Although extreme hypoxic conditions may compromise the health of the cells and lead to cell death, similar con fluencies between the normoxic and hypoxic condition at the conclusion of testing suggest that the 48 hour incuba tion prior to testing was sufficient U0126 order for cell adherence and equilibration. To support this observation, Pilch et al. found that hypoxia did not cause cell death decreased confluency, as dead cells were not observed in supernatant post hypoxia. Furthermore, Inhibitors,Modulators,Libraries the hypoxic oxygen concentration used in the study in vitro is similar to that reported by Hockel and Vaupel, Inhibitors,Modulators,Libraries 2001, in the core of solid tumors in vivo. Also, in addition to the 11 angiogenesis related analytes chosen for testing, a negative control unrelated to angio genesis was also assessed.

A chemotactic cytokine, RANTES, is responsible for recruiting leukocytes and acti vating natural killer cells. This cytokine was not expected to vary in a normoxic versus hypoxic environ ment, and we found similar expression levels of RANTES for both conditions validating our technical Inhibitors,Modulators,Libraries approach. Multiple techniques are available to assess VEGF expres sion. Some laboratories employ Inhibitors,Modulators,Libraries immunohistochemical analysis to determine VEGF receptor levels, usu ally for diagnostic and prognostic purposes. However, this study employed the Beadlyte CytokineProfiler Testing Service for two reasons. First, Inhibitors,Modulators,Libraries this service provides quanti tative analysis of the expression levels of the angiogenesis related factors, including VEGF. Second, testing was per formed on malignant epithelial cell cultures, rather than tissue sections.

Intact tissue sections that contain tumor CB-7598 cells as well as support tissue and vasculature are generally stained using IHC. VEGF receptors on endothelial cells and monocytes fluoresce. The described culture process selects specifically for malignant epithelial cells. Endothelial cells are selected against by culture condi tions, as the media employed do not promote the growth of these cells. monocytes are non adherent, so are rinsed away in routine media changes. Neither of these cell types is present in the described samples so IHC of the VEGF receptors was not possible. VEGF production was of most interest to this study due to its role in the mechanism of action of bevacizumab. The testing conditions were optimized to ensure that VEGF production was measurable, so VEGF results were availa ble for all samples tested. Table 3 includes the summary of all data in the All Samples field, a total of 50 samples. Results for the other ten analytes had detection levels out of range of the standard curve for at least two samples, if not more. As a result, the sample size for most of these angiogenesis related factors was less than 50.