CT26 Colon Adenocarcinoma Cells Research Articles

Babao Dan Alleviates Cancer Cachexia in Mice Via Inhibiting IL-6/STAT3 Signaling Pathway.

Cancer cachexia is a common but severe condition that causes muscle wasting, body weight loss, and progressive functional impairment, affecting over 50% of cancer patients. Currently, there are no effective treatments that can alleviate cachexia, and hence the discovery of new therapeutics that can effectively prevent or even reverse cancer cachexia is crucial. Babao Dan (BBD) is a Traditional Chinese Medicine (TCM) formula that has been used clinically in combating various cancers, however, its therapeutic potential in alleviating cancer cachexia remains unexplored. Our current study aims to determine the anti-cachectic effects of BBD treatment in alleviating cancer cachexia, as well as determining the underlying mechanisms involved. Mouse models of cancer cachexia were induced via implantation of CT26 colon adenocarcinoma cells, and the anti-cachectic effects and mechanisms of BBD were determined via examinations of body weight and muscle mass, as well as serum and muscle markers of cachexia and muscle atrophy. CT26 tumor implantation reduced in the rapid occurrence of cancer cachexia characterized by marked reductions in body weight and muscle mass, functional decrease in muscle function and accelerated deaths. BBD administration not only demonstrated robust anti-cachectic ability via preventing decreases in body weight, muscle mass, and muscle atrophy, but also markedly prolonged survival. The effects of BBD in alleviating cancer cachexia and its associated adverse effects were due to its ability in preventing the activation of IL-6/STAT3 signaling post-CT26 tumor implantation. Our findings demonstrated the robust ability of BBD in preventing cancer cachexia and alleviating the main cachexia-induced symptoms as well as prolonging survival via inhibiting activation of IL-6/STAT3 signaling pathway. Therefore, our study demonstrating the strong anti-cachectic effects of BBD in mice may provide a theoretical basis for the use of BBD as a safe and effective drug in the treatment of cancer cachexia.

Shed syndecan-2 enhances colon cancer progression by increasing cooperative angiogenesis in the tumor microenvironment

Although shed syndecan-2 potentiated the tumorigenic activities of colon cancer cells, how shed syndecan-2 increases this tumorigenic potential remains unclear. Using an orthotopic mouse model of colon cancer, we show that shed syndecan-2 increases colon cancer progression by cooperatively promoting angiogenesis. Co-administration with a synthetic peptide of shed syndecan-2 (S2LQ) enhanced the survival and tumor engraftment of luciferase-expressing CT26 colon adenocarcinoma cells orthotopically implanted into the cecum of BALB/c mice. Intravenous injection of S2LQ further enhanced the growth of orthotopic tumors in the cecum, with increases in the tissue infiltration of macrophages and the formation of blood vessels, mainly in peripheral layers of the tumor facing the stroma. Furthermore, S2LQ stabilized HIF1α and enhanced the VEGF expression in human colon cancer cell lines, and increased the migration of RAW 264.7 murine macrophage cells and bone marrow-derived macrophages. Finally, S2LQ increased the tube formation of vascular endothelial cells in vitro. Together, these data demonstrate that shed syndecan-2 enhances tumorigenic activity by increasing the crosstalk of cancer cells with tumor-associated macrophages and endothelial cells to enhance angiogenesis for colon cancer progression in the tumor microenvironment.

Immuno‐Reactive Cancer Organoid Models to Examine Microbiome Metabolite Effects on Immune Checkpoint Blockade Efficacy

