As expected, the metastatic foci consisted entirely of the high-grade dedifferentiated portion of the tumor, with no evidence of the low-grade chondrosarcoma component. The cellular compositon of the metastases correlates with the results of the invasion assay, where metastatic cell lines showed significantly higher percentage of invading cells in comparison with both NM cell lines.
Despite the fact that the metastatic cell lines were generated from the dedifferentiated cells, they exhibited expression of a number of genes that are considered to be chondrocyte markers [24
] (Supplementary Table 5a). As expected, however, the majority of the chondrocyte markers were downregulated in the metastatic cell lines in comparison with the two NM cell lines, consistent with the status of dedifferentiation. Additionally, we found expression of genes such as Collagen, type I, alpha 1 (COL1A1), Exostoses (multiple) 1 (EST1), Exostoses (multiple) 2 (EXT2), Vimentin (VIM), and Osteoprotegerin (TNFRSF11B), which are known to be involved in development of conventional chondrosarcomas [34
] (Supplementary Table 5a).
Despite the limitations arising from the fact that no samples were available for analysis of the primary tumor, and that only restricted amounts of bulk tissue could be obtained from the metastatic lesions, our findings have shed some light to the molecular mechanisms underlying metastasis in dedifferentiated chondrosarcoma.
For the first time we documented a high heterogeneity at the gene expression level among individual lung metastases of a dedifferentiated chondrosarcoma patient. The heterogeneity is even greater for the “metastasis associated” and the “multifunctional” genes (Supplementary Table 4A–C), thus suggesting that it is indeed of “functional nature”.
There is a possibility that the high degree of heterogeneity observed among the metastases might be due to the manipulations that they were subjected to after resection from the patient. We believe this not to be the case because the five metastases were treated identically and, importantly, their cells were cultured under the same conditions and were only allowed to go through four doublings. Moreover, all five cultures were initiated at the same time, following the same procedures and utilizing the same reagents. Accordingly, it is noteworthy that of the 59 genes encoding ribosomal proteins found to be expressed in each of the five metastases, there is a group of 26 genes that were consistently downregulated relative to the virtual NM-cell line (Supplemental Table 2). Such consistent downregulation of the ribosomal proteins in the metastases serves to validate both the experimental and the statistical methods utilized in this study.
There is also the possibility that the heterogeneity in gene expression observed among the metastases might be due to occurrence of different aberrations in the karyotypes of their cells. However, we did not find any significant karyotype aberrations in the Met.-cell lines, except for Met. 5, in which two translocations t (5; 13) (q11.2; q32) and t (17; 18) (q23; p11.31) were identified in about 25% of the cells (data not shown; Dr. Shivanand R Patil, personal communication).
Lastly, the heterogeneity of expression might have resulted from the fact that each of the five lung lesions had a different “traveling history”. They might have been derived from different parts of the primary tumor and hence exposed to different microenvironments, which may also have occurred during lung colonization. It is conceivable that such a high level of heterogeneity observed among the metastases might contribute at least in part to their notorious untreatability.
Remarkably, most of the genes in the “biased multifunctional signature” are known to be involved in metastatic dissemination in different types of cancer. In particular, upregulation of urokinase (PLAU) was associated with an increased rate of metastasis and a decreased metastasis-free survival in 114 cases of chondrosarcoma of bone [39
]. Our results, which revealed upregulation of PLAU and of tissue plasminogen activator (PLAT) in all Met.-cell lines, are in accordance with those reported by Häckel and colleagues [40
]. They found that the high-grade dedifferentiated components of the tumors—but not the low-grade components of the same tumors, nor conventional chondrosarcomas-exhibited robust, diffuse coexpression of PLAU and PLAT. Other genes such as IL8, ITGB1, MSN, TFPI2, CAV1, and TGFB1 are also in Section 2.8.3
“metastasis associated genes” (, Supplementary Table 2) [41
]. Furthermore, all the genes in the network shown in (Supplementary Materials Figure 1), with the exception of AP-1, are “multifunctional” and expressed in the Met.-cell lines. It is noteworthy that three of these genes—FN1, ILK, and CD44—are differentially expressed in four of the five Met.-cell lines, relative to the virtual NM-cell line. FN1, CD44, and CTNNB1 (beta-catenin) are also in the list of “metastasis-associated genes” (Supplementary Table 2) [19
]. Minn and colleagues [53
] found MMP1, CXCL1, and TNC among 18 of the most significant (P
< 0.05) genes in a lung metastasis signature. All three are in the “multifunctional” signature of dedifferentiated chondrosarcoma lung metastases—MMP1 and CXCL1 were significantly differentially expressed in 4 out of 5 metastases; TNC was differentially expressed in all 5 metastases and it is also a component of the “biased” signature). The authors also showed that combinations of MMP1 and CXCL1 could synergistically enhance lung colonization. Also, certain genes in the “biased” signature of dedifferentiated chondrosarcoma lung metastases, such as CCL2 and IL-8, were found to be significantly upregulated in primary tumors of nonsmall cell lung carcinoma with known history of lung metastases [54
