Murine Lung Metastasis Model Research Articles

Guanylyl cyclase C prevents colon carcinoma cell metastasis

Background/Aims Of patients with colorectal cancer, >50% die from metastatic progression of their disease. A novel paradigm is emerging in which guanylyl cyclase C (GC-C), the intestinal receptor for the diarrheagenic heat-stable enterotoxin (ST), serves as a tumor suppressor. While the importance of GC-C in regulating tumor cell proliferation has been defined, its role in mechanisms underlying metastasis remains unexplored. Methods The role of ST and cGMP, the intracellular mediator of GC-C, in regulating key in vitro indices of metastasis, including cell spreading, actin polymerization, detachment from collagen substrates, and matrix metalloproteinase-9 (MMP-9) activity was examined. The suitability of MMP-9 as a target for GC-C signaling in human colon tumors was confirmed, ex vivo, by RT-PCR employing laser capture microdissection and electron microscopy. Further, the impact of GC-C activation on the metastatic potential of human colon cancer cells was explored in vivo employing a murine model of lung metastasis. Results ST and cGMP negatively regulated all indices of metastasis examined, including tumor cell MMP-9 activity. Importantly, GC-C activation by ST remarkably reduced “seeding” of colon cancer cell metastases in mouse lung, in part, through an α3β1 integrin-dependent mechanism. Conclusions GC-C and its downstream effectors regulate the malignant progression of human colorectal tumors by inhibiting key processes underlying metastasis. Clinical Pharmacology & Therapeutics (2005) 77, P3–P3; doi: 10.1016/j.clpt.2004.11.016

Increased Gene Expression by Cationic Liposomes (TFL-3) in Lung Metastases Following Intravenous Injection

We recently showed that size, not surface charge, is a major determinant of the in vitro lipofection efficiency of pDNA/TFL-3 complex (lipoplex), even in the presence of serum. In this study, the effect of lipoplex size as a result of interaction with serum proteins on in vitro lipofection and the relationship of this with in vivo lipofection was examined in a murine lung metastasis model. As previously described, the pDNA to lipid ratio (P/L ratio) affected both the size and zeta potential of the lipoplex. In vitro studies also indicated that transgene expression in B16BL6 cells was largely dependent on the size of the lipoplex, both in the absence or presence (50% (v/v)) of serum. An in vivo lipofection experiment showed that predominant gene expression in lungs occurred only in tumor-bearing mice, not in normal mice. Based on the in vitro study, this tumor-related gene expression was not related to lipoplex size in the presence of serum (50% (v/v)), suggesting that the size alteration, as the result of interactions with serum proteins in the blood stream may not play an important role in the case of systemic injections. In addition, the efficient gene expression in tumor-bearing lung was not related to the progression of lung metastases. The area-specific gene expression in tumor-bearing lungs, which was largely dependent on the P/L ratio of the lipoplexes, was observed by fluorescent microscopy. Although the underlying mechanism for the area-specific transgene expression is not clear, it may be related to the interaction of lipoplexes with tumor cells, vascular endothelial cells under angiogenesis and normal cells in the lungs. The possibility that TFL-3 is a useful utility to the targeted delivery of pDNA to lungs and tumor-related lipofection is demonstrated. This result suggests that area-specific gene expression in lung metastases may be achieved by controlling the physicochemical properties of the lipoplex, i.e. the P/L ratio.

Cloning and characterization of angiocidin, a tumor cell binding protein for thrombospondin‐1

