Figure 1.
DAB-HRP tissue staining for BTF3, HINT1, NDRG1 and ODC1 in representative malignant (A,C,E,G) and non-malignant (B,D,F,H) prostate tissue cores
(of over 500 tissue cores stained for each antibody) from human prostate arrays. These are representatives of over 450-500 tissue core images analyzed for each biomarker antibody using the automated image analysis protocol (see Materials and Methods – Image particle analysis). Each core was imaged using a Leica upright microscope (10x) and saved as a jpg file. Each image was used to calculate protein expression (DAB label, brown, largely in acinar cells). Colored RGB images (A-H) were converted to corresponding grayscale images (I-P) for the quantification of the DAB signal using Analyze Particle protocol in ImageJ software to obtain Total Area stained, in pixel. Protein expression for BTF3, HINT1, NDRG1 and ODC1 was increased in malignant v benign cores (p<0.0001).
Figure 2.
Quantitation of DAB signal in prostate tissue array.
An unbiased, automated protocol was used to calculate Total Area stained (in pixel) standardized to amount of tissue in each core (see Methods). TA is converted to Area Fraction (total area divided by the total pixels in the image). Each bin is data for an individual, malignant (red) or non-malignant tissue (green) core. There is a significant increase in the AUC of protein expression in malignant v non-malignant prostate tissue (p<0.0001).
Figure 3.
ROC of putative prostate cancer biomarkers.
ROC curve demonstrates the discriminating performance of the protein expression in the differentiation between malignant and non-malignant tissue cores using the area fraction (probit) data for BTF3, HINT1, NDRG1 and ODC1. The operating characteristic values are given in Table S1. The solid line represents the ROC area of 0.5.
Figure 4.
Multi-labeled immunofluorescence for BTF3, HINT1 and NDRG1.
Co-expression of three proteins BTF3 (FITC-green), HINT1 (Cy3- blue) and NDRG1 (Cy5-red) in non-malignant (A) and malignant prostate tissue cores (B); images are representative tissue cores from the tissue array with over 200 samples. The three antibodies were incubated simultaneously and labeled with three Cy5 –red different fluorophores using Bond automated staining system. Whole tissue cores were imaged using a Leica confocal system and 4x4 tiling at exactly the same settings for comparison. Images were zoomed 3x using digital zoom in ImageJ. Scale bar =100µm.
Figure 5.
Differential pattern for BTF3, HINT1 and NDRG1 co-expression in malignant, biochemical relapse and non-relapse sample.
Co-expression of three proteins BTF3 (FITC-green), HINT1 (Cy3- blue) and NDRG1 (Cy5 –red) in malignant prostate tissue cores (A to D). Composite micrographs of immunofluorescently stained prostate tissue cores from patients with biochemical relapse (defined as PSA ≥ 0.2 ng/ml within 2 years of radical prostatectomy). Tissue cores from 4 patients (matched pairs for Gleason score and PSA), non-relapsed (A) and biochemical relapse (B) representative images used for quantitative co-localization analysis (see methods) are shown. Imaging was performed using a Leica confocal microscope; A and B are low magnification; C and D higher magnification; E is an example of the expression of the three markers in non-malignant tissue also at higher magnification. The expression of BTF3 (green), for example is evident in non-relapsed samples whereas it not very evident in cores from relapsed patients. Imaging was performed using an Olympus confocal microscope (40x tiled (A and B) or 40x with digital zoom 3.5-4 (C, D and E). High magnification images (C,D and E) were deconvolved using Huygens Profession software. 16bit tif files for each fluorophore (FITC, Cy3 and Cy5) were imported into ImageJ and pseudo colored (FITC, green, Cy3, blue and Cy5, red). Z-projections for up to 27 images are shown for each tissue core. Scale bar =100µm for A and B and 20µm for C and D. Quantitative colocalization analysis of images (e.g. Figure 5C and D) showed that most significant change in the pattern of expression, in non-relapsed vs relapsed samples, was due to the change in co-expression of BTF3 (FITC, green) and HINT1 (Cy3, blue) with Pearson coefficients for colocalization for non-relapse vs relapse = 0.73 ± 0.02 vs 0.60 ± 0.07 (means ± SD, n=4 , significance of difference p<0.02).