Introduction As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, a sizable percentage of patients are non-responders to all types of immunotherapy. Yet, there has been limited number of in vitro models to assess tumor immune-reactivity and enable directed study of the mechanisms of immunotherapy resistance in solid tumors. The goal of this project is develop a model in which we could validate cancer cell killing due to immune cell action in an easily modifiable environment. Towards that, we created 3D tumor tissue constructs (organoids) containing cancer cells paired with cytotoxic T-cells to model immunotherapy efficacy. Our choice of immune checkpoint inhibitor antibodies were a paired course of PD-1 and CTLA-4, which are used extensively in the clinic, or anti-CD47. Upon validation of our organoid model, they could address specific mechanisms behind certain tumor microenvironment elements, such as bacterial metabolites, on immunotherapy efficacy. Methods We created extracellular matrix like 3D structures using collagen/hyaluronic acid-based hydrogels into which tumor cell were mixed, along with T-cells. The cancer cells incorporated were either freshly-isolated CT-26 colon adenocarcinoma cells, 4T1 breast cancer cells, or cultured CT-26 cells. The T-cells were either derived from murine lymph nodes or were isolated from the murine tumor infiltrating mononuclear cell population. Organoids were treated with microbial metabolites and their response to therapeutic equivalent doses of anti- PD-1, CTLA-4 and CD47 antibodies was examined using cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry (IHC) staining. Results The results show that in our immune cell supplemented tumor organoid models the immune checkpoint inhibitor regimens stimulated internally localizing T-cells and inducing T-cell-mediated tumor cell killing. Checkpoint inhibitor treated samples showed proportionally greater loss of viability when compared to untreated controls (p<0.01). The results were corroborated by IHC, showing increased numbers of CD-4+ T-cells and cytotoxic proteins such as granzyme B in the stimulated samples. RT-qPCR results show an increase in key proteins for immune function when both immunotherapy and bacterial metabolites are administered simultaneously. Conclusion: We have created an ex-vivo immune-reactive tumor organoid model for studying immunotherapy. This model will allow us to modulate facets of the tumor cell component in the organoid system including cancer type and grade, tumor cell mutations, biochemical signals, and physical properties of the microenvironment. We can then observe the impacts of these changes on immunotherapy efficacy in order to identify factors that could be targeted to improve immunotherapy efficacy. In this study, we have focused on the effects of bacterial metabolites on immunotherapy efficacy. Preliminary data suggest that there may be a connection between these microbial factors and tumor sensitivity to immunotherapy.

Tumor cell endogenous HIF-1α activity induces aberrant angiogenesis and interacts with TRAF6 pathway required for colorectal cancer development

Hypoxia and inflammation are key factors for colorectal cancer tumorigenesis. The colonic epithelium belongs to the tissues with the lowest partial pressure of oxygen in the body, and chronic inflammation is associated with an increased chance to develop colon cancer. How the colonic epithelium responds to hypoxia and inflammation during tumorigenesis remains to be elucidated. Here we show, that murine colon adenocarcinoma cells with attenuated response to hypoxia, due to a knock-down (KD) of HIF-1α, produce smaller and less hypoxic tumors in an orthotopic mouse model when compared to tumors induced with control cells. HIF-1α-KD tumors showed more functional perfused vasculature associated with increased levels of vessel-stabilizing factors and reduced levels of proangiogenic factors, including extracellular matrix protein Cyr61/CCN1. Intratumoral injection of Cyr61 in HIF-1α-KD tumors revealed an in increased vessel permeability and tumor hypoxia. Further bioinformatics analysis identified a possible interaction between HIF-1α and TRAF6, an upstream effector of the NF-κB pathway that was confirmed by coimmunoprecipitation in MC-38 and CT26 colon adenocarcinoma cells and in situ by proximity ligation assay. Down-regulation of TRAF6 resulted in virtual abrogation of orthotopic tumor growth. Subcutaneous TRAF6-KD tumors were smaller and contained reduced vessel size and differently polarized macrophages. These data demonstrate that the tumor cell response to increased hypoxia in the colon leads to promotion of nonfunctional angiogenesis, regulated by both hypoxia and TRAF6 pathways.

