The purpose of the current study was to establish a platform to identify the nature of cancer stem cells which are known to be responsible for growth, recurrence, and distant metastasis of osteosarcoma by inducing cancer stem cells from human osteosarcoma cell lines.
It has been already proven that only some fractions of cells vigorously proliferate in in-vitro and in-vivo cultures of cancer cells [12]. From such findings, the concept that cancer is composed of both cancer stem cells with strong proliferative power and their progeny cells with limited proliferative power was on the rise. Consequently, stem-like cells needed to demonstrate their proliferative power, long-term self-renewability, and productive capacity of progeny cells with differentiability into primary cells [4, 13].
There have been numerous attempts to extract cancer stem cells out of solid tumor, and among those attempts, a number of experiments utilizing sarcospheres are well described [8, 14–17]. Most cells growing in non-adhesive culture media deprived of serum tend to perish or age out due to its harsh culture environment [18–20]. The fact that sarcospheres repeatedly succeed to be cultured through serial passages when migrated from non-adhesive culture media to adhesive culture media reflects its one to one relationship between sarcospheres and stem cells [13].
Sphere culture technique was used to formulate sarcospheres from human osteosarcoma cells lines in this study. The sarcospheres repeatedly formed their identical sarcospheres when cultured through serial passages more than seven generations. When monolayer cultivation of sarcospheres is performed on adhesive culture media, normal osteosarcoma monolayer cells were successfully cultured, and their cultivation time to reach enough cell concentrations was similar in each passage. When a monolayer of cultured cancer cells was transferred to non-adhesive culture media, formation of the sarcospheres were reproduced through serial passages. Above-mentioned results of the study confirm self-renewal and self-regeneration of sarcospheres originated from human osteosarcoma cells to prove that the sarcospheres contain similar phenotypes as their preceding stem cells.
ALP is normally present as various isoenzymes in various parts of body, such as hepatic tissues and bone tissues, and abundant in osteoblast; furthermore, pluripotent germ cells possess especially high activity of ALPs [21]. Consequently, activity of ALP, which represents cellular undifferentiated state, is widely used as a biomarker for embryonic stem cells and embryonic germ cells [22, 23].
Immunofluorescence staining, using ALP staining and anti-ALP antibodies in the current research, revealed that human osteosarcoma sarcospheres show extremely strong expression of ALP relative to cancer cell monolayer and bone marrow-derived mesenchymal stem cells. Such high activities of ALP show that cells within sarcospheres maintain similar undifferentiated state as seen in embryonic stem cells.
Oct3/4, homeoprotein transcription factor in POU family, is expressed within the inner cell mass in the early stage and essential in maintaining pluripotency; however, its expression decreases as differentiations progresses [16, 24, 25]. After maturation, Oct3/5 is not observed in somatic cells except for type A spermatogonium, testicular seminoma, teratoma and primitive progenitor cells [26]. Nanog is a newly discovered homeoprotein transcription factor, which is generally expressed in embryonic stem cells [27]. Expression of Nanog maintains its stem cell phenotype without leukemia suppressors and enables self-renewal and propagation of cell lines [[28, 29]]. Even though Nanog is expressed in not only embryonic stem cells but also teratoma and germ cell tumors, its expression in somatic cells has never been reported [16, 24, 28]. Transcriptions factors, such Nanog and Oct3/4, are essential in cell differentiation, and interactions between Nanog and Oct3/4 is critical to maintain pluripotency of stem [30]. SOX2, a SOX family transcription factor, is crucial in maintaining self-renewal of undifferentiated embryonic stem cells, regulates develops of embryos, and ultimately determines cell identity [31]. Furthermore, when SOX2 is co-expressed with OCT4 in suppression of Oct3/4, it is a central transcription factor associated with immortality in induced pluripotent stem cell (iPS) [32]. SOX2 is mostly expressed in embryonic stem cells as well as colon [33]. C-myc is a proto-oncogene, associated with self-replication process and also used as a biomarker for embryonic stem cells [34].
In the current study, expression of Oct3/4, Nanog, C-myc, and ALP was analyzed using RT-PCR. These four markers were strongly expressed in human osteosarcoma sarscospheres, compared to the control groups, which are cancer cell monolayer and bone marrow-derived mesenchymal stem cells. In study with sarcospheres, expression of Oct3/4, Nanog, and SOX2 confirmed pluripotency of stem cells, self-renewal and self-reproduction of embryonic stem cells, and self-regeneration of undifferentiated cells, respectively. In addition, expression of C-myc indicated undifferentiated state of the cells, and totipotency is confirmed by strong expression of ALP in ALP staining and immunofluorescence staining.
Osteosarcoma is a malignant cancer cell with bone-marrow mesenchymal origin, characterized by clinical, histologic, and molecular diversity and formation of abnormal bone matrix [1]. Bone marrow-derived mesenchymal stem cells can differentiate into all three germ layers and form bone matrix when induced for bone differentiation [35]. These similarities between osteosarcoma and bone marrow-derived mesenchymal stem cells infer to that development of osteosarcoma cell may be in response to abnormal differentiation of mesenchymal stem cell [36].
In the current study, comparing osteosarcoma sarcospheres to human bone marrow-derived mesenchymal stem cells as a control group, the result indicated that mesenchymal stem cells did not survive in non-adhesive culture condition and showed weaker activity in ALP staining and immunofluorescence staining than osteosarcoma sarcospheres; in addition, RT-PCR results demonstrated mild expression of Oct3/4 and ALP but a lack of Nanog expression in human bone marrow-derived mesenchymal stem cells. Therefore, such results indicated that bone marrow-derived mesenchymal stem cells are more committed adult stem cell relative to cancer stem cells; in other words, cancer stem cells are more primitive undifferentiated stem cells with capacity of self-replication, totipotency and pluripotency as seen in embryonic stem cells. Furthermore, de-differentiation process of bone marrow-derived mesenchymal stem cells is thought to contribute to development of osteosarcoma cancer stem cells, evidence of which is shown by a higher expression of SOX2, a pluripotency-specific transcription factor, in sarcospheres than the control group in our study.