In this study we aimed to identify the presence of putative CSCs in a human osteosarcoma cell line and to evaluate their role in resistance to chemo- and radiotherapy. Our findings provide evidence that this osteosarcoma cell line contains a subpopulation of cells with stem-like properties as demonstrated by the formation of spherical clones in serum-free medium under anchorage-independent conditions. These results are in accordance with those previous reported by Gibbs et al.
that demonstrated the presence of stem-like cells in distinct bone sarcomas when cultured in stressful growth conditions [16
]. This method has been widely used in the prospective isolation of cells with stem-like properties from several malignancies, and is particularly useful when specific markers have not been identified, as is the case of CSCs for most tumors [20
The isolated cells were found to express MSC's surface markers CD73, CD90 and CD13 and were able to differentiate into the osteoblastic, chondrocytic and adipocytic lineages under standard culture differentiating conditions, which indicates that they remain mesenchymal and preserve some degree of the MSC's plasticity. However, the expression pattern of MSC-related antigens cannot be used as a specific marker of stem-like cells in osteosarcoma, since parental MNNG/HOS cells exhibited an immunophenotype similar to that of sarcospheres. This is in line with the theory that osteosarcoma originates from a primitive MSC in a consequence of impaired differentiation into osteoblasts that undergo malignant transformation. Therefore it is conceivable that more differentiated parental MNNG/HOS cells retain some properties of MSC, including the expression of cell surface markers and that the overlap of immunophenotype markers is related with the stage of differentiation of MSCs at the time of the mutation [24
]. Moreover, after culturing in specific differentiation conditions, MNNG/HOS cells differentiated towards osteoblasts, which indicate these cells still have some propensity of the original lineage. In addition, we found that cells derived from sarcospheres expressed high levels of Oct4 and Nanog (Figure ), which are key transcription factors required for the maintenance of self-renewal and pluripotency of embryonic stem cells (ES). These attributes of ES cells have regularly been identified in subsets of stem-like cells derived from mesenchymal tumors and of many other solid tumors, and appear to be critical to the phenotype of tumor-initiating cells [25
Two other fundamental properties of cancer stem-like cells are their ability to self-renew and to generate differentiated progeny. The sphere-forming capacity of these cells that was observed following three serial passages under selective culture conditions demonstrated the presence of a self-renewing cell population within the MNNG/HOS cell line. Furthermore, when transferred to adherent plates and allowed to grow in serum-containing medium, sarcospheres were able to expand in monolayer acquiring the morphological features and biological behavior of parental MNNG/HOS cells. We noticed that after 19 days in differentiating culture conditions, sarcosphere-derived cells (SAR-OS cells) started to proliferate with the same doubling-time as the MNNG/HOS cells around 24 h (data not shown), displayed similar glucose uptake as assessed with FDG and showed a significant decrease in the expression levels of the proteins associated with a stem cell phenotype (Oct4 and Nanog) and of the ABC-related transporters (Pgp and BCRP), reaching the levels of parental MNNG/HOS cells. The degree of FDG accumulation is considered a good indicator of the metabolic status of tumor cells. This fluorinated glucose analogue is widely used for detecting and staging of malignant tumors based on the enhanced glucose utilization of tumor cells when compared to non-tumoral tissues [28
]. This increased accumulation of FDG is related to a change in the metabolism of tumor cells that switches from oxidative phosphorylation to glycolysis, even in the presence of high levels of oxygen. This process, the so-called Warburg effect, results in a much less efficient mechanism for energy generation, and thus an increase in the requirement for glucose uptake, but is important in providing building blocks to support cancer cell proliferation [30
We found that sphere-forming cells, in comparison with the parental MNNG/HOS cells, accumulated significantly lower amount of FDG (Figure ). Moreover, after being placed in differentiation culture conditions, we observed a marked and progressive increase in the cellular uptake of FDG until reaching the values of MNNG/HOS cells after 19 days (Figure ). This progressive increase in cellular FDG uptake indicates that there are dynamic changes in glucose metabolism occurring during the differentiation process and that undifferentiated cells, as stem like-cells, are likely to have low energy requirements. This could be related with the fact that this fraction of cells is entering into a quiescent status and divides infrequently. This is in line with previous studies suggesting that quiescent cells reduce their glucose uptake and metabolic rate in contrast with highly proliferative cells [32
Some studies have shown that stem cells can become quiescent without losing their proliferative potential [14
]. This has been referred as an intrinsic defense mechanism of CSCs that they use against chemotherapeutic drugs targeting rapidly dividing cells [33
]. As a result, at least some of the stem-like cells are able to survive and be responsible for tumor regrowth after therapy. The lower FDG accumulation by stem-like cells can have important clinical implications, as PET imaging with FDG is commonly used for monitoring tumor response to therapy, by measuring changes in FDG uptake. In general, tumors with low accumulation of FDG after therapy are considered to be a good prognostic factor. Based on our observations, this reading can be biased by the fact that surviving stem-like cells might be few in number and might not accumulate FDG efficiently due to their quiescence, a status that can be maintained for a defined period before they return to a proliferative state and initiate tumor recurrence.
The enhanced tumorigenic ability of sarcospheres was demonstrated in vivo
in immunocompromised mice. The animals injected with 100,000 sphere-derived cells developed massive tumors with approximately 150 mm3
of volume at 4 weeks, whereas the injection of the same number of MNNG/HOS cells induced tumors with a 7-fold lower volume (approximately 20 mm3
). Based on previous studies, the injection of few stem-like cells derived from mesenchymal neoplasms can be responsible to initiate a tumor due to their enhanced capacity for self-renewal and a more plastic capacity that enables them to adapt to the stringent environment of the xenografts [30
]. It seems reasonable that the tumors arising from the MNNG/HOS cells results from the presence of a subset of stem-like cells that are sufficient to initiate tumor formation.