Abstract 3350: Immuno-reactive cancer organoid models to examine immune checkpoint blockade efficacy

Abstract Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy. These differences in sensitivity can be either innate or acquired. Yet, there has been limited number of 3D in vitro models to assess tumor immune-reactivity. These models allow directed study of the mechanisms of immunotherapy resistance in solid tumors. Our goal was to create organoids containing cancer cells from a variety of origins paired with cytotoxic T-cells of various origins to model immunotherapy efficacy. We pursued a system in which we could validate cancer cell killing due to immune cell action in an easily modifiable environment. Our choice of immune checkpoint inhibitor antibodies were a paired course of PD-1 and CTLA-4, which are used extensively in the clinic for several cancer types, or anti-CD47. The immune reactive tumor organoid system could be more generally applied to answer questions about immunotherapy resistance. Methods: We created extracellular matrix (ECM)-like structures using collagen/hyaluronic acid-based hydrogels into which primary tumor cells or tumor cell lines were mixed, along with T-cells. The cancer cells incorporated were either MC-38 OVA cells for a positive T-cell-mediated cytotoxicity control, freshly-isolated CT-26 colon adenocarcinoma cells, 4T1 breast cancer cells, or cultured CT-26 cells. The T-cells were either derived from murine lymph nodes or were isolated from the murine tumor infiltrating mononuclear cell population. Organoids were treated with therapeutic equivalent doses. Additional factors such as microbial metabolites were evaluated for their effects on immunotherapy efficacy. We performed viability assays, flow cytometry, RT-qPCR, and immunohistochemistry (IHC) staining to characterize results. Results and Discussion: We have been able to successfully show that the immune checkpoint inhibitor regimens stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing our organoid model. Checkpoint inhibitor treated samples resulted in proportionally greater loss of viability with increased significance when compared to positive controls. The results were corroborated by IHC, showing increased numbers of CD-4+ T-cells and cytotoxic proteins such as granzyme B in the stimulated samples. Conclusion: We have created an ex-vivo tumor immune-reactive tumor organoid model for studying immunotherapy. This will allow us to modulate facets of the tumor in the organoid system including cancer type, tumor cell mutations, biochemical signals, and physical properties of the microenvironment. We can then observe the impacts of these changes on immunotherapy efficacy to determine what factors could potentially be contributing to differences in patient sensitivity. Citation Format: Ethan N. W. Shelkey, David Soto-Pantoja, Yong Lu, Shay Soker. Immuno-reactive cancer organoid models to examine immune checkpoint blockade efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3350.

Repression of osteopontin by IRF8 regulates a novel immunosuppressive checkpoint

Abstract While current immune checkpoint inhibitors (ICIs) have shown efficacy in a wide variety of human cancers, many patients and cancer types fail to respond to ICI immunotherapy, suggesting the presence of other immunoinhibitory checkpoints. We report here that a deficiency in the transcription factor interferon regulatory factor 8 (IRF8) led to a loss of function of cytotoxic T lymphocytes (CTLs) in both an allograft tumor and a peptide vaccine model. However, a T cell-specific knockout and a mixed chimera model demonstrated that IRF8 suppressed CTL activation through a CTL-extrinsic mechanism. This was found to be through IRF8-dependent suppression of the matricellular protein osteopontin (OPN). During colorectal cancer (CRC) development, IRF8 is downregulated in both tumor-associated CD11b+Ly6CloLy6Ghi myeloid cells and colon epithelial cells, where it represses OPN expression by binding to its promoter. Accordingly, in both mouse CRC models and human CRC patients, transcriptional downregulation of IRF8 was accompanied by an upregulation of OPN at the transcriptional and protein level. Enhanced levels of OPN were found to negatively correlate with patient survival. In vitro stimulation of mouse and human CTLs in the presence of OPN led to decreased activation and interferon gamma secretion. Similarly, deletion of spp1, which encodes for OPN, from murine CT26 colon adenocarcinoma cells caused increased susceptibility to CTL-mediated lysis and enhanced CTL activation. As such, tumor and myeloid-derived OPN may function as a novel immune checkpoint to restrain host CTL activation.

Selumetinib Attenuates Skeletal Muscle Wasting in Murine Cachexia Model through ERK Inhibition and AKT Activation.

