Sema4D promotes larger metastatic lesions, with fewer osteoblasts and more osteoclasts.

<p><b>A.</b> Hematoxylin and eosin (H&E) stained sections of control (C) tumors or those with silenced Sema4D (Sema4D shRNA) metastatic to the femur (top row; T, tumor; BM, bone marrow; original magnification 10x). Control tumors on average were larger than those from cells where Sema4D was silenced. Sema4D silencing was confirmed by immunohistochemistry in tumor tissues (second row; original magnification 40x). Osteoclasts attached to bone in the tumor microenvironment were identified by TRAP immunohistochemistry (TRAP, bottom row, positive cells indicated by arrows; original magnification 40x). <b>B.</b> Histomorphometric analysis for total tumor area in formalin fixed paraffin embedded specimens confirmed that control tumors were larger (tumor area in mm², Y-axis). <b>C.</b> Tumors made up of cells with silenced Sema4D exhibited greater preservation of bone volume (bone volume expressed as a percentage of total tissue volume, Y-axis). <b>D.</b> The number of osteoclasts present in lesional bone, as determined by TRAP positivity, was greater in control tumors (number of osteoclasts per mm² of bone surface, Y-axis). (E) The number of osteoblasts in lesional bone was higher in tumors where Sema4D was silenced (number of osteoblasts per mm² of bone surface, Y-axis; *, p ≤ 0.05).