Colon Cancer Metastasis Model Research Articles

A review of the anti-tumor effect of the combined administration of a cyclooxygenase-2 selective inhibitor and a non-specific immunostimulant protein-bound polysaccharide on an advanced colon cancer model using colon cancer cell lines

The anti-tumor effect of a cyclooxygenase (COX)-2 selective inhibitor or a non-specific immunostimulant (PSK) alone, as well as the anti-tumor effect of their combined administration were examined on a hepatic metastasis model of colon cancer using a colon 26 cell line (CT26) and its highly metastatic variant. Anti-tumor effects were assessed by the number of hepatic metastases. Serum MMP-9, TGF-β and IL-6 were also measured. In a preliminary experiment, cells (5×10(5)) of a mouse colon cancer 26 cell line (CT26) and its highly metastatic variant were implanted below the splenic capsule in BALB/c and CDF1 mice. The number of hepatic metastatic CT26 cell lesions in the CDF1 mice of the non-spleen-removed group at 2 weeks was found to be optimum for the experiments. Although no significant difference was found, etodolac treatment showed the highest inhibitory effect on the number of hepatic metastases at a concentration of 30 mg/kg. In contrast, intraperitoneal administration of 50 mg/kg PSK showed an inhibitory effect on hepatic metastases, but a significant difference was not observed. PSK (p=0.002) or the combined use of etodolac and PSK (p=0.001) exhibited a significant inhibition of the number of hepatic metastases. In addition, MMP-9 was significantly inhibited by the single use of etodolac or PSK, and was inhibited with an additive effect by the combined use of etodolac and PSK. IL-6 and TGF-β were significantly inhibited following the combined use of etodolac and PSK. In conclusion, etodolac did not exhibit any significant hepatic metastasis inhibitory effect, whereas it significantly reduced the MMP-9 level. PSK reduced both the number of hepatic metastases and MMP-9. Combined use of etodolac and PSK did not show any additive effect in the inhibition of the number of hepatic metastases, whereas it inhibited MMP-9, TGF-β and IL-6, suggesting the benefit of a combined effect.

Investigation of the Effective Dose of Agonistic 4-1BB Monoclonal Antibody in a Murine Colon Cancer Metastasis Model

Purpose: The aim of this study was to find the dose of agonistic 4-1BB monoclonal antibody (㎃b) that results in optimal T cell activation. Methods: Cancer was induced in mice by an intrahepatic parenchymal injection of 1×10? cells of CT26 cells. Cancer-carrying mice (n=84) were divided into seven groups and treated with either rat IgG or agonistic 4-1BB monoclonal antibody (㎃b) (5㎍, 10㎍, 20㎍, 100㎍, 200㎍, or 300㎍). All treatments were administered intraperitoneally on days 7, 9, and 11. Mice from each group were sacrificed on days 14, 28, and 42. Harvested livers were weighed and the numbers of T cells in the splenocytes were analyzed with a FACS Vantage flow cytometer. Results: Liver weights increased when 5㎍ of agonistic 4-1BB ㎃b was administered, but showed no additional weight increase for doses greater than 10㎍. The absolute numbers of CD4＋ and CD8＋ T cells increased in groups treated with low doses of agonistic 4-1BB ㎃b (5㎍, 10㎍, or 20㎍), but did not increase in the groups treated with high doses of ㎃b (100㎍, 200㎍, or 300㎍). The levels of CD4/annexin V and CD8/annexin V increased as the dose increased, and the absolute cell numbers of CD4/annexin V were greater than those of CD8/annexin V. Conclusion: Liver weight, including the cancer mass, failed to increase at agonistic 4-1BB ㎃b doses greater than 10㎍. A high dose (≥100㎍) of agonistic 4-1BB ㎃b resulted in lower counts of absolute T cells. This study suggests that a low dose (20㎍) of agonistic 4-1BB ㎃b can be used for optimal T cell activation in combination with other anti-cancer treatments.