Cancer cachexia is a multifactorial syndrome affecting the skeletal muscle. Previous clinical trials showed that treatment with MEK inhibitor selumetinib resulted in skeletal muscle anabolism. However, it is conflicting that MAPK/ERK pathway controls the mass of the skeletal muscle. The current study investigated the therapeutic effect and mechanisms of selumetinib in amelioration of cancer cachexia. The classical cancer cachexia model was established via transplantation of CT26 colon adenocarcinoma cells into BALB/c mice. The effect of selumetinib on body weight, tumor growth, skeletal muscle, food intake, serum proinflammatory cytokines, E3 ligases, and MEK/ERK-related pathways was analyzed. Two independent experiments showed that 30 mg/kg/d selumetinib prevented the loss of body weight in murine cachexia mice. Muscle wasting was attenuated and the expression of E3 ligases, MuRF1 and Fbx32, was inhibited following selumetinib treatment of the gastrocnemius muscle. Furthermore, selumetinib efficiently reduced tumor burden without influencing the cancer cell proliferation, cumulative food intake, and serum cytokines. These results indicated that the role of selumetinib in attenuating muscle wasting was independent of cancer burden. Detailed analysis of the mechanism revealed AKT and mTOR were activated, while ERK, FoxO3a, and GSK3β were inhibited in the selumetinib -treated cachexia group. These indicated that selumetinib effectively prevented skeletal muscle wasting in cancer cachexia model through ERK inhibition and AKT activation in gastrocnemius muscle via cross-inhibition. The study not only elucidated the mechanism of MEK/ERK inhibition in skeletal muscle anabolism, but also validated selumetinib therapy as an effective intervention against cancer cachexia. Mol Cancer Ther; 16(2); 334-43. ©2016 AACR.

Gamma-secretase inhibitor does not modulate angiogenesis in colon adenocarcinoma in obese mice.

Notch is a signaling molecule which plays a role in angiogenesis and γ-secretase is required for processing of Notch. In this study, we investigated the effect of γ-secretase inhibitor (DAPT) on tumor angiogenesis in diet-induced obese mice. 18 mice were divided into three groups; control, obese (diet-induced) and obese+DAPT. After 15 weeks, the obese mice were subjected for tumor induction of CT26 colon adenocarcinoma cells (5 x 105 cells). When the tumor size reached approximately 350 ± 50 mm3, half of the obese animals received DAPT (10mg/kg/day) subcutaneously. Blood samples were taken after 14 days and the tumors harvested for immunohistochemical staining and capillary density were reported as CD31 positive cells/mm2. The obese animals had higher serum leptin and NO concentrations, while, serum VEGF and VEGFR-1 concentrations were not different compare to control group. Administration of DAPT in obese mice significantly reduced serum VEGFR-1 and leptin concentrations and increased serum NO level (p < 0.05). Capillary density in the tumors of obese animals was not different compare to control groups. DAPT administration could not alter capillary density in the tumors. Administration of DAPT in obese mice altered serum angiogenic factors, however, it could not modulate tumor angiogenesis in diet-induced obese mice (Fig. 4, Ref. 26).

Abstract 2085: Interleukin 33 promotes tumor development, progression, and metastasis in colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States, and the major cause of death is metastasis which is frequently targeted to the liver. Metastasis progression typically occurs without symptoms, and is often diagnosed at the final stage when little can be done to help the patient. Understanding the genetic and molecular mechanisms at the early stages can lead to the identification of bio-molecular markers that might be useful for early diagnosis or as therapeutic targets to alleviate morbidity and mortality from the disease. To identify factors that promote CRC progression and liver metastasis, we utilized an orthotopic mouse model of CRC metastasis to the liver that we had developed by cecal implantation of CT26 colon adenocarcinoma cells followed by sequential in vivo selection to isolate a highly liver metastatic CRC cell line, CT26-FL3. A whole genome microarray analysis was performed to compare the genetic signature of CT26-FL3 to its isogenic parental cell line CT26. We identified interleukin (IL) 33 a one of the genes that was highly overexpressed in CT26-FL3 as compared to CT26 cells. We found that increased IL-33 expression was associated with advancing stages of CRC in patient samples and that its expression was already elevated in nascent adenomas in the ApcMin/+ mouse, a genetic mouse model of intestinal tumorigenesis. To determine its role in promoting tumor progression, the murine colon cancer cell line MC38 was stably transfected with an Il33-expressing plasmid. We found that overexpression of IL-33 in MC38 cells promoted faster growth of the primary tumor and enhanced its metastasis into the liver. Taken together, the data suggest that IL33 might play an important role in the early development and progression to metastasis of CRC. IL-33 can function as a conventional cytokine or as a transcriptional regulator. It signals through a membrane bound ST2 receptor, as well as a soluble ST2 receptor that can act as a molecular sink to block its signaling pathway. Its potential as a viable marker for diagnosis or as a therapeutic target will be further determined in this study. Citation Format: Yu Zhang, Celestia Davis, Maria Marjorette Pena. Interleukin 33 promotes tumor development, progression, and metastasis in colorectal cancer. [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 2085. doi:10.1158/1538-7445.AM2014-2085

