Cinomas of both mice. The transcription of several key CB 5083 Homatropine (methylbromide) site inflammatory factors, including IL1b, IL6 and TGFb1, was induced 
in the carcinomas of both mice. Nonetheless, extensive heterogeneity was observed between the two mice in other genes connected with inflammation (Table S3). In the carcinoma from Mouse ID7, there was strong induction at the level of transcription of numerous cytokines, chemokines and related genes. Mouse ID12 had induction of fewer genes, but these included major pro-inflammatory genes, such as IL1a and members of the TNF and TNFR families. A related hallmark, which also involves cytokines, is evasion of immune destruction of the tumor. IL10 is overexpressed in Mouse ID7, whereas VEGFA is overexpressed in Mouse ID12. Both of these can lead to immune suppression. In addition to their roles in tumor-promoting inflammation, the pro-inflammatory genes also activate invasion and metastasis. Many additional genes that are characterized as promoting invasion and metastasis were transcriptionally induced in the carcinomas from both or either of the mice. FN1, BMP1, COL1A2, COL5A2, MMP3 and MMP9 were induced in tumorsFigure 5. Heatmap of Mouse ID7 and Mouse ID12. A. Heatmap showing all transcripts that showed 4-fold change between carcinoma and normal skin in Mouse ID7 and their corresponding fold-change in Mouse ID12. B. Heatmap showing all transcripts that showed 4-fold change between carcinoma and normal skin in Mouse ID12 and their corresponding fold-change in Mouse ID12. C. Heatmap showing the fold-change of the transcripts that were significant in the average analysis, in Mouse ID7 and Mouse ID12. doi:10.1371/journal.pone.0057748.gHeterogeneous Gene Expression in SCC Developmentfrom both mice. Presumably, the fibronectins and collagens were induced in the tumor stroma. Thus, the tumors from both mice showed evidence of shared and mouse-specific transcriptional induction of genes promoting inflammation, invasiveness and metastasis. The TGFb pathway appears to be transcriptionally enhanced in both Mouse ID7 and ID12, which would be expected to lead to angiogenesis, another hallmark of cancer. It is notable that Mouse ID7 had increased PDGFR, whereas Mouse ID12 had an increase in VEGFR. These parallel pathways would both encourage angiogenesis. Overexpression of TGFb could also lead to EMT and changes in cell-cell contacts, which could cause another hallmark, namely, evasion of growth suppression [22]. An important hallmark of cancer is reprogramming energy metabolism. Many metabolic genes were induced or repressed in both mice, and in each mouse separately (Table S4). As an example of how the cancers in Mouse ID7 and ID12 could evolve due to both shared and specific changes in metabolism, we looked at enzymes involved in glycolysis. Hexokinase 3 (HK3) was induced in both mice. Enolase (ENO1 and ENO3) and phosphoglycerate mutase 2 (PGAM2) were induced only in Mouse ID7, whereas phosphofructokinase (PFKL) was induced only in Mouse ID12. There was a decrease in transcription of several members of the Cytochrome P450 family in both mice, which was even more striking in Mouse ID7. This decrease would be expected to result in a decrease 1313429 in aerobic respiration (Table S5). In the article by Quigley et al. [23], the authors analyzed copy number changes in the tumors and showed evidence of genome instability, another hallmark of cancer. In our analysis of gene transcription, we found an increase in transcription of BUB1B and BUB3.Cinomas of both mice. The transcription of several key inflammatory factors, including IL1b, IL6 and TGFb1, was induced in the carcinomas of both mice. Nonetheless, extensive heterogeneity was observed between the two mice in other genes connected with inflammation (Table S3). In the carcinoma from Mouse ID7, there was strong induction at the level of transcription of numerous cytokines, chemokines and related genes. Mouse ID12 had induction of fewer genes, but these included major pro-inflammatory genes, such as IL1a and members of the TNF and TNFR families. A related hallmark, which also involves cytokines, is evasion of immune destruction of the tumor. IL10 is overexpressed in Mouse ID7, whereas VEGFA is overexpressed in Mouse ID12. Both of these can 
lead to immune suppression. In addition to their roles in tumor-promoting inflammation, the pro-inflammatory genes also activate invasion and metastasis. Many additional genes that are characterized as promoting invasion and metastasis were transcriptionally induced in the carcinomas from both or either of the mice. FN1, BMP1, COL1A2, COL5A2, MMP3 and MMP9 were induced in tumorsFigure 5. Heatmap of Mouse ID7 and Mouse ID12. A. Heatmap showing all transcripts that showed 4-fold change between carcinoma and normal skin in Mouse ID7 and their corresponding fold-change in Mouse ID12. B. Heatmap showing all transcripts that showed 4-fold change between carcinoma and normal skin in Mouse ID12 and their corresponding fold-change in Mouse ID12. C. Heatmap showing the fold-change of the transcripts that were significant in the average analysis, in Mouse ID7 and Mouse ID12. doi:10.1371/journal.pone.0057748.gHeterogeneous Gene Expression in SCC Developmentfrom both mice. Presumably, the fibronectins and collagens were induced in the tumor stroma. Thus, the tumors from both mice showed evidence of shared and mouse-specific transcriptional induction of genes promoting inflammation, invasiveness and metastasis. The TGFb pathway appears to be transcriptionally enhanced in both Mouse ID7 and ID12, which would be expected to lead to angiogenesis, another hallmark of cancer. It is notable that Mouse ID7 had increased PDGFR, whereas Mouse ID12 had an increase in VEGFR. These parallel pathways would both encourage angiogenesis. Overexpression of TGFb could also lead to EMT and changes in cell-cell contacts, which could cause another hallmark, namely, evasion of growth suppression [22]. An important hallmark of cancer is reprogramming energy metabolism. Many metabolic genes were induced or repressed in both mice, and in each mouse separately (Table S4). As an example of how the cancers in Mouse ID7 and ID12 could evolve due to both shared and specific changes in metabolism, we looked at enzymes involved in glycolysis. Hexokinase 3 (HK3) was induced in both mice. Enolase (ENO1 and ENO3) and phosphoglycerate mutase 2 (PGAM2) were induced only in Mouse ID7, whereas phosphofructokinase (PFKL) was induced only in Mouse ID12. There was a decrease in transcription of several members of the Cytochrome P450 family in both mice, which was even more striking in Mouse ID7. This decrease would be expected to result in a decrease 1313429 in aerobic respiration (Table S5). In the article by Quigley et al. [23], the authors analyzed copy number changes in the tumors and showed evidence of genome instability, another hallmark of cancer. In our analysis of gene transcription, we found an increase in transcription of BUB1B and BUB3.