Experimentally Derived Metastasis Gene Expression Profile Predicts Recurrence and Death in Patients With Colon Cancer

Staging inadequately predicts metastatic risk in patients with colon cancer. We used a gene expression profile derived from invasive, murine colon cancer cells that were highly metastatic in an immunocompetent mouse model to identify patients with colon cancer at risk of recurrence. This phase 1, exploratory biomarker study used 55 patients with colorectal cancer from Vanderbilt Medical Center (VMC) as the training dataset and 177 patients from the Moffitt Cancer Center as the independent dataset. The metastasis-associated gene expression profile developed from the mouse model was refined with comparative functional genomics in the VMC gene expression profiles to identify a 34-gene classifier associated with high risk of metastasis and death from colon cancer. A metastasis score derived from the biologically based classifier was tested in the Moffitt dataset. A high score was significantly associated with increased risk of metastasis and death from colon cancer across all pathologic stages and specifically in stage II and stage III patients. The metastasis score was shown to independently predict risk of cancer recurrence and death in univariate and multivariate models. For example, among stage III patients, a high score translated to increased relative risk of cancer recurrence (hazard ratio, 4.7; 95% confidence interval, 1.566-14.05). Furthermore, the metastasis score identified patients with stage III disease whose 5-year recurrence-free survival was >88% and for whom adjuvant chemotherapy did not increase survival time. A gene expression profile identified from an experimental model of colon cancer metastasis predicted cancer recurrence and death, independently of conventional measures, in patients with colon cancer.

Antibody-mediated FOXP3 protein therapy induces apoptosis in cancer cells in vitro and inhibits metastasis in vivo

In addition to its immune suppressive function in T-regulatory cells, the nuclear transcription factor, FOXP3, has been identified as a tumor suppressor. To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated FOXP3 protein therapy of cancer, the Fv-FOXP3 fusion protein produced in Pichia pastoris was tested on breast, ovarian, and colon cancer cells in vitro, and with colon cancer cells in vivo in a mouse model of colon cancer metastasis to liver. Treatment with Fv-FOXP3 resulted in dose-dependent cell death of cancer cells in vitro. Apoptosis was established as a mechanism of cell death by demonstrating increased production of the p17 activated fragment of caspase-3 by cancer cells in response to Fv-FOXP3 and inhibition of cell killing by the caspase inhibitor, Z-VAD-FMK. Fv-FOXP3 treatment resulted in clinically significant reduction in tumor burden in a syngeneic model of colon cancer metastasis to liver in Balb/c mice. These results represent the first demonstration of effective full-length FOXP3 protein therapy and emphasize the clinical potential of mAb 3E10 as an intracellular and intranuclear delivery vehicle of FOXP3 for prevention and treatment of cancer metastasis.

Inhibition of the Tumor Necrosis Factor-α Pathway Is Radioprotective for the Lung

Radiation-induced lung toxicity limits the delivery of high-dose radiation to thoracic tumors. Here, we investigated the potential of inhibiting the tumor necrosis factor-alpha (TNF-alpha) pathway as a novel radioprotection strategy. Mouse lungs were irradiated with various doses and assessed at varying times for TNF-alpha production. Lung toxicity was measured by apoptosis and pulmonary function testing. TNF receptor 1 (TNFR1) inhibition, achieved by genetic knockout or antisense oligonucleotide (ASO) silencing, was tested for selective lung protection in a mouse lung metastasis model of colon cancer. Lung radiation induced local production of TNF-alpha by macrophages in BALB/c mice 3 to 24 hours after radiation (15 Gy). A similar maximal induction was found 1 week after the start of radiation when 15 Gy was divided into five daily fractions. Cell apoptosis in the lung, measured by terminal deoxyribonucleotide transferase-mediated nick-end labeling staining (mostly epithelial cells) and Western blot for caspase-3, was induced by radiation in a dose- and time-dependent manner. Specific ASO inhibited lung TNFR1 expression and reduced radiation-induced apoptosis. Radiation decreased lung function in BALB/c and C57BL mice 4 to 8 weeks after completion of fractionated radiation (40 Gy). Inhibition of TNFR1 by genetic deficiency (C57BL mice) or therapeutic silencing with ASO (BALB/c mice) tended to preserve lung function without compromising lung tumor sensitivity to radiation. Radiation-induced lung TNF-alpha production correlates with early cell apoptosis and latent lung function damage. Inhibition of lung TNFR1 is selectively radioprotective for the lung without compromising tumor response. These findings support the development of a novel radioprotection strategy using inhibition of the TNF-alpha pathway.