KITENIN-targeting MicroRNA-124 Suppresses Colorectal Cancer Cell Motility and Tumorigenesis

MicroRNAs are increasingly implicated in the modulation of the progression of various cancers. We previously observed that KAI1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in sporadic human colorectal cancer (CRC) tissues and hence the functional KITENIN complex acts to promote progression of CRC. However, it remains unknown that microRNAs target KITENIN and whether KITENIN-targeting microRNAs modulate CRC cell motility and colorectal tumorigenesis. Here, through bioinformatic analyses and functional studies, we showed that miR-124, miR-27a, and miR-30b negatively regulate KITENIN expression and suppress the migration and invasion of several CRC cell lines via modulation of KITENIN expression. Through in vitro and in vivo induction of mature microRNAs using a tetracycline-inducible system, miR-124 was found to effectively inhibit the invasion of CT-26 colon adenocarcinoma cells and tumor growth in a syngeneic mouse xenograft model. Constitutive overexpression of precursor miR-124 in CT-26 cells suppressed in vivo tumorigenicity and resulted in decreased expression of KITENIN as well as that of MYH9 and SOX9, which are targets of miR-124. Thus, our findings identify that KITENIN-targeting miR-124, miR-27a, and miR-30b function as endogenous inhibitors of CRC cell motility and demonstrate that miR-124 among KITENIN-targeting microRNAs plays a suppressor role in colorectal tumorigenesis.

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 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.

Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity and reactive oxygen species production in MDCK-I renal cells and intact mice

Cisplatin-induced oxidative stress can cause liver and kidney damage, thus limiting therapeutic efficacy. Thus, in the present study, since Rhus verniciflua Stokes (RVS) containing flavonoids has antioxidant effects, we investigated whether it can protect cisplatin-induced toxicity in vitro and in vivo, The in vitro effects of RVS on the cell viability and reactive oxygen species (ROS) production were investigated using cisplatin-treated Madin-Darby Canine kidney (MDCK)-I renal cells. Its in vivo effects were also studied in BALB/c mice inoculated with CT-26 colon adenocarcinoma cells and treated with cisplatin with or without RVS. Liver and renal functions were assessed together with indices of tissue oxidation. RVS prevented cisplatin-induced cytotoxicity and ROS release against MDCK-I cells. RVS alone exerted modest antitumor activity against CT-26 cells. When used concurrently with cisplatin, RVS prevented the increases in serum blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and NO, while reducing liver and kidney tissue MDA content, and increasing catalase, glutathione (GSH), and superoxide dismutase (SOD) activities. Moreover, the antitumor efficacy of cisplatin was not altered by concurrent administration of RVS. These findings demonstrate that RVS prevents cisplatin-induced toxicity in vitro and in vivo via an antioxidant activity without hurting its antitumor effectiveness, suggesting that RVS can be usefully applied to the neoplastic patients as a combined chemopreventive agent with cisplatin.

Targeting transforming growth factor-β signaling in liver metastasis of colon cancer

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-β can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-β receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-β type I and type II receptors, LY2109761, inhibits TGF-β-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-β on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.

