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. 2012 May 15;109(22):8699–8704. doi: 10.1073/pnas.1017909109

Fig. 2.

Fig. 2.

Stable down-regulation of HSP27 in angiogenic cancer cells induces long-term dormancy in vivo, and overexpression of HSP27 in nonangiogenic cells induces expansive tumor growth in vivo. Five pools of the HSP27-expressing angiogenic breast cancer cell line transduced with different shRNA sequences against HSP27—HSP27KD-1, KD-2 and KD-3—were generated. (A) Their HSP27 protein expression was confirmed by Western blot and compared with the NT control. (B–D) Down-regulation of HSP27 protein expression in HSP27KD-3 cells was quantified by immunohistochemistry and compared with that in NT control cells using in vitro cell pellets (B). NT control cells are shown in C; HSP27KD-3 cells, in D. (E) HSP27 tagged with GFP was overexpressed (HSP27OE+GFP) in the parental nonangiogenic MDA-MB-436-NA (with intrinsically low HSP27 (Fig. 1A) cells and confirmed by Western blot analysis. (F) In vivo s.c. xenograft growth curves of HSP27KD-3, HSP27KD-1, and the NT control (estimated as mean ± SE tumor volume). (G) In vivo s.c. xenograft growth curves of HSP27OE+GFP, the control vector, and the parental unaltered nonangiogenic cells (estimated as mean ± SE tumor volume). (H and I) The NT control tumors showed vivid neo-angiogenesis within and around the growing tumors. (J and K) In contrast, unaffected normal-appearing s.c. vessels surround a small HSP27KD-3 tumor (J), whereas early angiogenic activity can be observed in a late (day 70) HSP27KD-3 tumor (K). (M–P) HSP27 protein staining was consistently high in the NT control tumors (M and N) but was low or absent in microscopic dormant tumors formed by the HSP27KD-3 cells (O and P). (L and Q) A mouse inoculated with HSP27KD-3 cells spontaneously initiated tumor growth (L), and was confirmed by immunohistochemistry to have regained HSP27 protein expression (Q). (R) The mean HSP27 staining index was 2.6-fold higher for the NT control tumors than for tumors formed by the HSP27KD-3 cells in vivo. (Original magnifications: 400× in C, D, M, O, and P; 200× in N and Q.) The Student t test was used to assess the statistical significance of differences (*).