Hepatic Ischemia-Reperfusion Increases Vascular Endothelial Growth Factor and Cancer Growth in Rats

In liver surgery, the hepatic pedicle often is clamped to reduce blood loss, and later unclamped, representing hepatic ischemia and reperfusion (I/R) with induction of hypoxia. Vascular endothelial growth factor (VEGF) expression reportedly is induced by hypoxia; further, some cancer cells express the VEGF receptor (flt-1, flk-1/KDR). We hypothesized that I/R-induced VEGF expression could enhance growth of microscopic tumor via VEGF receptors on tumor cells, thus promoting liver metastasis in a rat model. Time-dependent VEGF expression in liver and plasma was determined by enzyme-linked immunosorbent assay in rats subjected to 60 min of 70% hepatic I/R (I/R group). Other rats given an intrasplenic inoculation of a rat colon adenocarcinoma cell line (RCH-H4) were divided 3 days later into three groups: group A, untreated; group B, sham operation; group C, 70% I/R for 60 min. Liver metastasis was evaluated on day 14. Expression of flt-1 and flk-1/KDR was examined in RCN-H4 cells, and effects of exogenous VEGF on RCN-H4 cell proliferation were determined by MTT assays. Hepatic VEGF expression increased significantly in the I/R group compared to the control group. Liver metastasis was more extensive in group C than in groups A and B. RCN-H4 cells expressed flt-1 and flk-1/KDR, while exogenous VEGF increased RCN-H4 cell proliferation. Hepatic ischemia reperfusion leads to induction of VEGF and this is associated with increased tumor burden in an animal model of colon cancer metastasis.

Liver metastasis models of colon cancer for evaluation of drug efficacy using NOD/Shi-scid IL2Rγnull (NOG) mice

To examine the drug efficacy of a novel farnesyltransferase inhibitor (FTI), CH4512600, in vivo, we developed a reliable liver metastasis model of human colon cancer using NOD/Shi-scid IL2Rgamma(null) (NOG) mice. Eleven human colon cancer cell lines were examined for their ability to form diverse metastatic foci in the livers of NOG mice. When inoculated with 10(4) COLO320DM, HCT 116, HT-29, WiDr, LoVo and LS174T cells, liver metastasis was evident in 100% (6/6), 100% (6/6), 88.9% (8/9), 87.5% (7/8), 83.3% (5/6) and 50.0% (3/6) of the NOG mice, respectively. CaCo2, COLO201, LS123, SW48 and SW1417 showed no metastasis when seeded at 10(4) cells even in NOG mice. The mRNA expression levels and genetic mutations of N, H and K-RAS genes, which directly affect the levels of cellular RAS protein that would be molecular target for FTI, were also examined in these six metastatic human colon cancer cell lines for molecular biological and genotypic characteristics. Only three cell lines had a point mutation in the RAS oncogene. LS174T cell line had a point mutation of the K-RAS gene at codon 12 (gly12 --> asp; G12D), and HCT 116 and LoVo cell lines had a point mutation of the K-RAS gene at codon 13 (gly13 --> asp; G13D). Relative gene expression levels of N, H and K-RAS genes in the HCT 116 cell line were 2.6-5.0-fold lower than that of LS174T and LoVo cell lines. We selected HCT 116 cell line from our liver metastasis model for evaluation of FTI CH4512600 efficacy in vivo. Using the NOG mouse liver metastasis model, we demonstrated the effectiveness of FTI CH4512600 to suppress tumor growth in vivo and to prolong mouse survival significantly from 36.9+/-2.9 to 50.3+/-9.4 days.

Therapeutic Efficacy of Replication-Competent Retrovirus Vector–Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.

The effects of surgical trauma on colorectal liver metastasis

The majority of cancer mortality is associated with metastatic disease. Therefore, determining a strategy to decrease the time to metastasis after surgery may increase patient survival and quality of life. Indeed, many studies have shown no significant difference between open and laparoscopic resection of colon cancer [3, 4]. However, the long-term incidence of metastasis after laparoscopic versus open colon resection has not been reported. Previous studies demonstrated that increased trauma leads to increased growth of tumors in mice [1, 2]. We therefore investigated whether the extent of operative trauma has an impact on tumor metastasis in a murine colon cancer metastasis model.

Antibody-Mediated p53 Protein Therapy Prevents Liver MetastasisIn vivo

To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.

ANTIBODY-MEDIATED P53 PROTEIN THERAPY PREVENTS LIVER METASTASIS IN VIVO.

