Figure 3

Nestin KD is associated with stromal invasion in vivo. (a) Tumors derived from A2058 nestin KD (NesKD, solid line) and vector control (Vec, dash line) cells had comparable growth rate in NOD-SCID mice at 3 × 10⁶ cells/injection/mouse (N=5). (b) Representative immunohistochemical staining of nestin and MMP3 in A2058 xenograft tumors at 28 days post grafting. Nestin KD tumors (bottom) had significantly enhanced MMP3 staining in the tumors confirmed with efficient nestin KD when compared with vector control tumors (top) where nestin was abundantly expressed (nestin KD, black bar; vector control, white bar; both N=5, P<0.001). Scale bars: 50 μm. (c) Representative H&E and immunohistochemistry staining of vector control (top) and nestin KD (bottom) tumors at 7 days post xenografting. In contrast to well-circumscribed subcutaneous nodules of vector control tumors (N=4), nestin KD tumors (N=4) had significant stromal invasion in multiple areas. Immunoreactivity of SOX2 stained both A2058 vector control and nestin KD tumor cells. Efficient KD was confirmed in the nestin KD tumors. In contrast to dense cytoplasmic nestin staining in vector control tumors, a submembranous pattern of nestin distribution was observed in localized, spindle cells scattered in nestin KD tumors (inset). Nestin KD tumors had enhanced MMP3 staining. CD31⁺ vessels and F4/80⁺ dendritic cells appeared at peritumoral stroma of vector control tumors, whereas these cells diffusely infiltrated nestin KD tumors. Scale bars: 50 μm. (d) Examples of patterns of nestin expression that may be detected in tumorigenic (nodular, top) and infiltrative (desmoplastic, bottom) regions of certain human primary melanomas. Nodular regions by H&E staining are well-circumscribed (broken line: border between tumor (right) and adjacent stroma (left)) and composed of coalescent, rounded cells that express abundant cytoplasmic nestin by immunohistochemical staining. Infiltrative regions, in comparison, show diffuse permeation of stroma by elongated spindle-shaped cells that express quantitatively less nestin immunoreactivity that is localized to sub-plasma membrane regions. Scale bars: 50 μm.