A Manual ROI Selection was used for normal, PIN, cancer, and perineural invasion areas to separately analyze the epithelial compartment. In normal, basal cells, which were identified by position, morphology and according to the CK5-stained serially adjacent tissue section, were selected separately from the luminal cells for analysis. In PIN, cancer, and perineural invasion lesions, the whole epithelial/cancer compartment was selected for analysis. After regions of interest were selected, separate nuclei and cells were identified using manually set parameters and thresholds, followed by classification of nuclei based on manually set thresholds. To identify nuclei and cells, ��membrane�� was chosen for the IHC marker. For nucleus detection, hematoxylin and IHC thresholds were set at 0.055 arbitrary unit and 1.02 a.u., respectively. For detection of membranes and cells, the IHC threshold was set to 0 a.u., and the membrane thickness to 1 pixel. Nuclei classification thresholds were set to classify a nucleus as having either no, low, medium or high immunohistochemical staining. These thresholds were chosen individually based on slides from a control prostate tissue sample used in every staining run, by identifying the darkest and lightest nuclear staining. The lightest staining was used to set the no/low staining threshold, the highest staining was used for the medium/high threshold, and the value in between those two thresholds were used for the low/medium threshold. To determine the potential functionality of cilia that are not lost in prostate cancer cells we measured their lengths. Abnormally short cilia have been shown to correlate with loss of function in signal transduction pathways such as Hedgehog signaling. This suggests that abnormal cilia lengths are a convenient indirect measure of the functional state of the cilia. A total of 5,941 cilia were 
measured on all epithelial and cancer cells, with an average of 730 cilia per tissue type. A median cilia length was calculated per patient. Given that cilia are known to suppress the canonical Wnt signaling pathway, we investigated if the loss of primary cilia in prostate cancer correlates with increased canonical Wnt signaling. Nuclear ��-catenin is a marker of activated canonical Wnt signaling. AbMole Povidone iodine Therefore, we measured nuclear ��-catenin in the tissue types representative of increasingly severe stages of prostate cancer. We predicted that primary cilia loss or shortening would correlate with increased nuclear ��-catenin. The tissue sections adjacent to the sections stained for primary cilia were stained for ��-catenin and scored for percentage of positively stained nuclei and staining intensity. In normal prostate tissue, luminal cells had higher nuclear ��-catenin than basal cells. 57.7% of luminal cells had high nuclear ��-catenin scores. Therefore, we found an inverse correlation between presence of cilia and nuclear ��-catenin in normal prostate.