Molecular profiling of giant cell tumor of bone and the osteoclastic localization of ligand for receptor activator of nuclear factor kappaB

Abstract

Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor kappaB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease.

Figure 1

Gene expression heatmaps for leiomyosarcoma, synovial sarcoma, malignant fibrous histiocytoma, liposarcoma, and giant cell tumor using both unsupervised (A) and supervised (B) analysis.

Figure 2

Cell fractionation studies. A: Light microscopy showing typical cultures of primary disaggregated giant cell tumor (GCT; left panel), osteoclast-enriched cultures (middle panel), and stromal cell cultures (right panel). Arrows indicate giant cells. B: RT-PCR for expression of regulatory genes, osteoclast and osteoblast markers in primary giant cell tumor cultures, and osteoblast- and osteoclast-enriched fractions of GCT. This experiment was performed three times with similar results using independent samples to those used in Table 4.

Figure 3

Tabular representation of genes most highly expressed in osteoclast- and stromal cell-enriched fractions of giant cell tumor of bone. Genes highlighted in red are genes reported to be expressed in osteoclasts; in blue, expressed in stromal cells; in green, genes implicated in signaling of osteoclast formation or function (see text for details).

Figure 4

Flow phenotyping of GCT with FITC and PerCP (controls) and RANKL and CD45. There is an increase in the cell population staining positive for both RANKL and CD45, and there are no cells staining RANKL positive and CD45 negative compared with the control samples with FITC and PerCP.

Figure 5

Confirmation of expression profiling data by immunohistochemistry of paraffin-embedded fixed giant cell tumor of bone. a: Conventional H&E section; b: CD4 staining for osteoclasts; c: CD45, which can be also used as an osteoclast cell surface marker; and d: CD68, which is a hemopoietic cell or osteoclast marker. Bar = 50 μm in a, and 25 μm in b to d.

Figure 6

RANKL antibody staining of giant cell tumor of bone. Antibody control GCT section (a) and RANKL antibody staining in parallel sections (b) showing specific staining adjacent to osteoblasts clustering at a site of newly forming bone. Antibody control (c) and RANKL staining (d) of parallel sections from a tumor showing specific staining of osteoclasts (low magnification). e and f: Higher magnification of RANKL-stained osteoclasts, showing granular cytoplasmic staining. Scale bar = 25 μm.