Signature in Renal Carcinoma Figure 5. Adipogenic differentation in clear cell renal cell carcinoma. a. Cellular differentiation experiments showing that KIJ-308 and KIJ- 265 ccRCC cells are capable of adipogenic differentiation and become lipid-laden in adipogenic media, as indicated by Oil Red ‘O’ staining. Normal patient-matched cells were unable to differentiate. b. A498 ccRCC cells are also capable of adipogenic differentiation, as indicated by Oil Red ‘O’ staining, unlike normal renal canine MDCK cells. c. Under adipogenic media conditions, ccRCC also produces glycogen, as shown by a PASH stain. doi:10.1371/journal.pone.0010696.g005 a loss of DTFs that controls renal differentiation and is followed by EMT and adipogenic transdifferentiation, which leads to ccRCC (Fig. 8). Discussion Our study links biological pathways in ccRCC using gene array and pathway analyses of patient-matched normal renal and ccRCC tissues. Furthermore, we have validated a number of genes that are altered in ccRCC that were previously undefined. The ccRCC pathway signature is largely characterized by three major biological alterations: 1) Loss of renal function that is reflected by down-regulation of pathways related to normal renal function, such as excretion and electrolyte and ion channels. It is likely linked to a loss of renal epithelial differentiation since the pathway-related renal functions are performed by renal epithelial cells. 2) Down-regulation of various metabolic pathways, which indicates a significant metabolic alteration in ccRCC compared to normal renal cells. The metabolic pathways at the top of our pathway list include butanoate, propanoate, and valine, leucine and isoleucine degradation. These were also down- regulated in another study [29], in which analysis of ccRCC was performed using proteomics and metabolic profile. These pathways are also down-regulated in metastatic colon cancer [30]. Some of these metabolic changes are likely related to VHL gene mutation and/or the Warburg effect [31,32]. 3) The activation of immune pathways, especially antigen presenting and processing pathways, is quite striking. The same finding is seen when ccRCC is compared to papillary RCC (data not shown). In addition, an overexpression of immune genes has been previously reported [33]. This immune gene expression signal is likely coming from tumor infiltrating immune cells; however, aberrant immune gene expression by ccRCC cells is also contributory, shown by our elevated TLR2 and CXCR4 expression data. Increased expression of both of these proteins has been linked to progressive renal disease and tumor formation/angiogenesis [34];[35];[36]. It is common for ccRCC to contain areas of necrosis, especially when the tumor grows faster PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10696