Table 1.
RANKL overexpressing LNCaP cells induced high incidences of bone and soft tissue metastases.
Figure 1.
RANKL overexpression induced epithelial to mesencymal transition.
RANKL-transfected LNCaP cells induced EMT in histomorphology (A) and gene expression in mRNA (B) and protein (C). Data represent one of 3 RANKL-stably transfected and 2 neo-stably transfected LNCaP cell clones.
Figure 2.
RANKL overexpression activated c-Met signaling components detected by single quantum dot labeling, SQDL.
Differential QD labeling of proteins was performed in LNCaP-neo (A) and LNCaP-RANKL (B) cells using DAPI nuclear staining as reference. For each analysis, QD labeled protein expression is presented in pseudocolor, with the overlaid image of DAPI and QD signals (Merged). ×400.
Figure 3.
Quantification of differential gene expression subsequent to RANKL overexpression in LNCaP cells.
Cell-based average intensity counts from 1,000 each of neo- and RANKL-transfected LNCaP clones were quantified using inForm software. Statistically significant changes in protein expression were observed (P<0.05).
Figure 4.
Castration induced c-Met activated and EMT associated proteins.
The c-Met activated and EMT associated proteins were detected in the CRPC LTL-313 xenograft model by conventional IHC (A) and SQDL (B). Castration increased a panel of genes in LTL-313 xenograft prostate cancer and decreased AR expression compared to xenografts obtained from intact hosts by both IHC and SQDL. ×400.
Figure 5.
Multiplexed quantum dot labeling (MQDL) simultaneously detected c-Met activated and EMT genes in a CRPC LTL-313 xenograft model.
Single tumor tissue sections from intact and castrated hosts were subjected to sequential MQDL. Enhanced expression of c-Met activated genes was detected in tumor tissues from castrated hosts. (A) A stack of multiple images (Cube), nuclear staining (DAPI), and individual gene expression (in pseudocolors) are shown. Gene expression signals were superimposed with DAPI nuclear signals to yield a composite image for analysis. ×400. (B) Significant differences in gene expression were found by quantification analysis of 1,000 cells in each tumor specimen using inForm software (P<0.05).
Figure 6.
MQDL assesses differential gene expression in clinical primary prostate cancer specimens.
C-Met-activated and EMT protein expressions in 5,000 cells of each representative prostate cancer tissues of Gleason scores 7 (3+4) and 10 (5+5) were compared. Expression intensities were quantified with the inForm software and statistically compared (P<0.05).
Figure 7.
MQDL detects EMT biomarkers in CRPC LTL-313 prostate tumors harvested from castrated hosts and from clinical prostate cancer.
(A) The CRPC LTL-313 model showed androgen deprivation induced more abundant expression of EMT markers in a cluster of cells within the tumor region, as revealed by QD labeling of EpCAM, N-Cad, and RANKL. (B) Similar EMT biomarkers were detected in a clinical primary prostate cancer specimen (Gleason score 6; 3+3) with documented bone metastasis. M denotes cells that completed EMT and E/M indicates cells undergoing partial EMT. ×400.
Figure 8.
MQDL detects EMT biomarkers in clinical bone tissue specimens.
A representative specimen of human prostate cancer bone metastasis co-expressed high levels of epithelial EpCAM, and mesenchymal RANKL and vimentin proteins. ×400.