Confocal images of bone sections (20 μm-thick) from sham mice (A) or mice sacrificed at days 35 (B) or 70 (C,D) post-injection. GFP+ cancer cell (green)-bearing bone sections were incubated with an antibody against 200 kD neurofilament (NF200, a marker of myelinated nerve fibers; white). Note that in the sham mice (A), NF200+ nerve fibers that innervate the healthy marrow space appear as single nerve fibers with a highly linear morphology. In contrast, as GFP+ prostate tumor cells proliferate and form tumor colonies at day 35 post-cell injection (B), the NF200+ sensory nerve fibers undergo marked sprouting characterized by a highly branched morphology and increased density as compared to sham mice. With time, older parent colonies show a dramatic loss of GFP+ cancer cells, as well as a decrease in the density of NF200+ nerve fibers (C), whereas immediately adjacent new daughter cancer cell colonies show robust expression of GFP as well as sprouting and formation of neuroma-like structures (arrow) by NF200 nerve fibers (D). Images were acquired at the metaphyseal region of the bone marrow and were projected from 40 optical sections at 0.5 μm intervals.