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Keywords:

  • aldehyde dehydrogenase;
  • cancer stem cells;
  • OS99-1;
  • osteosarcoma;
  • tumorigenesis

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDHbr cells) from the OS99-1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99-1 cell line. ALDHbr cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDHlo cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDHbr cells illustrated the stem cell characteristics of self-renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma.

Over the past decade, increasing evidence has supported the notion that tumors are organized by a hierarchy of heterogeneous cell populations with different proliferation potentials in which the capability to initiate tumor formation and promote tumor growth exclusively resides in a small subpopulation of tumor cells termed cancer stem cells (CSCs) or tumor-initiating cells.1, 2 According to the CSC model, CSCs may originate from stem cells or progenitor cells in transformed tissues through deregulated self-renewal. Like normal stem cells, CSCs have self-renewal ability, which drives tumorigenicity. Moreover, these cells have the capability to differentiate, albeit aberrantly, giving rise to a heterogeneous population of differentiated cells that are non-tumorigenic, lack the ability to self-renew, have limited proliferation potential and constitute the bulk of the tumor. The stem cell-like phenotype of CSCs and their rare number within the tumor may account for their ability to escape from conventional therapies, thus leading to tumor recurrence and eventually metastasis, even when the primary lesion has been eradicated.3, 4 Although the specific markers may differ from one tumor to another, CSCs are primarily characterized by the ability to form new tumors through serial transplantation in immunodeficient nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice, as well as by the capacity to display stem/progenitor cell properties such as self-renewal and the ability to reestablish tumor heterogeneity.5 The identification of CSC populations has significantly increased understanding of tumor biology and has important implications for new cancer therapies.

Osteosarcoma is the most common primary tumor of bone, with peak incidence in the teenage years. Approximately 400 new pediatric cases occur per year in the United States.6 Recently, the existence of stem-like cells in primary osteosarcomas and cell lines derived from human osteosaracoma was demonstrated in a subpoplation of cells capable of self-renewal. These cells have been detected in spherical clones under anchorage-independent, serum-starved culture conditions, as side population (SP) cells based on efflux of Hoechst 33342 dye or as CD133+ cells sorted using cancer stem cell marker CD133.7–9 Our previous study also confirmed the presence of a stem-like cell population in 4 separate human osteosarcoma cell lines, as characterized by sphere-forming capacity and by expression of the stem cell markers.10 Most recently, Levings et al. reported expression of an exogenous human Oct-4 promoter that was used to identify tumor-initiating cells in osteosarcoma.11 However, no study has reported the identification of cancer stem-like cells in human osteosarcoma cell lines based on high aldehyde dehydrogenases (ALDH) activity.

Cytosolic ALDHs are a group of enzymes involved in oxidizing a wide variety of intracellular aldehydes into their corresponding carboxylic acids.12 Among these enzymes, ALDH1 is thought to have an important role in oxidation of alcohol and vitamin A and in cyclophosphamide chemoresistance.13 Elevated levels of ALDH activity (ALDHbr cells) have been found in murine and human hematopoietic and neural stem and progenitor cells compared to other cells.14–16 More recently, high ALDH activity has been used to define stem cell populations in many cancer types including breast cancer,17 liver cancer,18 colon cancer19 and acute myeloid leukemia.20 In this study, we investigated whether high ALDH activity can be used to identify CSCs in human osteosarcoma cell lines.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell culture

Human osteosarcoma cell lines MG63 and Saos-2 were purchased from American Type Culture Collection (Manassas, VA). Human osteosarcoma cell line Hu09 was purchased from Health Science Research Resources Bank (Osaka, Japan). Human osteosarcoma cell line OS99-1 was obtained from Dr. Sheila M. Nielsen-Preiss (Montana State University). It should be noted that the OS99-1 cell line was originally derived from a highly aggressive primary human osteosarcoma.21 All cells were routinely cultured in Dulbecco's Modified Eagle Medium (DMEM)/F12 medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Gibco) in a humidified atmosphere of 5% CO2 in air at 37°C. For consistent results, cells for ALDEFLUOR cell analysis were used within 2 to 3 passages of each cell line and repeated at least 3 times as described previously.22 This was performed to verify the consistency of the percentage of ALDHbr cells in each cell line and confirmed that no significant changes between P2 and P3 occurred in each cell line.

