Injection Of Colon Carcinoma Cells Research Articles

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.

Micro-CT Based Experimental Liver Imaging Using a Nanoparticulate Contrast Agent: A Longitudinal Study in Mice

BackgroundMicro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging.MethodologyExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano.Principal FindingsAfter a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks.ConclusionsThe nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.

Evaluation of immunological escape mechanisms in a mouse model of colorectal liver metastases

BackgroundThe local and systemic activation and regulation of the immune system by malignant cells during carcinogenesis is highly complex with involvement of the innate and acquired immune system. Despite the fact that malignant cells do have antigenic properties their immunogenic effects are minor suggesting tumor induced mechanisms to circumvent cancer immunosurveillance. The aim of this study is the analysis of tumor immune escape mechanisms in a colorectal liver metastases mouse model at different points in time during tumor growth.MethodsCT26.WT murine colon carcinoma cells were injected intraportally in Balb/c mice after median laparotomy using a standardized injection technique. Metastatic tumor growth in the liver was examined by standard histological procedures at defined points in time during metastatic growth. Liver tissue with metastases was additionally analyzed for cytokines, T cell markers and Fas/Fas-L expression using immunohistochemistry, immunofluorescence and RT-PCR. Comparisons were performed by analysis of variance or paired and unpaired t test when appropriate.ResultsIntraportal injection of colon carcinoma cells resulted in a gradual and time dependent metastatic growth. T cells of regulatory phenotype (CD4+CD25+Foxp3+) which might play a role in protumoral immune response were found to infiltrate peritumoral tissue increasingly during carcinogenesis. Expression of cytokines IL-10, TGF-β and TNF-α were increased during tumor growth whereas IFN-γ showed a decrease of the expression from day 10 on following an initial increase. Moreover, liver metastases of murine colon carcinoma show an up-regulation of FAS-L on tumor cell surface with a decreased expression of FAS from day 10 on. CD8+ T cells express FAS and show an increased rate of apoptosis at perimetastatic location.ConclusionsThis study describes cellular and macromolecular changes contributing to immunological escape mechanisms during metastatic growth in a colorectal liver metastases mouse model simulating the situation in human cancer.

Pathological animal models in the experimental evaluation of tumour microvasculature with magnetic resonance imaging

The purpose of this study was to evaluate the applications of magnetic resonance imaging (MRI), and in particular, dynamic contrast-enhanced MRI (DCE-MRI), in the assessment of tumour microvasculature by means of animal tumour models evaluated before and after antiangiogenic treatment. Forty-two MRI exams were performed with intravascular contrast media in 21 rats: tumours were induced by subcutaneous injection of colon carcinoma cells in 7 rats and mammary adenocarcinoma cells in 14 rats. Perfusion and permeability parameters of the implanted tumours were evaluated by using two contrast media (B22956/1 and Gd-DTPA37-albumin) to establish response to treatment with two different antiangiogenic drugs (tamoxifen and SU6668). These parameters were correlated with histology to obtain a radiological-histological map of tumour microvasculature. DCE-MRI revealed greater enhancement in the peripheral area than in the central area in all the examined animal models. In the mammary carcinoma experiment, vascular permeability measured by means of B22956/1 in the animals treated with the antiangiogenic drug (0.0043317+/-0.0040418 ml/min(-1)/ml(-1)) was significantly less than in untreated animals (0.0090460+/-0.0043680 ml/min(-1)/ml(-1)), whereas no significant difference was observed with Gd-DTPA-albumin (13.14+/-13.94 ml/min(-1)/ml(-1) in treated animals and 18.07+/-11.92 ml/min(-1)/ml(-1) in untreated animals). In the colon carcinoma experiment, mean permeability and perfusion decreased by 51% (from 5.2+/-1.1 to 2.5+/-0.8 ml/100 ml) and 59% (from 0.00165+/-5.1 to 0.0067+/-4.8 ml/min(-1)/ml(-1) of tissue), respectively, in all animals after antiangiogenic drug administration. DCE-MRI permits a noninvasive evaluation of tumour microcirculation and in particular of its dynamic characteristics and vascularity before and after antiangiogenic treatment.

Early Events of Hepatic Metastasis Formation in Mice: Role of Kupffer and NK-Cells in Natural and Interferon-γ-Stimulated Defense

Surgical manipulation of a tumor may result in increased influx of tumor cells into the systemic and portal circulation and give rise to formation of metastases. In addition, major surgery has been reported to cause profound immunosuppression. In an attempt to increase the host–antitumor immune mechanisms following surgery we have studied the effect of preoperative administration of interferon-γ, related to the antimetastatic effects of Kupffer cells (KC) and natural killer cells (NK-cells) in the early phase of liver metastasis formation. Colon carcinoma cells were injected into the superior mesenteric vein of syngeneic mice and after 17 days metastases were quantified by weight, number, and uptake of [125I]iododeoxyuridine. Unstimulated control mice developed 10.5 surface nodules per liver 17 days following injection of colon carcinoma cells into the superior mesenteric vein of syngeneic mice. This figure was only 2.6 in mice stimulated with a single dose of 1000 IU IFN-γ 4 h prior to inoculation of tumor cells. Administration of GdCl3, which is reported to deplete and block the function of Kupffer cells, 24 h prior to tumor cell inoculation resulted in a 5-fold tumor mass increase relative to control. Injection of anti-asiolo-GM1antiserum, which eliminates the hepatic NK-cells, induced a 10-fold increase in tumor mass. These results indicate an important early antimetastatic function of hepatic NK-cells and KC and that presurgical administration of IFN-γ may be important for eliminating circulating tumor cells and inhibiting development of residual tumors.

Microvascular architecture of hepatic metastases in a mouse model.

Development of effective treatment for hepatic metastases can be initiated by a better understanding of tumour vasculature and blood supply. This study was designed to characterise the microvascular architecture of hepatic metastases and observe the source of contributory blood supply from the host. Metastases were induced in mice by an intrasplenic injection of colon carcinoma cells (106 cells/ml.) Vascularization of tumours was studied over a three week period by scanning electron microscopy of microvascular corrosion casts. Metastatic liver involvement was observed initially within a week post induction, as areas approximately 100 μm in diameter not perfused by the casting resin. On histology these spaces corresponded to tumour cell aggregates. The following weeks highlighted the angiogenesis phase of these tumours as they received a vascular supply from adjacent hepatic sinusoids. Direct sinusoidal supply of metastases was maintained throughout tumour growth. At the tumour periphery most sinusoids were compressed to form a sheath demarcating the tumour from the hepatic vasculature. No direct supply from the hepatic artery or the portal vein was observed. Dilated vessels termed vascular lakes dominated the complex microvascular architecture of the tumours, most tapering as they traversed towards the periphery. Four vascular branching patterns could be identified as true loops, bifurcations and trifurcations, spirals and capillary networks. The most significant observation in this study was the direct sinusoidal supply of metastases, together with the vascular lakes and the peripheral sinusoidal sheaths of the tumour microculature.