Establishment from a human chondrosarcoma of a new immortal cell line with high tumorigenicity in vivo, which is able to form proteoglycan-rich cartilage-like nodules and to respond to insulin in vitro.

The human chondrosarcoma cell line (HCS-2/8) established by our group expresses cartilage phenotypes such as production of cartilage-type proteoglycans and collagen type II, but its tumorigenicity is low. To develop an in vitro experimental system for studies of human chondrosarcomas, a new immortal cell line of human chondrosarcoma, named HCS-2/A, was established from the same tumor. HCS-2/A cells proliferated with a doubling time of 3 1/2 days in a medium containing 20% fetal bovine serum (FBS). This growth rate was comparable to that of HCS-2/8 cells. However, HCS-2/A cells proliferated more rapidly than HCS-2/8 cells in the presence of 2-10% FBS. Like HCS-2/8 cells, HCS-2/A cells had a polygonal shape in sparse cultures and became spherical as they reached confluence, after which they formed nodules composed of multilayered cells and a large quantity of extracellular matrix showing strong metachromasia. The nodules formed by HCS-2/A cells were thicker and also larger in diameter than those formed by HCS-2/8 cells. Electron microscopically, the cells in the nodules resembled chondrocytes in vivo, but each cell had an irregular-shaped nucleus which is a characteristics of tumor cells. The cells actively synthesized "cartilage-specific" large proteoglycans and their level of proteoglycan synthesis was comparable to that of HCS-2/8 cells. Insulin, which stimulates proteoglycan and DNA syntheses in cultured chondrocytes, markedly increased proteoglycan synthesis in HCS-2/A cells. On the other hand, the hormone only slightly increased proteoglycan synthesis in HCS-2/8 cells. Insulin also stimulated DNA synthesis in cultured HCS-2/A cells, but not in HCS-2/8 cells. Immunostaining revealed that HCS-2/A cells produced type-II collagen but not type-I collagen. However, the level of collagen synthesis of HCS-2/A cells was lower than that of HCS-2/8 cells. Inoculation of HCS-2/A cells into athymic mice resulted in the formation of chondrosarcomas that grew faster than those arising from HCS-2/8 cells.