ALDEFLUOR cell analysis and flow cytometry sorting

To characterize the population that expresses high levels of ALDH (ALDHbr) in 4 cell lines, the ALDEFLUOR kit (Stemcell Technologies, Vancouver, BC, Canada) was used to isolate ALDHbr cells, as described previously.17 Incubation of cells with ALDEFLUOR in the presence of the specific ALDH-inhibitor dimethylaminobenzaldehyde (DEAB), which results in decreased fluorescence, was used as a negative staining control for the assay.14–17

Implantation of sorted cells into NOD/SCID mice

To assess the difference in tumorigenicity of ALDHbr and ALDHlo cells sorted from the OS99-1 cell line, freshly sorted cells were washed and suspended in 200 μl of serum-free HBSS/Matrigel (BD Biosciences, San Jose, CA) mixture (1:1 volume) and then injected subcutaneously into the right and left lower abdominal area of NOD/SCID mice. Our previous experience indicated that a high degree of cellular dispersion would be produced when sorted cells were subcutaneously injected into NOD/SCID mice without stromal support; therefore, we used Matrigel to act as a physical barrier to prevent cellular dispersion and the subsequent loss of injected cells. Coinjection with Matrigel has been shown to increase tumor formation and yield optimal tumor growth.23 Tumor growth was monitored weekly for 32 weeks. Tumors formed were removed and a portion of each tumor was processed for histological analysis. All animal studies were performed according to protocol approved by the Institutional Animal Care and Use Committee of the University of Michigan.

Xenograft enrichment of human osteosarcoma OS99-1 cells

It has been shown that cancer stem-like cells can be enriched with their tumorigenic capability in the reconstituted, xenografted tumor. To assess whether tumorigenicity of cells isolated from xenografts can be enhanced in xenograft compared to those directly isolated from the cell line, unsorted OS99-1 cells were injected into NOD/SCID mice to establish xenograft tumors. Tumors formed were removed and harvested for preparation of single cell suspensions and histology.

Injection of single cell suspensions of sorted tumor cells and serial transplantation

Xenografted tumors formed by injection of either unsorted or sorted OS99-1 cells were minced with scissors, mixed with 1 mg/ml collagenase Type II (Sigma-Aldrich Co., St. Louis, MO), incubated for 3–4 hr, passed through a 70-μm cell strainer and then washed twice with DMEM/F12/10% FBS medium. Cells were resorted into ALDHbr and ALDHlo fractions as described above. During flow cytometry, mouse H-2Kb antibody (BD Biosciences Pharmingen, San Jose, CA) was used to eliminate cells of mouse origin from the xenograft tumors. Sorted cells were reinjected into the animals as the primary transplantation. The entire procedure was repeated with the formed tumors to conduct the secondary and tertiary transplantations.

Immunohistochemistry/immunofluorescence

For immunohistochemistry, formalin-fixed paraffin sections of tumor samples were stained with goat anti-ALDH1 antibody (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) using standard procedures. Immunofluorescent staining was performed as we previously reported.10

Cell proliferation assay

The freshly sorted ALDHbr and ALDHlo cells from xenografts were cultured at a density of 2 × 103 cells per well in a 96-well plate triplicates with 100 μl of culture medium and allowed to grow for 10 days. Cell proliferation was assessed by using Celltiter96 AQueous One Solution reagent as described previously.24 Briefly, at specific time point during cultivation, the medium was discarded and replaced with 100 μl of PBS and then 20 μl of Celltiter96 AQueous One Solution reagent (Promega, Madison, WI) was added to each well and incubated at 37°C for 2 hr. The absorbance at 490 nm was recorded using Bio-TEK ELx 800 Plate Reader (Bio-Tek Instruments, Inc., Wilrijk, Belgium).

Soft agar assay

Freshly sorted ALDHbr and ALDHlo cells from xenografts were seeded in 6-well plates coated with a 1% agarose bottom layer in culture medium containing 10% FBS, a middle layer of 0.6% agarose including 5 × 103 cells and a top layer of culture medium only.25 The plates were incubated at 37°C for 4 weeks and fixed with formalin for 20 min. Plates were then stained with 0.005% crystal violet for 1 hr, and colonies were visualized by trans-UV illumination and counted using analysis software QuantityOne (Bio-Rad, Hercules, CA).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Oct3/4A, Nanog and Sox-2 mRNA

To assess mRNA expression levels of embryonic stem cell markers Oct3/4A, Nanog and Sox-2 in the ALDHbr and ALDHlo cells from xenografts, qRT-PCR assay was performed as we previously reported.10 The Eppendorf Mastercycler Realplex Detection System (Eppendorf, Germany) was used for conducting qRT-PCR.

