Fig 1.
Flow chart and summary of cross-species analyses.
Step-wise depiction of sample processing and cross-species analyses that were performed.
Fig 2.
Canine and human iUC samples cluster together.
A list of genes that are commonly annotated and significantly expressed (between normal and iUC, p<0.05, FC2) in dogs and humans, was generated. Hierarchical clustering was performed on these genes (n = 436) using Euclidean distance metrix. Figure illustrates that canine and human normal controls cluster together and these cluster separately from canine and human iUC samples. The iUC samples from dogs and humans clustered together. The color codes are: (1) red bar denoting canine normal bladder, (2) brown bar denoting normal human bladder, (3) blue bar denoting canine iUC samples, and (4) grey bar denoting human iUC samples.
Fig 3.
Canine iUC samples cluster as two distinct groups.
Hierarchical clustering illustrates the differential expression of genes (p<0.05, FC2) between canine iUC samples vs. normal canine bladder. Furthermore, the canine iUC samples clustered in two distinct groups.
Fig 4.
Principal Component Analyses (PCA) plot of normal canine bladder and iUC samples.
A PCA plot was generated using normalized data. The PCA plot shows clear separation between normal samples and canine iUC samples. In addition, the PCA plot also demonstrates clear segregation of the canine iUC samples into two groups i.e., group I and group II.
Fig 5.
Analyses of canine iUC samples reveal enrichment for basal and luminal subtypes and also for genes in P53 pathways.
A list of genes representing basal and luminal subtypes and those involved in P53 pathways was manually generated from published human iUC dataset (Cancer Genome Atlas Research Network. 2014a; Choi et al 2014a and Damrauer et al 2014). A second list was generated, using these genes as a reference, which were also significantly expressed in canine iUC samples and hierarchical clustering was performed using Euclidean distance metrix. Heat maps indicate genes enriched for basal (A) and luminal (B) subtypes of breast cancer in the canine iUC samples and also indicate enriched genes in the P53 pathways (C). Red and blue bars above the heat map indicate the clean separation of samples into the previously observed groups. There was no clear segregation of basal and luminal genes.
Fig 6.
Immunohistochemical detection of EGFR expression in normal and iUC canine tissues.
Photomicrographs of canine normal bladder (A and B) and canine iUC samples (C and D) demonstrating immunoreactivity to EGFR. Paired negative controls were used for each specimen (B and D). Please note membrane immunostaining of tumor cells (C) and normal urothelium (A).