Detection And Characterization Of Side Population From Human Bone Sarcomas

Bone sarcomas in general are rare tumors, which are derived from mesenchymal tissues-nonepithelial tissues originated from the embryonic mesodermal or ectodermal germ layers. Most bone sarcomas are of high grade and tend to develop pulmonary metastases. Despite improvements in surgery and multi-agent chemotherapy, in patients with metastatic or recurrent disease the 5-year survival rate is not significantly increased. The poor survival rate of these patients is largely due to their lack of responsiveness to chemotherapy. The mechanisms underlying sarcoma drug resistance and recurrence are poorly understood. One possible cause of therapeutic failure is that tumors contain cells with stem-like properties, such as self-renewal, multidrug resistance, quick expansion and invasive growth, which ultimately give rise to metastases and recurrences. Recently, new concepts of“cancer stem cell (CSC) or tumor initiating cell”have been proposed that tumors contain a small subset of cells with high resistance to chemotherapy and radiation, which both self renew and yield phenotypically diverse progeny to maintain tumor tissues, just like normal adult stem cells. The CSC hypothesis helps explain the drug resistance and relapse of tumors after the current cancer therapies. Furthermore, the CSCs may be the new target in cancer therapies.Numerous investigations have provided evidence for the existence of CSCs, which has already been identified in a large variety of human cancers. However, studies of bone sarcoma CSCs have seldom been reported, perhaps due to few recognized cell surface makers proven successfully to select and identify the bone sarcoma initiating cells. Fortunately, other properties of stem cells can be used to isolate cells with progenitor characteristics. Dye exclusion is a valuable technique successful in isolating and identifying CSCs, based on stem cells possessing a high ability to exclude fluorescent DNA-binding dye, Hoechst 33342. A small subset of cells designated as a“side population (SP)”has been isolated using this technique by fluorescence-activated cell sorting (FACS). SP cells have been identified in several mammalian malignant tumor tissues and well-established cancer cell lines. The studies revealed that SP cells have stem-cell–like characteristics, which suggests SP cells are enriched with CSCs. However, few studies of bone sarcoma SP cells have been reported.Hence, The present study was undertaken to isolate SP fraction from human osteosarcoma and Ewing's sarcoma(human osteosarcoma cell line MG63, human Ewing's sarcoma cell line SK-ES-1 and primary human osteosarcoma samples) and characterize several properties of SP cells such as repopulating capacity, tumorigenicity,self-renewal, clonogenicity, invasiveness, multi-drug resistance and the expression of“stemness”genes.1. Isolation of SP cells from human osteosarcoma and Ewing's sarcomaThe presence of SP cells in human Ewing's sarcoma cell line SK-ES-1 was examined by staining cells with Hoechst 33342 dye and SK-ES-1 cells (1.2%) were classified as SP cells, whereas none of SP fraction was found in human osteosarcoma cell line MG63. Moreover, among 6 primary human osteosarcoma samples tested, SP cells were detected in 5 samples(OS1–OS5) with the diverse amount, ranging from 3.7% to 10.2%.2. Differences of properties between SP and non-SP cells1) Repopulating capacitySP cells derived from SK-ES-1 generated both SP and non-SP with a fraction size comparable with the original population, whereas the non-SP produced mainly non-SP cells. In OS1–OS5, SP cells also produced both SP and non-SP cells, while non-SP produced non-SP cells on the whole, however, with a small fraction of SP cells.2) Growth curveBoth in SK-ES-1 and OS1–OS5, the growth curve of SP populations was similar in shape to that of non-SP population and both had an exponential growth phase. The proliferation rates were not signicantly different between SP and non-SP cells deriveded from SK-ES-1. Howerer, in OS1–OS5, it seemed that the growth of non-SP cells was faster than that of SP cells in the first 3-4 days.3) Clonogenicity and invasivenessBoth SK-ES-1 and OS1–OS5 SP cells were more clonogenic than non-SP cells and there was no signicant difference between non-SP cells and total unstained cells. Furthermore, SP cells of SK-ES-1 were more invasive than non-SP cells.4) TumorigenesisOS1 and OS5 SP cells exhibited high tumorigenicity in nude mice while non-SP cells still maintained some level of tumorigenicity in spite of large numbers of cells needed to form tumor. However, only SP fraction had the capacity to self-renew in vivo, for the secondary sorted SP cells from primary xenografted tumors that formed from SP population had the capacity to form tumors in secondary mice, whereas non-SP cells from non-SP tumors failed to regenerate tumors and the pathologica results revealed that both primary and secondary xenografted tumors had identically histological appearances to original tumors.5) Drug resistanceIn SK-ES-1 group, IC50 values of SP cells for 48-h treatment of doxorubicin and cisplatin were significantly increased. In osteosarcoma group, IC50 values for 48-h treatment of doxorubicin, cisplatin and methotrexate were significantly increased (except Cisplatin to OS1, cisplatin and methotrexate to OS5) in SP cells compared with non-SP cells. The data revealed that SP cells showed higher resistance to chemotherapeutic drugs than non-SP cells.6) Expression of“stemness”geneBoth in SK-ES-1 and OS1–OS5, the expression levels of ABC transporters genes were higher in SP cells compared with non-SP cells, especially for ABCG2, which was up-regulated significantly in SP fraction of all tested cell samples. Furthermore, The level of Oct4 mRNA expression was obviously up-regulated in osteosarcoma SP cells compared with non-SP cells. The similar result was observed in Nanog.In conclusion, our studies showed that a small fraction of cells with SP phenotype existed in bone sarcomas and SP cells exhibited significantly tumorigenicity and several properties of stem sells such as repopulating capacity, elevated clonogenic ability, self-renewal, multi-chemoresistance and high expression of“stemness”genes. All these results suggest that SP cells derived from bone sarcomas are enriched with stem-like cells, which are responsible for tumor initiation and maintenance. Our studies fill a gap in research of bone sarcoma CSCs and lay the foundation for bone sarcoma treatment by targeting SP cells.