Statistical analysis

Data were expressed as mean ± SD. Statistically significant differences were determined by 1-way ANOVA analysis, Student t-test and χ2 analysis, where appropriate, and defined as p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Human osteosarcoma cell lines contain ALDHbr population

Using the marker ALDH that was previously used to identify CSCs in breast and colon cancer,17, 19 we identified a similar population in human osteosarcoma by exposing 4 human osteosarcoma cell lines to ALDEFLUOR and then analyzing by flow cytometry. In each experiment, a sample of cells was stained simultaneously with ALDEFLUOR and DEAB, a specific inhibitor of ALDH used to identify the ALDHbr population. As shown in Figure 1a, all 4 cell lines tested demonstrated ALDH enzymatic activity, although the levels of activity were different. The OS99-1 cell line contained 45.07% ± 3.75% ALDHbr cells, whereas the Hu09 cell line contained 1.84% ± 0.31% ALDHbr cells, which was close to that found in the Saos-2 cell line (1.56% ± 0.34%), and much higher than that found in the MG63 cell line (0.59% ± 0.16%). The level of ALDH in OS99-1 was significantly higher compared to the other 3 cell lines (p < 0.001), whereas no statistically significant differences were detected among the other 3 cell lines (Fig. 1b). As ALDHbr cells were more abundant in OS99-1 cells, attention was focused on OS99-1 cells.

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Figure 1. (a) Representative flow cytometric analysis of cells with high ALDH activity in 4 human osteosarcoma cell lines: OS99-1, 45.07% ± 3.75%; Hu09, 1.84% ± 0.31%; Saos-2, 1.56% ± 0.34% and MG63, 0.59% ± 0.16%. Cells incubated with ALDEFLUOR substrate and specific inhibitor of ALDH, DEAB, were used to establish the baseline fluorescence of these cells (R0) and to define the ALDHbr population (R1). (b) Evaluation of ALDHbr cell percentage indicated OS99-1 cells highly expressed ALDH statistically, as compared to other cell lines (*p < 0.001). Data represent mean ± SD and are representative of 3 independent experiments.

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Tumor-initiating cells are rare in the ALDHbr population sorted directly from the OS99-1 cell line

To determine whether ALDHbr cells sorted from the OS99-1 cell line are more tumorigenic than their ALDHlo counterparts in vivo, we injected both types of cells separately into NOD/SCID mice, in addition to injection of unsorted cells alone. The sorted ALDHbr and ALDHlo cells, as well as unsorted cells, were all maintained subcutaneously in mice, and tumor growth was monitored weekly for 32 weeks, when animals were sacrificed. The absence or presence of tumor was confirmed by histological examination. Tumor formation with unsorted OS99-1 cells was highly dependent on the number of injected cells. No tumors developed in NOD/SCID mice until at least 1 × 105 unsorted OS99-1 cells were injected (Table 1). For the sorted cells from the OS99-1 cell line, surprisingly, no statistically significant difference in tumor formation was noticed between injections with ALDHbr and ALDHlo cells (p > 0.05; Table 1). Tumor formation could occasionally be found with injection of 1 × 104 and 1 × 103 ALDHbr cells in 1 of 6 mice, respectively, whereas no tumors were observed with 5 × 104 ALDHbr cells in all 6 mice (Table 1). The size of the tumors grew moderately so that the injection of 1 × 104 and 1 × 103 ALDHbr cells developed palpable tumors after 11 weeks and 18 weeks, respectively. In comparison, tumor was formed in 1 of 6 mice 16 weeks after injection with ALDHlo cells when the injected cell number was 1 × 104.

Table 1. Ability for tumor formation in sorted cells from human osteosarcoma OS99-1 cell line based on ALDH activity
  1. Note: OS99-1 cells were isolated by flow cytometry as described in Figure 1a based on high ALDH activity and assayed for the ability to form tumors after injection into subcutaneum of NOD/SCID mice. Mice were examined for tumor formation by palpation and subsequent autopsy. Data are expressed as number of tumors formed/number of injections. Statistically significant difference in tumor formation between ALDHbr cells and ALDHlo cells was determined by χ2 analysis.

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The results revealed that tumorigenic cells that exist in the ALDHbr population sorted directly from the OS99-1 cell line are rare and these few cells are not able to consistently give rise to tumors in NOD/SCID mice.

