ABSTRACT
Whole-body optical imaging of small animals has emerged as a powerful, user friendly, and high-throughput tool for assaying molecular and cellular processes as they occur in vivo. As with any imaging method, the utility of such technology relies on its ability to provide quantitative, biologically meaningful information about the physiologic or pathologic process of interest. Here we used an animal tumor model to evaluate the extent of correlation between noninvasively measured fluorescence and more traditional measurements of biomass (tumor volume and tumor weight). C57/BL6 mice were injected subcutaneously with murine colon adenocarcinoma cells that were engineered to express GFP. Serial measurements of fluorescence intensities were performed with a macroscopic in vivo fluorescence system. The progressive increases in intensity correlated strongly with growth in tumor volume, as determined by caliper measurements (R2 = 0.99). A more stringent correlation was found between fluorescence intensity and tumor weight (R2 = 0.97) than between volume and weight (R2 = 0.89). In a treatment experiment using tumor necrosis factor-alpha, fluorescence intensity (but not tumor volume) was able to differentiate between treated and control groups on day 1 post-treatment. These results validate the ability of noninvasive fluorescent imaging to quantify the number of viable, fluorescent cells in vivo.
Subject(s)
Adenocarcinoma/pathology , Colonic Neoplasms/pathology , Fluorescent Dyes/analysis , Fluorometry/methods , Genes, Reporter , Luminescent Proteins/analysis , Animals , Cell Count , Female , Genetic Vectors/genetics , Green Fluorescent Proteins , Injections, Subcutaneous , Luminescent Proteins/genetics , Mice , Mice, Inbred C57BL , Neoplasm Proteins/analysis , Neoplasm Proteins/genetics , Neoplasm Transplantation , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Retroviridae/genetics , Transduction, Genetic , Tumor Cells, Cultured/transplantationABSTRACT
Expanding applications of cDNA microarrays such as fine needle aspiration biopsy and laser capture microdissection necessitate the ability to perform arrays with minute starting amounts of RNA. While methods for amplifying RNA have been advocated, the fidelity of array results using amplified material has not been fully validated. Here we demonstrate preserved fidelity in arrays using one or two rounds of mRNA amplification, validated by downstream real-time quantitative PCR. In addition, the quality of the array data was superior to that obtained using total RNA. Based on these results, we recommend routine mRNA amplification for all cDNA microarray-based analysis of gene expression.
Subject(s)
Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/isolation & purification , Animals , Base Sequence , DNA Primers , Mice , Polymerase Chain Reaction/methods , Tumor Cells, CulturedABSTRACT
The rat aortic ring assay has been previously described as a useful ex vivo model for analyzing the biological activity of various inhibitors of angiogenesis. Rat aortic rings are exposed to antiangiogenic agents for a five-day incubation period. Then, the degree of microvessel outgrowth from the rings is analyzed and quantified. In contrast to most in vitro angiogenesis assays, the rat aortic ring model provides a unique microenvironment to evaluate the interaction of various cell types and biological factors for their influence on angiogenesis. Microarray analysis is an accepted method for the evaluation of gene expression profiles and can be used to better understand changes in gene expression that occur when rat aortic rings are exposed to a particular biological agent. Here we describe a method of using microarray technology to evaluate the modulation of gene expression in angiogenesis using the rat aortic ring assay.
Subject(s)
Angiogenesis Inhibitors/analysis , Angiogenesis Inhibitors/genetics , Gene Expression Profiling/methods , Oligonucleotide Array Sequence Analysis/methods , Angiogenesis Inhibitors/administration & dosage , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/physiology , Endothelial Growth Factors/administration & dosage , Gene Expression Regulation , Male , Neovascularization, Physiologic/drug effects , Neovascularization, Physiologic/genetics , Rats , Rats, Sprague-Dawley , Triazoles/administration & dosageABSTRACT
BACKGROUND: Circulating inhibitors of angiogenesis have been suggested to affect the growth of distant micrometastatic disease in patients with cancer. This study was designed to evaluate circulating endostatin levels in colorectal cancer patients with liver metastases. METHODS: Plasma samples from 30 colorectal cancer patients with liver metastases were analyzed for endostatin and vascular endothelial growth factor (VEGF) by using competitive enzyme immunoassays. Samples were compared with plasma from age- and sex-matched healthy controls; values >2 SD above the control mean were considered elevated. RESULTS: Plasma endostatin levels were significantly higher in the 30 cancer patients than controls (P < .0001) and correlated with preoperative VEGF levels (P = .0008). Eighteen patients underwent surgical treatment (liver resection, n = 10; or isolated hepatic perfusion with melphalan, n = 8). Seventeen treated patients were available for follow-up. Eight of 11 patients who progressed had elevated plasma endostatin levels at the time of progression. None of six patients who remained progression free had elevated endostatin levels at last follow-up (P = .02). CONCLUSIONS: Plasma endostatin levels are elevated in colorectal cancer patients with liver metastases and correlate with VEGF levels. Elevated endostatin levels during follow-up are associated with disease progression. Understanding the role of endogenous endostatin in cancer patients may lead to novel strategies to inhibit tumor angiogenesis.
