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Angiogenesis is essential for solid tumor growth. Although successful antiangiogenic therapies have been demonstrated in animal models, a systematic comparison of the efficacy of different antiangiogenic factors has not been described in the hepatic environment. To address this issue, CT26 murine colon carcinoma cells were transfected with retroviral vectors encoding murine endostatin (mEndostatin), human angiostatin (hAngiostatin), murine-soluble vascular endothelial growth factor receptor-2, (msFlk-1), or murine-soluble Tie2 (msTie2). The transfected cells were then subjected to another round of transfection with a luciferase cDNA-encoding retroviral vector. Expression of these putative antiangiogenic proteins inhibited the proliferation of human umbilical vein endothelial cells in vitro but not tumor cells. To examine effects on tumor growth in vivo, modified cells were delivered via intrasplenic injection into BALB/c mice to induce liver metastases. Tumor burden was measured weekly by bioluminescence. Growth of hepatic metastases in vivo was significantly reduced in mice that were administered cells expressing msTie2 (76% reduction compared with control cells 21 days after intrasplenic inoculation; P < 0.05). Similar results were observed with cells that expressed msFlk-1 and hAngiostatin. However, expression of mEndostatin had no significant effect on the growth of liver metastases compared with control animals. These findings indicate that multiple antiangiogenic pathways are necessary for the growth of hepatic metastases, and each of these pathways is a potential clinically relevant antiangiogenic target for the treatment of this disease.

Hepatic metastasis is a primary cause for failure of locoregional therapy in colorectal cancer. Increased expression of osteopontin (OPN), a ligand for alpha(v)beta3 integrin and CD44 receptors, is associated with metastasis in several types of cancer. However, the mechanism by which OPN mediates metastasis in colorectal cancer remains unknown. We hypothesized that OPN mediates invasion of colon cancer cells through basement membrane and migration through extracellular matrix (ECM). In this study, we used CT26 murine colon adenocarcinoma cells syngeneic to BALB/c mice to generate cell lines (pS-OPN) in which OPN expression was suppressed through small interfering RNA (siRNA) plasmids. CT26 wild-type cells (WT) and CT26 cells stably expressing murine-mismatch siRNA (pS-MM) served as controls. Western blotting quantified OPN protein levels and our most downregulated clone, pS-OPN-A4, demonstrated a mean 3.0-fold decrease in OPN protein expression versus WT. In vitro cell motility and invasiveness were decreased in pS-OPN-A4 by 3.6-fold (P = 0.004 versus WT) and 4.1-fold (P = 0.01 versus WT), but proliferation was similar amongst cell lines. We demonstrated that OPN suppression significantly correlates with MMP-2 downregulation. In vivo hepatic metastasis was assessed by quantifying liver weights and surface tumor nodules in 33 BALB/c mice (11/group) subjected to intrasplenic injection of tumor cells. pS-OPN-A4 resulted in a 50.4% decrease in mean liver weight compared with WT (3.79 +/- 1.49 g versus 1.88 +/- 1.34 g, P = 0.009). Only 18% of pS-OPN-A4 livers had >20 metastatic surface nodules compared with 89% for WT and 75% for pS-MM-V6. This study demonstrates that RNA interference stably reduces CT26 tumor expression of OPN and significantly attenuates CT26 colon cancer metastasis by diminishing tumor cell motility and invasiveness.

Pathologic angiogenesis induced by a tumor is essential for its survival. The promise of tumor inhibition by targeting angiogenesis over the past several years has translated into numerous ongoing clinical trials. Recently, in a phase III trial involving patients with metastatic colorectal cancer, Bevacizumab (Genentech, Inc, San Francisco, CA), a recombinant humanized monoclonal antibody against vascular endothelial growth factor used in conjunction with standard chemotherapy was shown to increase survival, progression-free survival, response rate, and duration of response compared to chemotherapy alone. Thus far, duration of the increased response remains less than 6 months. The majority of deaths in patients with colorectal cancer are related to hepatic metastases. It is hoped that novel approaches directed at the complex interactions between tumor and microenvironment in the angiogenic process will strengthen the therapeutic armamentarium against hepatic malignancies.

Background: We used bioluminescence imaging (BLI) to validate serial assessment of neoplastic growth within the murine liver. We hypothesized that, in mice bearing luciferase-expressing liver metastases, bioluminescence would reflect neoplastic burden estimated by liver weight., Materials and Methods: Murine colon carcinoma cells were infected with a retroviral vector encoding luciferase. Bioluminescence was measured in vitro, and in vivo following intrasplenic tumor cell inoculation into Balb/C mice. At varying time intervals, mice were imaged and immediately killed to determine correlation of in vivo and ex vivo BL with liver weight. To examine sensitivity in vivo, we performed direct intrahepatic inoculation., Results: In vitro, photon emission correlated with increasing cell numbers. In the metastatic model, a statistically significant increase was depicted in both liver weight and bioluminescence 20 days after tumor inoculation compared with earlier time points (P < 0.0001). With progressively increasing tumor burden, however, a poor correlation between in vivo BL and liver weight was observed. When livers with very large tumors were excluded, R(2) = 0.90. In contrast, correlation between ex vivo BL and liver weight remained high throughout the study period (R(2) = 0.94)., Conclusions: When imaging tumors at end-stages of disease, in vivo BL underestimates actual tumor burden. If context-specific limitations are recognized, however, BLI is a sensitive approach to temporal monitoring of tumor progression in preclinical models.