Micro-CT Imaging with a Hepatocyte-selective Contrast Agent for Detecting Liver Metastasis in Living Mice

Micro-computed tomography (CT) is a important tool for longitudinal imaging of tumor development. The detection and monitoring of tumors in the liver in live animals using micro-CT is challenging. We evaluated the feasibility of high-resolution micro-CT enhanced with a hepatocyte-selective contrast agent for detecting liver metastases in a live murine model. Hepatic metastases were induced in 10 BALB/C mice. Two mice each were randomly selected on days 3, 5, 7, 10, and 13 after CT26 colon adenocarcinoma cells were injected into the portal vein; micro-CT imaging was performed at 10 minutes and 4 hours after intravenous administration of a hepatocyte-selective contrast agent at a dose of 0.4 mL/mouse. The attenuation values of the normal liver and the tumors were obtained. The number of metastases was counted and their sizes were measured on the micro-CT images. Gross or histopathologic evaluation was performed for correlating the liver tumors with the micro-CT images. A total of 74 separate tumor sites larger than 300 microm in diameter were detected on pathologic examination of the mice that were sacrificed 7 days after cell injection. On micro-CT, 66 of 74 tumors were detected (83.8%). The smallest tumor detected on micro-CT was 300 microm. There were eight false-negative readings on micro-CT. The sizes of the individual liver metastases measured by micro-CT and on the excised specimen were highly correlated (P < .001). The correlation between the CT scan measurement and the actual measurement was r = 0.8354 (P < .0001). High-resolution micro-CT enhanced with a hepatocyte-selective contrast agent can be a promising tool for detecting liver metastases in a live murine model.

Mycophenolate Mofetil Inhibits Tumor Growth and Angiogenesis In Vitro but Has Variable Antitumor Effects In Vivo, Possibly Related to Bioavailability

Identifying immunosuppressive agents with antitumor effects could help address the problem of posttransplant malignancy. Here we tested for potential inhibitory effects of mycophenolate mofetil (MMF) on tumors in vitro and in vivo. Mouse CT26 colon adenocarcinoma, B16 melanoma, and human TMK1 gastric adenocarcinoma cells were tested for in vitro growth in the presence of MMF. In vitro angiogenesis was tested with a rat aortic-ring assay. Tumor cells were implanted into dorsal skinfold chambers (DSFC) to assess in vivo angiogenesis. Subcutaneous tumor growth was determined in mice receiving MMF. MMF caused a dose-dependent reduction in tumor cell numbers in vitro, starting between 0.1 to 1 microM. Vessel sprouting from aortic rings was markedly blocked by similar concentrations of MMF. In vivo, however, DSFC results showed a marginal reduction in CT26 tumor angiogenesis with MMF doses of 40 or 80 mg/kg/day, although MMF did inhibit TMK1 vascularity. Moreover, 80 mg/kg/day MMF did not reduce subcutaneous CT26 tumor volumes, but did slightly inhibit B16 and TMK1 expansion. Interestingly, the mycophenolic acid (MPA) blood level 2 hr after 80 mg/kg/day MMF bolus dosing was near 14 mg/L, but decreased dramatically thereafter, suggesting a drug availability issue. Indeed, intermittent 2-hr MMF pulses in tumor-cell cultures substantially reduced the antiproliferative effect of MPA. MMF strongly inhibits tumor cell growth and angiogenesis in vitro, but only marginally inhibits tumors in vivo. These contrasting results may relate to drug availability, where intermittent exposure of tumor cells to immunosuppressive doses of MMF substantially reduce its potential antitumor effects.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse beta-glucuronidase (mbetaG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-beta-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional beta-glucuronidase (betaG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (betaG) on CT26 tumors. The fluorescent intensity in betaG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral betaG vector into carcinoma xenografts. Importantly, mbetaG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human betaG also allowed in vivo imaging, demonstrating that human betaG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