Thrombospondin-1 (TSP-1) is a matrix protein that has been implicated in mechanisms of tumor progression. Our laboratory previously showed that the CSVTCG (cys-ser-val-thr-cys-gly) sequence of TSP-1 functioned as a tumor cell adhesion domain and CSVTCG peptides as well as an anti-peptide antibody possessed anti-metastatic activity in a murine model of lung metastasis. In a subsequent study, a putative TSP-1 binding protein from lung carcinoma was isolated by CSVTCG-peptide affinity chromatography. In this study, we present the full-length cDNA of this binding protein isolated from a prostate cancer cell (PC3-NI) cDNA library. The purified recombinant protein, termed angiocidin, is a potent inhibitor of tumor growth of Lewis Lung carcinoma in vivo and tumor invasion and angiogenesis in vitro. In addition, the recombinant protein inhibits tumor and endothelial cell proliferation and induces apoptosis. The activity of angiocidin both in vivo and in vitro is partially dependent on its TSP-1 binding activity, since an angiocidin deletion mutant missing a high affinity-binding site for TSP-1 failed to inhibit tumor growth in vivo and was less active in its anti-tumor and anti-angiogenic activities in vitro. These results suggest that the anti-tumor activity of TSP-1 reported in many studies may be mediated in part by binding proteins such as angiocidin. Such proteins may function as tumor-suppressor proteins, which limit the growth of tumors by inhibiting angiogenesis and cell matrix interaction.

Therapeutic effect of intravenous delivery of lipoplexes containing the interferon-beta gene and poly I: poly C in a murine lung metastasis model.

We have evaluated and compared the efficacy of systemic administration of lipoplex formulations containing plasmids encoding IFN-beta or IFN-gamma, and a synthetic double-strand RNA poly I:poly C (pI:pC), a type I IFN inducer, in a lung metastasis model in which colon carcinoma CT-26 cells were inoculated intravenously into immunocompatible mice. Injection of lipoplexes containing plasmid DNA, regardless of IFN gene insertion, stimulated a transient increase in the serum concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and IFN-gamma, while injection of lipoplexes containing pI:pC led to a low level of TNF-alpha and undetectable IFN-gamma production. Furthermore, injection of these lipoplexes containing plasmids resulted in the production of a mixture of type I and type II IFNs, partly derived from the inserted IFN genes, in lung tissue cultures. In tumor-prophylactic experiments, intravenous injection of lipoplexes containing plasmid, regardless of IFN gene insertion, showed a significant reduction in lung metastatic nodules probably due to proinflammatory cytokines such as TNF-alpha and IFN-gamma nonspecifically induced by the CpG motifs in the plasmid and the type I IFNs produced. On the other hand, the antimetastatic effect of pI:pC-lipoplex seemed to be due mainly to IFN-beta induced by pI:pC. In established lung metastasis experiments, a single intravenous administration of lipoplexes containing IFN-beta gene or pI:pC, but not other lipoplexes, showed a significant therapeutic effect on the tumor metastasis: reduction in tumor nodules and prolongation of survival time of tumor-burden mice. The therapeutic effects were specifically impaired by anti-IFN-beta antibody treatment, indicating that IFN-beta produced by the lipoplexes played an important role in the suppression of established metastatic lung tumors. Thus, the local IFN-beta in the lung delivered by intravenous administration of lipoplex containing IFN-beta gene or pI:pC may be a convenient and useful method of inhibiting established metastatic lung tumors.

Inhibition of lung metastases of murine renal cell carcinoma by the combination of radiation and interferon-alpha-producing tumor cell vaccine.

We have previously demonstrated in a murine lung metastasis model that local sublethal radiation of tumors can synergistically enhance their sensitivity to immunotherapy with either systemic high-dose interleukin-2 (IL-2) or vaccination with autologous tumor cells expressing IL-2, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF). Host antitumor activity was mediated in large part by natural killer cells, which can be activated by IFN-alpha. In the present study, we used this lung metastasis model to investigate the efficacy of combined therapy with local tumor radiation and vaccination with IFN-alpha-secreting tumor cells (Renca/IFN-alpha). The in vitro and in vivo growth rates of Renca/IFN-alpha cells were significantly reduced relative to normal controls. Subcutaneous vaccination with Renca/IFN-alpha or selective X-irradiation of the left lung (300 rad) reduced the number of lung tumors by 40% and 27%, respectively. The combination of lung irradiation plus vaccination reduced the number of lung metastases by 60%, and the net tumor volume by 95%. The reductions in tumor volume in both irradiated and non-irradiated lungs were comparable. These results indicate that host antitumor response to subcutaneous vaccination with Renca/IFN-alpha was systemic, and was significantly enhanced by radiation of tumor-bearing lungs. A regimen based on enhancement of IFN-alpha immunotherapy by local tumor radiation may be useful in the treatment of metastatic renal cell carcinoma.