Typically, the isolation of TICs from solid tumors relies on the identification of a combination of cell surface markers often associated with normal stem cells. However, a single phenotype represented by a defined set of markers cannot account for the cellular heterogeneity encountered in OS.30
To overcome these difficulties, we have selected OS TICs by functionally labeling slowly dividing/quiescent cells using the fluorescent dye, PKH26. In addition, we have identified several genes and molecular pathways that are dysregulated in OS TICs compared with the bulk of human OS cells.
Our data demonstrated that human OS cell lines contain a slowly dividing cell subpopulation that retains the red fluorescent dye PKH26 (PKH26Hi) and possesses TIC features. PKH26Hi cells form sarcospheres more efficiently than the rest of the tumor cells. Like breast or glioma CSCs, OS TICs overcome anoikis and serum deprived conditions to form spherical clones. The fact that OS cells can grow as sarcospheres and can be serially passaged demonstrates the presence of cells with self-renewal ability. Sarcosphere formation capacity in established OS cell lines and tumor samples have been demonstrated previously in references 17, 18
. Interestingly, we also observed a correlation between sarcosphere number and size with tumorigenicity in OS. 143B and MNNG/HOS are two of the most tumorigenic OS cell lines, and they constitute the group with the highest number and largest sarcospheres in our experiments. This phenomenon could be due to an increased proportion of OS TICs in these cell lines. Our results also showed that the most tumorigenic tumor samples (obtained directly from metastatic pulmonary nodules) also follow this pattern. A similar observation has been reported in malignant brain tumors.31
While sarcosphere formation capacity is a good in vitro assay to study OS TICs, the gold-standard to demonstrate functional TICs in any particular cancer is the formation of tumors after limiting dilution transplantation in immunodeficient mice. The interplay of tumor-initiating cells with their niche is critical for cancer growth.32
OS is a primary malignant tumor of the bone and ideally xenograft transplantation of putative TICs should be performed orthotopically in the bone of NOD/SCID mice. However, performing limiting dilution transplantations into the bone of immunodeficient mice is technically very challenging. CD133+
was identified as a marker of OS TICs in established cell lines in vitro but lacked confirmation in animal experiments.18
Subcutaneous injection of human OS TIC expressing the surface markers CD117+
or containing the Oct-4 activated promoter has been used as an alternative to orthotopic transplantation.19,20
In our studies, we observed the formation of bone tumors after injecting as few as 500 cells of OS TICs (PKH26Hi) directly into the tibia of NOD/SCID mice. To our knowledge, this is the first time that human OS TICs are defined by limiting dilution transplantation in an orthotopic mouse model. The CSC model posits that only this tumor cell subpopulation should have self-renewal capacity to generate tumors and reconstitute the original tissue cell heterogeneity. Although we observed that non-OS TICs were able to form bone tumors after orthotopic injection, this required ten times more tumor cells than OS TICs. This finding may be due to the intrinsically high-tumorigenic capacity of established OS cell lines or some contamination with OS TICs after FACS analysis. Nevertheless, our approach shows that even established OS cell lines are composed of different cell subpopulations with varied tumorigenic capacities. Furthermore, this method provides an alternative for identifying OS TICs using a more biological approach instead of relying in the expression of cell surface markers. In our hands, the expression of CD117 (c-kit) by immunohistochemistry in several 143B xenograft bone tumors was negative.
The isolation of OS TICs (PKH26Hi) led us to investigate the genes and molecular pathways that are differentially expressed in this tumor cell subpopulation. Labeling OS cells with ZsGreen significantly reduced the chance of specimen contamination with non-tumor cells. This is particularly important in OS that lack unique tumor markers. OS TICs (PKH26Hi) and PKH26Lo subpopulations isolated from the appropriate bone “niche” microenvironment were employed for gene expression analysis directly without the cells being cultured or passaged in vitro which may significantly influence genotype characterization.33
We found that genes involved in bone/skeletal development are upregulated in OS TICs. From this group of genes, IGF-1 is particularly interesting because it has been reported to play a key role in the tumorigenesis of several sarcomas besides being involved in the formation of trabecular bone and longitudinal bone growth.34,35
The activation of IGF-1R by IGF1 in OS stimulates cell growth in vitro and in vivo.36,37
Moreover, IGF-1R inhibition by monoclonal antibodies resulted in a delay in tumor growth and increased the overall survival in a xenograft OS model.38
Our analysis did not reveal that genes involved in embryogenesis or “stemness” such as NANOG or OCT-3/4 were significantly upregulated in OS TICs.17,29
There was only a modest increase in the expression of NANOG and OCT-3/4 observed by both microarray and qPCR. We believe that these differences as compared with prior reports could be due to the experimental conditions of the sarcosphere assays, which enhance the development of OS TICs and may increase the expression of these “stemness” genes. The expression of NANOG and OCT-3/4 seems to be increased to a much smaller extent in primary TICs isolated from OS orthotopic bone tumors. However, we did find that IHH, a member of the Hedgehog family, is enriched in this cell compartment. The Hedgehog signaling pathway has a role during embryogenesis, bone development and also has been implicated as one the mechanisms dysregulated in CSCs in several tumors.39,40
However, the role of Hedgehog signaling in TICs and in the formation of distant tumors or metastasis is not fully understood. We observed that genes involved in cell migration were overexpressed in OS TICs, as might be expected if this cell subpopulation is involved in the metastatic process. Particularly, upregulation of MMP14, a member of the matrix metalloproteinase family, may be critical for OS progression and metastasis as has been shown in other tumors.41
We also observed that several genes that play a role in intracellular acetylation are downregulated in OS TICs. Histone acetylation can result in epigenetic gene silencing and chromatin remodeling which may allow the cell to remain in a stem-like state.42
Ultimately, more detailed functional studies will be necessary to determine the involvement of these cellular processes in OS pathogenesis.
In summary, we demonstrated that established OS cell lines contain TICs that efficiently generate bone tumors after orthotopic injection in immunodeficient mice. We identified this cell subpopulation using the long-term label retention membrane dye PKH26 that functionally labels slowly dividing/quiescent cells. This characteristic may account for OS TIC's resistance to most of the anticancer drugs that target rapidly dividing cells, and may explain later relapse. The genomic profiling of OS TICs reveals some pathways specifically dysregulated in this tumor cell subpopulation. Understanding OS heterogeneity may help in the design of improved treatments that combine cytotoxic drugs and agents that selectively target TICs.