BMC Genomics 2007, 8:117 http://www.biomedcentral.com/1471-2164/8/117 on this pathway involving HeLa cells as a model may draw inconclusive results. Care must be taken to identify which cell line and growth condition would yield the most appropriate model. The results here can not be quantita- tively represented by cluster analyses such as SVD. There- fore, the pathway analysis was of great use when determining which cell line should be used to model cer- vical cancer or normal cervix at a pathway-specific level. Highest and lowest pathway correlations to cervical tissue In order to identify pathways that require more attention when selecting a cell line model, the correlations of all cell lines to cervical cancer for a specific pathway were aver- aged to generate a single correlation that represented all cell lines and growth conditions (Figure 5A). Biologically relevant pathways that had the highest and lowest correla- tion to tumor tissue were plotted. Examples include the JNK cascade, which was modeled well by all cell lines, whereas the "RNA Processing" pathway and several cell cycle pathways were the most poorly represented by most cell lines, compared against cervical cancer. These exam- ples are plotted in detail in Additional file 4. A pathway analysis of normal cervix versus cervical cancer is shown in Figure 5B. Pathways that share similar gene expression between normal and cancer include the regula- tion of cytokines, the JNK cascade, and a few metabolic pathways; the JNK cascade is modeled well in most cell lines studied as mentioned above (Figure 5A). Pathways with a low correlation between normal and tumor cervical tissue include mitosis, G-protein signaling, and regulation of development. The "mitosis" pathway is modeled poorly by most cell lines when compared to cervical can- cer (Figure 5A), and has a low correlation between normal and tumor tissue (Figure 5B). Further, the correlation to normal tissue by most cell lines is -0.3, indicating that mitosis is poorly modeled by cell lines in general, but model tumor tissue much better than normal tissue, as expected. Most of these cell lines have been outside the in vivo envi- ronment for decades and as a consequence have adapted to their new environment, resulting in changes of gene expression. We expected to see many important pathways with low correlations to tissue, and this was the case. We observed poor correlation to the cell cycle, RNA process- ing, and cell signaling pathways in cell lines compared to cervical cancer. This was due perhaps to both accumulated mutations and the fact that cultured cell lines may have different modes of cell-to-cell communication. These pathways, with low correlation to tissue (typically below 0.3), are extensively studied in cancer research [35-37]. There were pathways that retained a high correlation to tissue across many cell lines, including the JNK cascade, positive regulation of cell proliferation, and other tran- scriptional regulation pathways, which indicates they are still relevant to study by researchers using current cell lines. Media effect on correlation HeLa and SiHa cell lines were cultured in a different type of media to assess whether small changes to the environ- ment could have a large impact on the correlation to tis- sue. The effect of different culture media on the correlation to cervical tissue was assessed at the pathway level using both SiHa and HeLa cell lines. An example of three biologically relevant pathways that changed in cor- relation between the two media is shown in Figure 6, though the changes were not restricted to metabolic path- ways. The correlation to cervical cancer increased by changing the medium to DMEM; the increased correlation to cervical tissue was expected after the transition from a minimal medium (MEM) to a richer medium (DMEM). We hypothesized that the increase in correlation was due to the addition of glucose in DMEM, which is absent from MEM. After ranking the genes based on their impact on the correlation, the third highest gene that lowered the correlation of HeLa cultured in MEM was PDK4. Pyruvate dehydrogenase kinase has been found to increase its expression during starvation [38]. In our experiments, PDK4 was found to have a 4-fold decrease in expression in cervical cancer, a 2-fold increase in expression in HeLa cul- tured with DMEM, and a 20-fold increase in expression in HeLa cultured with MEM. As the function of PDK4 is the regulation of glucose metabolism, this indicates HeLa cul- tured in MEM experience starvation-like conditions, whereas HeLa cultured in DMEM have an environment similar to in vivo cancer conditions. This example of PDK4, along with the three metabolic pathways shown in Figure 6, provided examples of how an increase in the cor- relation to normal cervix was achieved by simply chang- ing the medium. Simple changes to the culturing environment can therefore have a dramatic affect on the relevance of some cell lines as models of cervical cancer. Organotypic effect on correlation Organotypic culture physically resembles the structure of cervical epithelium. HeLa and SiHa cell lines increased the correlation to tissue when cells were cultured in organo- typic cultures versus monolayer. The organotypic control, which consists of the same culture environment minus the fibroblasts, also increased the correlation over the cell lines, indicating a simple collagen bed was sufficient to increase the global modeling of cell lines to cervical can- cer. In the case of the organotypic and organotypic con- trol, the two environments did not differ significantly in their correlations (Figure 3). Further evidence that organotypic cultures were better models over monolayer culture was provided by calculat- Page 7 of 13 (page number not for citation purposes)