Giant cell tumors of bone are common but unusual tumors that are comprised of multiple cell types. Most attention has been focused on the giant cells, which resemble osteoclasts morphologically and functionally. This study examines the properties of a cell line derived from mononuclear cells from one of these tumors, since it appears likely that these cells may be able to influence the activities of cells with the osteoclast phenotype. This cell line, C433, has the following characteristics: (1) it represents undifferentiated cells, not recognized by any known antigenic markers for leukocytes; (2) it contains tartrate-resistant acid phosphatase; (3) it responds to the osteotropic factors 1,25 dihydroxyvitamin D3, insulin-like growth factor I and II, but not to parathyroid hormone; (4) it forms sarcomas in nude mice; and (5) it produces an activity that stimulates isolated avian and rat osteoclasts to resorb bone. This cell line may be useful in examining interactions between osteoclasts and accessory cells involved in bone resorption.

It has long been known that complex interactions occur between tumors and normal host immune cells. The human melanoma cell line A375 has been used previously as an indicator cell for tumor cell cytotoxicity mediated by monocytes. During other studies on this tumor cell line, we noted that the conditioned media harvested from A375 cultures induced both the human monocytoid cell line U937 and human blood monocytes to release the cytokine tumor necrosis factor (TNF). We characterized this tumor factor which induced TNF release by monocytic cells. Purification was performed using ammonium sulfate precipitation, ion exchange (DEAE) chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The factor copurified with granulocyte-macrophage colony-stimulating factor (GM-CSF). The purified material caused the release of TNF by U937 cells and stimulated formation of granulocyte-macrophage colonies in methyl cellulose. TNF release by U937 cells in response to A375-conditioned medium was inhibited by neutralizing antibodies to GM-CSF. The TNF-inducing activity in A375-conditioned medium was completely removed by an anti-GM-CSF affinity column. Western blotting using antibodies to GM-CSF confirmed a single Mr27,000 band in A375-conditioned medium. We found that recombinant human GM-CSF stimulated TNF production by the same cells as the tumor-conditioned medium. These data show that A375 human melanoma cells produce GM-CSF, which in turn causes TNF production by cells in the monocyte lineage. The combination of GM-CSF production by the tumor and TNF production by immune cells may influence not only tumor growth but also some of the paraneoplastic syndromes associated with malignancy such as hypercalcemia, cachexia and leukocytosis.

The rat Leydig cell tumor is a well characterized model of the humoral hypercalcemia of malignancy. The studies reported here were provoked by the observation that tumor-bearing rats become extremely cachectic and develop hypertriglyceridemia as they become hypercalcemic. Since the bone resorbing cytokine tumor necrosis factor (TNF)/cachectin is associated with cachexia and hypertriglyceridemia, we examined hypercalcemic tumor-bearing rats for evidence of increased TNF production using a TNF radioimmunoassay. We found that immunoreactive TNF was increased in the plasma of tumor-bearing rats. The increase in plasma TNF was comparable to that previously shown in hypercalcemic nude mice bearing Chinese hamster ovarian cell tumors transfected with the human TNF gene. There was no detectable TNF activity in tumor culture media which suggested that the tumor itself was not the source of excess TNF production. However, we found that tumor cell conditioned media enhanced the production of TNF activity by normal macrophages in vitro, indicating that increased TNF production in vivo may result from a tumor factor(s) which stimulates TNF production by normal immune cells. When TNF was added together with tumor products to organ cultures of fetal rat long bones, osteoclastic bone resorption was potentiated. These data are consistent with the concept that in this model of the humoral hypercalcemia of malignancy, increased TNF production by normal immune cells is increased, has systemic effects as suggested by cachexia and hypertriglyceridemia, and may work in concert with factors produced directly by tumor cells to overwhelm normal calcium homeostasis.