Regulation of the mouse organic solute transporter α-β, Ostα-Ostβ, by bile acids

The mechanisms responsible for bile acid regulation of mouse intestinal organic solute transporter alpha-beta (Ostalpha-Ostbeta) expression were investigated. Expression of Ostalpha-Ostbeta mRNA was increased in cecum and proximal colon of cholic acid-fed mice and in chenodeoxycholate-treated mouse CT26 colon adenocarcinoma cells. Sequence analysis revealed potential cis-acting elements for farnesoid X receptor (FXR) and liver receptor homolog-1 (LRH-1) in the mouse Ostalpha and Ostbeta promoters and reporter constructs containing Ostalpha and Ostbeta 5'-flanking sequences were positively regulated by bile acids. Expression of a dominant-negative FXR, reduction of FXR with interfering small RNA (siRNA), or mutation of the potential FXR elements decreased Ostalpha and Ostbeta promoter activity and abolished the induction by chenodeoxycolic acid. Negative regulation of the Ostalpha and Ostbeta promoters by bile acids was mediated through LRH-1 elements. Ostalpha and Ostbeta promoter activities were increased by coexpression of LRH-1 and decreased by coexpression of SHP. Mutation of the potential LRH-1 elements and siRNA-mediated reduction of LRH-1 expression decreased basal promoter activity. As predicted from the promoter analyses, ileal Ostalpha and Ostbeta mRNA expressions were increased in wild-type mice administered the FXR agonist GW4064 and decreased in FXR-null mice. Immunoblotting analysis revealed that Ostalpha and Ostbeta intestinal protein expressions correlated with mRNA expression. The mouse Ostalpha and Ostbeta promoters are unusual in that they contain functional FXR and LRH elements, which mediate, respectively, positive and negative feedback regulation by bile acids. Although the positive regulatory pathway appears to be dominant, this arrangement provides a mechanism to finely titrate Ostalpha-Ostbeta expression to the bile acid flux.

Rapamycin Protects Allografts From Rejection While Simultaneously Attacking Tumors in Immunosuppressed Mice

No AccessJournal of UrologyUrological Survey: Abstracts: Renal Transplantation and Renovascular Hypertension1 Dec 2004Rapamycin Protects Allografts From Rejection While Simultaneously Attacking Tumors in Immunosuppressed Mice David A. GoldfarbM.D. David A. GoldfarbDavid A. Goldfarb More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)61483-8AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "Rapamycin Protects Allografts From Rejection While Simultaneously Attacking Tumors in Immunosuppressed Mice." The Journal of Urology, 172(6 Part 1), p. 2506 References 1 : Ten years of sirolimus therapy for human renal transplantation: the University of Texas at Houston experience. Transplant Proc2003; 35: 25S. Google Scholar © 2004 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 172Issue 6 Part 1December 2004Page: 2506 Advertisement Copyright & Permissions© 2004 by American Urological Association, Inc.MetricsAuthor Information David A. Goldfarb More articles by this author Expand All Advertisement PDF downloadLoading ...

Rapamycin protects allografts from rejection while simultaneously attacking tumors in immunosuppressed mice.

Cancer is an increasingly recognized problem associated with immunosuppression. Recent reports, however, suggest that the immunosuppressive agent rapamycin has anti-cancer properties that could address this problem. Thus far, rapamycin's effects on immunity and cancer have been studied separately. Here we tested the effects of rapamycin, versus cyclosporine A (CsA), on established tumors in mice simultaneously bearing a heart allograft. In one tumor-transplant model, BALB/c mice received subcutaneous syngenic CT26 colon adenocarcinoma cells 7 days before C3H ear-heart transplantation. Rapamycin or CsA treatment was initiated with transplantation. In a second model system, a B16 melanoma was established in C57BL/6 mice that received a primary vascularized C3H heart allograft. In vitro angiogenic effects of rapamycin and CsA were tested in an aortic ring assay. Results show that CT26 tumors grew for 2 weeks before tumor complications occurred. However, rapamycin protected allografts, inhibited tumor growth, and permitted animal survival. In contrast, CsA-treated mice succumbed to advancing tumors, albeit with a functioning allograft. Rapamycin's antitumor effect also functioned in severe combined immunodeficient BALB/c mice. Similar effects of the drugs occurred with B16 melanomas and primary vascularized C3H allografts in C57BL/6 mice. Furthermore, in this model, rapamycin inhibited the tumor growth-enhancing effects of CsA. Moreover, in vitro experiments showed that CsA promotes angiogenesis by a transforming growth factor-beta-related mechanism, and that this effect is abrogated by rapamycin. This study demonstrates that rapamycin simultaneously protects allografts from rejection and attacks tumors in a complex transplant-tumor situation. Notably, CsA protects allografts from rejection, but cancer progression is promoted in transplant recipients.