]. PLAU, another gene in the dedifferentiated chondrosarcoma- “biased” signature, was found to be involved in dissemination of bladder cancer lung metastases [55
Noteworthy, majority of the genes from the network (Supplementary Figure 1)—PLAU, PLAT, TFPI2, ITGB1, CCL2, IL8, Cav1, FN1, ILK, and CD44—are known to be expressed in mesenchymal stem cells (MSCs) [56
], as well as in other adult stem cells. Moreover, around 36% of the genes differentially expressed during chondrogenic differentiation of MSC [62
] also had altered expression in the dedifferentiated chondrosarcoma metastases (27/74 genes differentially expressed in MSC) (Supplementary Table 5b). Some of these genes were found to be expressed at high level in both studies. Interestingly, the majority of the downregulated genes listed in this table are considered to be chondrocyte markers (AGC1, COL1a1, COMP, SPARC, Col3a1, BGN, FMOD) [24
]. Hence, as expected, they did become downregulated during the dedifferentiation of chondrosarcoma and/or upregulated during the chondrocytic differentiation of MSC. FN1 was upregulated during the chondrogenic differentiation of MSC but in our study it was upregulated in the metastases (Supplementary Table 5b). In concordance with our data, FN1 was also upregulated in different metastatic chondrosarcomas [28
Nonetheless, it remains to be determined if stem-like cells are indeed involved in the process of metastatic dissemination in dedifferentiated chondrosarcomas, and if “multifunctional” genes enable the rate-limiting steps of metastatic dissemination.
Although unlikely, it is conceivable that the fact that patient A had already been exposed to cytotoxic chemotherapy when the lung lesions were resected might have influenced our results. We find it to be unlikely because—with the exception of vinculin—none of the genes in the “biased signature” was upregulated in a SAGE library derived from the metastasis-free lung tissue sample obtained from the same dedifferentiated chondrosarcoma patient (Malchenko, S. and Soares, MB, personal communication), relative to the nonmetastatic tumor. Moreover, in other large-scale gene expression studies, none of the genes in the “biased multifunctional signature” exhibited altered expression upon exposure to a similar treatment regimen [63
This is the first report of a macrophage infiltration in lung metastases of dedifferentiated chondrosarcoma. It has been shown that macrophages, derived from circulating monocytes, represent a major component of the leukocyte infiltration in a tumor microenvironment [20
]. An increase in the density of tumor-associated macrophages (TAMs) was correlated with poor prognosis in the majority of clinical studies in different types of cancer, including sarcomas [20
]. Also, tumor overexpression of macrophage chemoattractants has been shown to correlate with poor prognosis [23
]. Notwithstanding these findings, we cannot exclude that the observed macrophage infiltration represents either a specific antitumor defense mechanism or a general “physiological” reaction on local microenvironmental changes caused by the metastases [65
]. However, the evenly high macrophage density that was observed all throughout the metastatic nodules is arguably not consistent with such interpretation.
Functional redundancy plays an important role in cancer development [66
]. It is conceivable that coexpression of certain genes in the “multifunctional” signature of dedifferentiated chondrosarcoma metastases—such as CD44, PLAU, CXCL1, CCL2, and IL8—might increase the level of functional redundancy in the process of macrophage recruitment to the dedifferentiated chondrosarcoma metastases [16
]. Functional synergy might also play an important role in cancer development. Quite intriguing are the examples of synergy between some of these “multifunctional” genes—CCL2 and IL8, MMP1 and CXCL1 [16
], and PLAT and PLAU [69
]—suggesting that the synergizing capacity of “multifunctional” genes might bear significance to the process of metastatic dissemination. Unfortunately, due to the restricted amount of tissues, as it was mentioned previously, we did not have an opportunity to directly correlate protein expression of the “multifunctional” genes with macrophage recruitment in the metastatic lesions. Also, since there is no in vivo
model of dedifferentiated chondrosarcoma lung metastasis, we did not have an option to analyse the involvement of the “multifunctional” genes in the process of metastatic dissemination experimentally.
In summary, we provide evidence for the first time of high heterogeneity at the gene expression level among individual lung metastases of a dedifferentiated chondrosarcoma patient. Despite this heterogeneity, we identified a set of “multifunctional” genes that are commonly expressed in the metastases. Also for the first time, we documented massive macrophage infiltration in the dedifferentiated chondrosarcoma lung metastases. It remains to be determined if the same phenomena will be observed in lung metastases of other dedifferentiated chondrosarcoma patients. Albeit derived from a single case, our findings have shed some light to the molecular mechanisms underlying metastasis in dedifferentiated chondrosarcoma.