Chondrosarcoma is the second most common primary sarcoma of bone and to date unresectable chondrosarcomas have a poor outcome [3
]. Grade III and dedifferentiated chondrosarcomas are extremely aggressive in nature and there is an urgent need for model systems facilitating research in order to develop novel therapeutic strategies. Growing chondrosarcoma cells in culture, however, is a challenge and well growing chondrosarcoma cell lines are sparse. We present here the establishment and characterization of three new chondrosarcoma cell lines originating from grade III and dedifferentiated chondrosarcoma.
Recently chondrosarcoma has been found to harbor IDH1 and IDH2 mutations (5;15) and we published that the mutation is retained in a subset of chondrosarcoma cell lines [15
]. In glioma IDH mutations seem to be the earliest event in gliomagenesis even before TP53 mutations occur [20
]. In conventional chondrosarcoma we observe a similar phenomenon, where IDH mutations are present already in a high percentage of low-grade tumors and TP53 mutations are observed to increase with grade (4;5;15). Cell lines created from IDH mutant gliomas have been reported to eliminate their IDH mutation under standard culture conditions [7
]. Recently, however, a glioma cell line carrying an endogenous IDH1 R132H mutation was published, but this cell line showed a slow growth rate in culture [21
]. We here present three chondrosarcoma cell lines, one carrying an IDH1 R132C mutation, one carrying an IDH2 R172W mutation, and one wild type for IDH mutations with stable karyotypes and steady growth patterns. These cell lines show numerical changes and additional mutations. We speculate that in IDH mutant chondrosarcoma the acquisition of additional mutations as we have shown here have facilitated their growth in culture.
The inactivation of tumor suppressor genes is a well-known phenomenon in cancer and p16 mutations have been reported in 20-41% of human chondrosarcomas [22
]. Interestingly, all studies observed loss of p16 to be correlated with increasing histological grade in conventional chondrosarcoma. Recently, we showed inactivation of p16 in 30/38 (79%) dedifferentiated chondrosarcoma cases [26
]. We previously published three chondrosarcoma cell lines to be negative for p16 using western blot [18
] and upon overexpression of p16 using lentiviral vectors the metabolic activity and cell viability of these cell lines was decreased, indicating loss of p16 to play a role in the proliferative capacity of chondrosarcoma cells. Introduction of p16 in the endogenously TP53 mutant HT-1080 fibrosarcoma cell line, which was recently reported to carry an IDH1 R132C mutation [5
], also led to cell cycle arrest and growth inhibition [27
]. We report here three new chondrosarcoma cell lines lacking p16 expression based on a homozygous deletion of the CDKN2A locus as shown by aCGH analysis, and confirmed loss of p16 expression using immunohistochemistry. Moreover, aCGH analysis showed a copy number loss around the 17p13.1 locus in L835, whereas a copy number gain was observed in L2975 and L3252. However, mutation analysis for TP53 showed no activating mutations in exons 5–8, and immunohistochemistry showed no p53 overexpression. Together, our data suggest that while IDH mutations are important as early events in a subset of chondrosarcomas, additional inactivation of p16 may be crucial for acquiring a more aggressive phenotype.
The literature presents us with 5 conventional chondrosarcoma cell lines that have been well characterized using pathological, immunohistochemical, and molecular genetic methods [8
], and we here present L835 as an additional cell line. We previously published L835 to be able to form 3D pellets [33
] and we now show it to be highly stable in culture. L835 cell line showed a slower growth rate compared to the dedifferentiated chondrosarcoma cell lines. In all cases complex genome alterations were observed. Dedifferentiated chondrosarcoma is comprised of two separate components, a high grade anaplastic component and a low to intermediate grade cartilaginous component [2
]. The histogenesis has been under debate but evidence points to a single precursor cell with early separation of the two components as a small number of genetic changes is identical in both components, with additional genetic alterations in the anaplastic component [26
]. Indeed, 3 out of 3 dedifferentiated chondrosarcomas with IDH1 mutations carried the mutation in both components [26
]. Moreover, 79% of the anaplastic and 82% of the cartilaginous components show loss of p16 expression [26
]. L2975 and L3252 were both derived from the recurrence of a dedifferentiated chondrosarcoma; both cell lines exhibited a higher growth rate in vitro, than the L835 cell line, but the cells in culture expressed chondrogenic markers. L2975 proved to be the most aggressive cell line both in culture and in our in vitro migration assay, which may explain why this was the only cell line to be successfully xenografted. We show here the use of L2975 dedifferentiated chondrosarcoma cells with an IDH2 R172W mutation in mouse models, which can be an important asset in the research for new treatment strategies.