The chemo- and radiosensitivity assays clearly demonstrated the higher resistant profile of spherical clones when compared with parental MNNG/HOS cells. The mean IC50
values of all tested drugs (DOX, CIS and MTX) in sarcospheres were significantly higher (up to three to four-fold) than those obtained in adherent MNNG/HOS and SAR-OS cells (Table ). One of the mechanisms that have been proposed to explain the chemoresistance of CSCs is the activity of certain ABC transporters that mediate drug efflux, preventing the intracellular accumulation of chemotherapeutic agents at toxic levels. The up-regulation of these transporters has been observed in CSCs of several malignancies and is also the basis for the Hoechst-33324 dye exclusion assay to isolate a side-population (SP) enriched with cancer stem-like cells in cell lines and tumor samples [35
]. Our data demonstrated a significant high expression of Pgp and BCRP in spherical colonies compared with parental cells (Figure ), which might explain the higher resistance of CSCs to DOX and MTX, since these drugs are substrates of those transporters [12
]. The reversal of resistance to DOX that was observed in sarcospheres after co-incubation with VER sustains this hypothesis. It has long been known that VER restores drug accumulation sensitizing resistant tumor cells through inhibition of drug efflux pumps [37
]. VER is a non-specific inhibitor of ABC transporters and is itself a transport substrate of Pgp and BCRP competing with drugs for the transporter, blocking the efflux of the chemotherapeutic agent. Therefore is seems reasonable that both proteins are functionally active and contribute for the drug resistance phenotype of CSCs, at least to DOX which is the main chemotherapeutic drug used in the treatment of osteosarcoma.
In addition to an increased capacity for drug efflux, other mechanisms may co-exist, like alterations in cell cycle, enhanced DNA repair capacity, reduced apoptosis and expression of specific drug-detoxifying enzymes [38
]. High activity of the detoxification enzyme aldehyde dehydrogenase (ALDH1) has been found in osteosarcoma CSCs as well as in other solid tumors, and is referred as a possible drug resistance mechanism for both normal and malignant stem cells [39
]. This enzyme is responsible for the oxidation of intracellular aldehydes, thereby mediating self-protection and resistance to some alkylating agents (e.g. cisplatin) used in cancer treatments [41
The ability of stem-like cells to enter in a quiescent or slow-dividing state can also contribute for their resistance to conventional therapies that target proliferating cells. The low energy requirement evidenced by the small accumulation of FDG in sarcospheres is consistent with a slow dividing rate of these cells. Likewise, the mean LD50
values obtained from irradiation cell-survival curves were significantly superior for sarcospheres as compared with adherent cells (Table ). The survival curves of sarcospheres clearly showed a shoulder at lower and, therefore, clinically relevant doses of irradiation, which is probably due to an enhanced capacity to repair potential lethal damages. In contrast, no obvious shoulder was observed in both adherent MNNG/HOS and SAR-OS cells. This is consistent with the higher α/β ratio that was obtained for sarcospheres (18.16 Gy) compared to that of the MNNG/HOS cells (1.25 Gy). In general, cells displaying high α/β ratios are more resistant to cell dead induced by lethal DSB and have an enhanced capacity of DNA repair [42
]. It is well established that cell killing after exposure to ionizing radiation is partially mediated by free radicals. Consistent with the increased radioresistance, were the decreased production of ROS levels in sarcosphere-derived cells, compared with adherent counterparts (Figure ), potentially as a result of increased levels of free radical scavengers (e.g., glutathione and superoxide dismutases). These results are in agreement with previous data reported by others in cancer-initiating cells of brain tumors [43
] and breast cancer [44
]. They found that stem cell-enriched subpopulations contained low levels of ROS and developed less DNA damages compared with non-stem counterparts, and that those low levels of ROS were associated with increased expression of free radical scavenging systems. Notably, the depletion of ROS scavengers, via pharmacological depletion of glutathione, increased the radiosensitivity of breast CSCs, which demonstrated the importance of anti-oxidative defenses in radioresistance and survival of stem-like cells. The general low ROS concentration found in normal tissue stem cells, compared with their cellular descendants suggests that stem cells have conserved this attribute for protecting their genome from endogenous and exogenous ROS-mediated damage [45
The absence of significant alterations in cell cycle progression of sarcospheres following irradiation and drug exposure as well, suggests that they possess highly activated basal DNA repair mechanisms and possibly enhanced efficiency on DNA damage response activity that restrain them from undergoing apoptosis. In fact, we observed that sarcospheres are less susceptible to apoptosis as compared with parental MNNG/HOS cells, since the typical signs of apoptosis were only visible for higher doses of irradiation and drugs. Both treatments induced apoptosis and a G2/M cell cycle arrest in a dose-dependent manner in adherent cells indicating a cellular response to DNA-induced damages.
Our results are in line with previous findings observed in CSCs isolated from mesenchymal neoplasms showing increased chemoresistance with respect to their adherent counterparts [46
]. Such limited effectiveness of standard therapies suggests that they possess innate resistance mechanisms allowing them to survive and initiate tumor recurrence. The high levels of Pgp/BCRP expression and the relative quiescence observed in sarcospheres compared with the bulk population come out as potential resistance mechanisms operating in osteosarcoma stem-like cells. Nevertheless we cannot exclude other mechanisms such as the up-regulation of anti-apoptotic and down-regulation of pro-apoptotic pathways, as well as active DNA repair that can contribute to the overall resistance of CSCs to standard therapies.