Decreased ALDHbr cells in OS99-1 xenografts

To further validate whether tumorigenicity of cells in the reconstituted tumors differs from that of cells in the cell line, we next isolated ALDHbr cells from xenograft tumors based on previous findings that xenografts retained many features of the primary tumor on multiple passages,26 and using a xenograft model to isolate CSCs has been validated in several cancers.27–32 Surprisingly, we found that the percentage of ALDHbr cells derived from OS99-1 xenografts was 3.13% ± 0.53% (Fig. 2a), which was around a 15-fold reduction (Fig. 2b) compared to that of cells directly isolated from the OS99-1 cell line (Fig. 1a), as described earlier. Immunostaining results further confirmed that ALDH-positive cells dramatically decreased in OS99-1 xenografts compared to that of the OS99-1 cell line (Figs. 2c and 2d).

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Figure 2. Decreased number of ALDHbr cells shown in xenografts formed by injection of OS99-1 cells. (a) Representative flow cytometric analysis of ALDHbr cells in OS99-1 xenografts; percentage of ALDHbr cells was 3.13% ± 0.53% in xenograft tumors. Inset shows negative controls when cells were treated with ALDH-inhibitor DEAB. Data represent mean ± SD and are representative of 3 independent experiments. (b) Percentage of ALDHbr cells in OS99-1 xenografts had around 15-fold reduction compared with ALDHbr cells isolated from OS99-1 cell line, as described in Figure 1a. (c) Immunofluorescent staining shows ALDH expression clearly detected in the cytoplasm of OS99-1 cells (red areas), and nuclei counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) (blue areas). (d) Immunohistochemical staining indicated sparse ALDH-positive cells detected in OS99-1 xenografts using horseradish peroxidase, with brown staining indicating ALDH-positive cells. Bar = 50 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Enhanced cell growth rate and colony-forming ability on soft agar with ALDHbr cells sorted from OS99-1 xenografts

To determine whether ALDHbr cells are more proliferative and clonogenic than their ALDHlo counterparts after xenograft enrichment, we collected ALDHbr and ALDHlo cells from the OS99-1 xenografts and compared their proliferative and clonogenic abilities using cell proliferation assay and soft agar colony formation assay, respectively. After sorting by flow cytometry, ALDHbr and ALDHlo cells were immediately cultured in DMEM/F12 medium containing 10% FBS to perform the proliferation assay. After 6 to 10 days, ALDHbr cells reached a logarithmic growth phase, whereas the ALDHlo cells still grew slowly. The cell proliferation of ALDHbr cells in cell culture was evaluated to be 4- to 8-fold faster than that of ALDHlo cells (p < 0.001; Fig. 3a).

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Figure 3. Increased cell growth rate, clone formation ability and stem cell marker gene expression in vitro in ALDHbr cells sorted from OS99-1 xenografts. (a) Freshly isolated ALDHbr and ALDHlo cells were cultured in 96-well plates at a density of 2,000 cells per well and allowed to grow for 10 days, at specific time point during cultivation, the medium was discarded and replaced with 100 μl of PBS, and then 20 μl of Celltiter96 AQueous One Solution reagent was added to each well and incubated at 37°C for 2 hr. The absorbance at 490 nm was recorded. ALDHbr cells grew faster than ALDHlo cells (p < 0.001). (b, c) Freshly isolated ALDHbr and ALDHlo cells were cultured in 6-well plates at a density of 5,000 cells per well and allowed to grow for 4 weeks. More cell colonies could be found in ALDHbr cells than in ALDHlo cells (p < 0.001). (d) Relative quantitative mRNA expression of Oct3/4A, Nanog and Sox-2 genes in ALDHbr and ALDHlo cells freshly isolated from OS99-1 xenografts. Gene expression levels were normalized to β-actin. Enriched ALDHbr cells expressed significantly higher Oct3/4A, Nanog and Sox-2 compared to their ALDHlo counterparts from the xenografts (*p < 0.05; **p < 0.001). Each experiment was performed 3 times; representative examples are shown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Clone formation assays were also performed after cell sorting. Four weeks after culture, mean clone formation was 25% ± 2.5% and 3% ± 1.2% in ALDHbr and ALDHlo cells, respectively. Statistical analysis showed significant differences in clone formation between the 2 populations (p < 0.001; Figs. 3b and 3c).