Purpose To test the clinical efficacy of antibody-mediated protein transduction of p53 into cancer cells in vivo in a mouse model of colon cancer metastasis to liver. Methods An Fv fragment of mAb 3E10 was previously shown to penetrate into cancer cells and localize in the nucleus. A His6-tagged Fv-p53 fusion protein was produced in Pichia pastoris X-33 yeast cells transfected with pPICZA-Fv-p53. Fv-p53 was isolated on NiNTA-agarose and assayed for killing CT26.CL25 colon cancer cells in vitro and CT26.CL25 cells injected into the portal vein of syngeneic Balb/c mice. Results Experiments in vitro demonstrated killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by portal vein injections of 100 nM Fv-p53 10 minutes and 1 week after injection of cancer cells. Mice were sacrificed 2 weeks after the second injection of Fv-p53 and assigned metastasis scores based on the following criteria: 0 - no liver metastasis; 1 - minimal infiltration (less than 1 cm2 area of metastasis); 2 - mild to moderate infiltration (1-2 cm2 area of metastasis); 3 - severe infiltration (greater than 2 cm2 area of metastasis but the liver was not entirely infiltrated); 4 - complete infiltration. As shown in Table 1, Fv-p53 prevented liver metastasis. Conclusion Fv-p53 treatment had a profound impact on liver metastasis. These results demonstrate the clinical potential of an antibody Fv fragment as a protein transduction domain for intracellular/intranuclear delivery of p53 for prevention of cancer metastasis in vivo.

A retrogen plasmid-based vaccine generates high titer antibody responses against the autologous cancer antigen survivin and demonstrates anti-tumor efficacy

We describe the use of retrogen plasmid-based vaccine technology to break tolerance and to generate a robust, dose-dependent antibody response against the self cancer antigen, survivin. We further demonstrate that this phenomenon is due to the incorporation of the survivin antigen into the retrogen system rather than to some peculiarity unique to survivin. In contrast to other genetic immunization methods designed to produce antibody responses, the retrogen system results in a broad range of antibody isotypes, indicative of both a Th-1 and a Th-2 CD4 + response. Additional evidence of a Th-1 response is demonstrated by tumor growth inhibition in a mouse model of colon cancer metastasis. We speculate that this cost-effective technology could one day bolster or even supplant the use of monoclonal antibodies in the targeting of cell surface cancer antigens.

In situ localization of gelatinolytic activity in the extracellular matrix of metastases of colon cancer in rat liver using quenched fluorogenic DQ-gelatin.

Matrix metalloproteinases (MMPs) such as gelatinases are believed to play an important role in invasion and metastasis of cancer. In this study we investigated the possible role of MMP-2 and MMP-9 in an experimental model of colon cancer metastasis in rat liver. We demonstrated with gelatin zymography that the tumors contained MMP-2 and MMP-9, but only MMP-2 was present in the active form. Immunolocalization of MMP-2 showed that the protein was localized at basement membranes of colon cancer cells and in intratumor stroma, associated with extracellular matrix (ECM) components. However, zymography and immunohistochemistry (IHC) do not provide information on the localization of MMP activity. Therefore, we developed an in situ zymography technique using the quenched fluorogenic substrate DQ-gelatin in unfixed cryostat sections. The application of DQ-gelatin in combination with a gelled medium allows precise localization of gelatinolytic activity. Fluorescence due to gelatinolytic activity was found in the ECM of tumors and was localized similarly to both MMP-2 protein and collagen type IV, its natural substrate. The localization of MMP-2 activity and collagen type IV at similar sites suggests a role of MMP-2 in remodeling of ECM of stroma in colon cancer metastases in rat liver.

Promotion of colon cancer metastases in rat liver by fish oil diet is not due to reduced stroma formation.

Recently, it was demonstrated that dietary omega-3 polyunsaturated fatty acids (PUFAs) induce 10-fold more metastases in number and 1000-fold in volume in an animal model of colon cancer metastasis in rat liver. It was observed that tumors of rats on a fish oil diet lacked peritumoral stroma unlike tumors in livers of rats on a low fat diet or a diet containing omega-6 PUFAs. In the present study, only one-third of the tumors in livers of rats on omega-3 PUFA diet contained peritumoral stroma, whereas peritumoral stroma was present in 87% of the tumors in livers of rats on low fat diet. To explain these findings, we tested the hypothesis that fish oil exerts a direct inhibiting effect on the formation of extracellular matrix in tumor stroma as a consequence of blocking transformation of fat storing cells into myofibroblasts. It was found with immunohistochemical analysis of desmin as marker for fat storing cells and alpha-smooth muscle actin as marker for myofibroblasts that numbers of myofibroblasts were higher in tumors containing intratumoral stroma only than in tumors containing both peritumoral and intratumoral stroma. As most of the tumors in fish oil-treated rats contained intratumoral stroma only, this suggests that transformation of fat storing cells into myofibroblasts was highest in tumor stroma of fish oil-treated rats. Therefore, it is unlikely that the lack of stroma around tumors in fish oil-treated rats is due to inhibition of transformation of fat storing cells into myofibroblasts, but lack of peritumoral stroma is rather a consequence of rapid development of tumors in livers of fish oil-treated rats.