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. 2014 Nov 25;6(2):1202–1216. doi: 10.18632/oncotarget.2841

Figure 2. ADT sequentially alters the cancer stem-like cell populations and promotes EMT in TRAMP PCa tumors.

Figure 2

(a) The protocol of TRAMP mice experiments was illustrated. TRAMP mice were castrated at 12-week-old. Tumor samples were collected and analyzed at 12wks, 16wks, 20wks, 24wks and 32wks. (b) The growth rates of prostate epithelium were demonstrated by BrdU incorporation in TRAMP prostate before and after castration. The mice were i.p. injected with BrdU (10 ug/g body weight) every 6hr, and killed 24hr later. Paraffin-fixed tissue sections were stained by the BrdU detecting kit (Zymed Laboratories). The castration decreased the BrdU incorporation rates in 16wks-old mouse prostates, while the BrdU incorporation rates began to increase from 20wks and even higher than pre-castration from about 24wks (P<0.05), indicating the happen of CRPC. (c) Considerable metastatic foci in lung kidney and liver of were gradually increased from 24-wk-castration TRAMP mice. (d) From 20wks to 24wks, the castration resistant tumor continually grew bigger in gross (the first and second upper row). H&E staining showed that castration resistant tumors are poorly differentiated from 16wks to 24wks tumor samples (the third row). When double staining CK5 (green, marker for basal epithelial cell) and CK8 (red, marker for luminal epithelial cell) in immunofluorescence examination, the decreasing CK8+ staining following castration were detected, and the increasing CK5+/CK8+(yellow color, overlapped by green and red signals) cells, which indicated the expansion of intermediate cell population, were noticed in 16wks and 20wks tumors (the fourth row). Interestingly, the staining for epithelial markers, both CK5 and CK8, was significantly diminished in 24wks tumors. The CD44 expression significantly increased in 20wks and 24wks of castration resistant tumors (the fifth row). And the expression of E-Cadherin significantly decreased in 20wks and 24wks of castration resistant tumors (the last row). Significance was defined as p<0.05(*). (e) To summarize the sequential events following castration. After the TRAMP mice were castrated in 12wks, the dedifferentiation of epithelium, characterized by expanded CK5+/CK8+ cell population, happened in 16wks, followed by the EMT transition on 20wks and metastasis on 24wks.