migration from the spleen into the liver. Results: Luciferase imaging performed 4 weeks post-injection showed that mice treated with anti-miR-182 had an appreciably lower burden of macroscopic liver metastases compared to the control. Treatment with anti-miR-182 reduced the number of mice with > 50 macroscopic liver metastases by half. We also confirmed that the anti-miR treated cohort displays lower levels of miR-182 compared to the scrambled treated cohort both in the tumor tissue and in the liver parenchyma. Moroever, we demonstrated that miR-182 targets ADCY6 and FOXO3 were up-regulated in the tumor cells of anti- miR-182 treated mice. Conclusions: These results indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of target levels. These results suggest that anti-miR-182 is a promising therapeutic strategy for metastatic melanoma. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Regulus Therapeutics Isis Pharmaceuticals

Abstract 527: Pyrrole-Imidazole polyamide inhibits MMP-9 transcription and induces suppression of tumor metastasis

Abstract Pyrrole-Imidazole (PI) polyamide that targets the AP-1-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. MMP-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. The synthesized MMP-9 PI polyamide showed selective DNA binding ability and significantly inhibited MMP-9 mRNA and protein expression and enzymatic activity in both MDA-MB-231 and Hela cells. Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 minutes of incubation with an MDA-MB-231 cell and remained in the nuclei up to 96 hours after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis of xenografted tumor cells and host angiogenesis in tumors in a mouse model of liver metastasis. Our results suggest that this PI polyamide that targets the MMP-9 gene promoter can be a novel MMP-9 down-regulating molecule for anti-metastasis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 527.

Inhibition of MMP‐9 transcription and suppression of tumor metastasis by pyrrole‐imidazole polyamide

Matrix metalloproteinase (MMP)-9, the 92-kDa type IV collagenase, contributes to tumor invasion and metastases, and strategies to down-regulate its expression could ultimately be of clinical utility. A pyrrole-imidazole (PI) polyamide that targets the activator protein-1 (AP-1)-binding site of the MMP-9 promoter was designed and synthesized as a gene-silencing agent for tumor metastases. The synthesized product showed selective DNA binding ability. The MMP-9 PI polyamide significantly inhibited MMP-9's mRNA expression, protein level, and enzymatic activity in human breast adenocarcinoma cells (MDA-MB-231). Furthermore, the MMP-9 PI polyamide inhibited migration and invasion by in vitro wound-healing and matrigel-invasion assay. The FITC-labeled PI polyamide was localized in nuclei in 45 min of incubation with an MDA-MB-231 cell and remained in the nuclei for up to 96 h after incubation in vitro. It was also quickly localized in the mouse cellular nuclei of many tissues, including liver, kidney, and spleen, after intravenous injection without using any drug-delivery system. Moreover, the polyamide treatment significantly decreased metastasis in a mouse model of liver metastasis. Our results suggest that this PI polyamide, which targets the MMP-9 gene promoter, can be a novel MMP-9 down-regulating molecule for antimetastasis.