Increased gene expression of stem cell markers in ALDHbr cells sorted from OS99-1 xenografts

Oct3/4, Nanog and Sox-2 are essential transcription factors that play a critical role in maintenance of self-renewal and pluripotency of embryonic stem cells.33 To determine whether ALDHbr cells of OS99-1 enriched in xenograft would display properties of stem cells and preferentially express those genes, total RNA was extracted from freshly sorted ALDHbr and ALDHlo cells, and qRT-PCR was performed to assess gene expression by the 3 stem cell markers. As shown in Figure 3d, Oct3/4, Nanog and Sox-2 in ALDHbr cells were all consistently higher than those in ALDHlo cells (p < 0.05; p < 0.001).

Enriched propensity of tumor initiation in ALDHbr OS99-1 cells

To determine whether ALDHbr cells sorted from xenografts are more tumorigenic than their ALDHlo counterparts in vivo, we transplanted 100 to 10,000 freshly sorted ALDHbr and ALDHlo cells from OS99-1 xenograft subcutaneously into NOD/SCID mice. Experiments were performed in triplicate, as previously described.17 Only the injection of ALDHbr cells gave rise to visible tumors, whereas no tumors were observed with ALDHlo cells, even when as many as 10,000 cells were injected (Table 2). The size and latency of tumor formation with ALDHbr cells highly correlated with the number of injected cells, where the injection of as few as 100 ALDHbr cells resulted in tumor growth in 2 of 3 mice (Fig. 4a). Injection with cell numbers greater than 1,000 was capable of developing tumors in all experimental animals. Hematoxylin and eosin staining of the tumor sections confirmed that tumors formed by ALDHbr cells contained malignant cells (Fig. 4b), whereas only residual Matrigel and apoptotic cells were seen at the sites of injection with ALDHlo cells (Fig. 4c).

Table 2. Ability for tumor formation in sorted cells from human osteosarcoma OS99-1 xenografts based on ALDH activity
  1. Note: Cells from OS99-1 xenografts were isolated by flow cytometry based on high ALDH activity as described in Figure 2a and assayed for the ability to form tumors after injection into the subcutis of the NOD/SCID mice. Mice were examined for tumor formation by palpation and subsequent autopsy. Data were expressed as number of tumors formed/number of injections.

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Figure 4. Tumor formation in NOD/SCID mice injected with highly tumorigenic cells from OS99-1 xenografts. (a) Representative tumor growth in a NOD/SCID mouse at the injection site, even with as few as 100 ALDHbr cells. No tumor formation seen at the injection site of 100 ALDHlo counterpart. (b) H&E staining of tumor generated from ALDHbr cells reveals presence of malignant cells. (c) Injection site of ALDHlo cells contained only residual Matrigel and apoptotic cells. Bar = 50 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The ability for self-renewal and recapitulation in diverse phenotype of ALDHbr cells in OS99-1 enriched xenograft

Normal stem cells are defined by their ability to both self-renew and generate phenotypically diverse progeny. To test if our highly tumorigenic cells also exhibited these properties, we performed serial transplantation with ALDHbr cells derived from primary tumors that grew from an initial injection of 10,000 ALDHbr cells. ALDHbr cells isolated from the primary OS99-1 xenografts were injected into mice to form secondary tumors, followed by the development of tertiary tumors with the secondary-derived ALDHbr cells. The resultant tumors were reanalyzed for ALDH-marked cells. The percentage of ALDHbr cells in the primary, secondary and tertiary tumors were 3.35% ± 0.79%, 3.45% ± 1.21% and 3.39% ± 0.82%, respectively, which were all close to the original xenograft from which they were derived (Fig. 5a–c).

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Figure 5. The highly tumorigenic osteosarcoma cells display CSC properties. (ac) Phenotypic diversity in tumors arising from ALDHbr cells, confirmed by flow cytometric analysis of cells with high ALDH and by immunostaining of ALDH expression (d). (a–c) Tumorigenic ALDHbr cells from OS99-1 xenografts were then isolated and re-injected into NOD/SCID mice. Percentages of ALDHbr cells in 3 rounds of passages were similar to that observed in the tumor from which they were derived. Insets in a–c show negative controls when cells are treated with the ALDH-inhibitor DEAB. Tumorigenic cells formed tumors that contained phenotypically diverse cells similar to those seen in the original xenografted tumor. (d) Expression of ALDH in tumors derived from ALDHbr cells. Tissues were examined for the presence of ALDH in the 3 rounds of tumor passages. Antibody localization was done using horseradish peroxidase, with brown staining indicating the presence of ALDH antigen. Bar = 50 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The highly tumorigenic ALDHbr cells produced additional ALDHbr cells as well as phenotypically diverse nontumorigenic cells, recapitulating the same phenotypic complexity of the OS99-1 xenografted tumors from which the tumorigenic cells were derived. The histological appearance of the serially transplanted tumors was identical, and immunohistochemical staining with ALDH1 antibody further confirmed that distributions of ALDH expressed in sections of the serially transplanted tumors were also very similar (Fig. 5d). These data suggest that ALDHbr cells in OS99-1 or, even more generally, osteosarcoma, represent CSCs that are capable of undergoing self-renewal to maintain the ALDHbr population while producing differentiated progeny that are nontumorigenic and similar to ALDHlo cells.

Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Our investigation indicates that in osteosarcoma, high ALDH activity can be used to identify a subpopulation of cells that are highly tumorigenic. Specifically, results from the highly aggressive human osteosarcoma OS99-1 cell line suggest that a subpopulation of cells play a role in tumor malignancy and expansion. Although the ALDHbr cells derived from OS99-1 xenografts represented a small fraction of the tumor population, the cells were able to initiate new tumors in NOD/SCID mice. In addition, ALDHbr cells displayed several features typically seen in CSCs, including the ability for self-renewal, generation of differentiated progeny, recapitulation of the tumor phenotype from which they were derived, and increased expression of stem cell marker genes.

The concept of a CSC was originally introduced based on the observation that when cancer cells of many different types were assayed for their proliferative potential in various assays in vitro and in vivo, only a minority of cells showed extensive proliferation.1 Although CSCs have been identified in a variety of malignancies,27–32 the identification of CSCs in human osteosarcoma has been more difficult than in tumors originating from other types of tissues. Because of differences in mesenchymal origin, the markers that have been identified and developed for hematologic, neural and epithelial cancers are not necessarily applicable for isolation of CSCs from osteosarcoma. To date, the existence of such a stem-like cell population in human primary osteosarcomas and osteosarcoma cell lines has been mostly detected by the expression of stem cell marker genes as well as their ability to form spheroids in vitro,7, 10 but cells with these stem cell attributes have not been characterized by their ability to form tumor in vivo. Recently, a separate study has isolated SP cells from human primary osteosarcomas using efflux of Hoechst 33342 dye and has demonstrated that SP cells could initiate tumors in immunodeficient mice.8 However, it has been suggested that identification of the CSCs cannot solely rely on SP sorting, as the possessed SP phenotype is not universally presented in all CSCs and there may exist other defensive mechanisms for CSCs to evade drug therapies that cannot be identified by Hoechst dye staining.34 Therefore, the SP cells identified by Hoechst dye do not fully represent all CSCs. Seeking a reliable marker to screen tumor-initiating cells while preserving most of their functionality and viability thus becomes extremely critical.

We chose the marker ALDH as a starting point based on prior work on breast CSCs, in which cells with high ALDH activity were identified as putative CSCs.17 Our results show that all 4 cell lines contained different percentage of ALDHbr cells, but OS99-1 cell line contained the highest percentage of ALDHbr cells compared to the other 3 cell lines, where Saos-2 and MG63 cells were reported as non-tumorigenic cells,35–37 which implies that ALDH activity may be related to the aggressiveness of tumors, as the OS99-1 cell line was originally established from a high-grade human osteosarcoma.21

It is interesting to note that the retained ALDHbr cells were dramatically decreased when OS99-1 cells grew in reconstituted xenografts. This result is consistent with a previous report showing that ALDH activity in a lung cancer cell line had 3-fold reduction occurring in vivo during growth of the tumor.38 A possible explanation for the variation in ALDH activity may be due to different growth conditions. Changes in phenotypical characteristics and a multitude of genetic aberrations could take place within cancer cell lines passaged in vitro.39 Thus, it is probably more reliable to use an in vivo model that provides a physiologic environment to develop candidate CSCs that can be isolated in human osteosarcoma.

Using freshly sorted ALDHbr and ALDHlo cells from OS99-1 xenografts, our in vitro experiments revealed that ALDHbr cells grew faster and had a greater ability to form cell colonies than ALDHlo cells, supporting the theory that ALDHbr cells possess stem cell properties of self-renewal and high proliferation potential. Using xenograft to derive the ALDHbr cells, we further discovered that these cells had much greater tumorigenicity. The subpopulation of ALDHbr cells that remained (an average of 3%) gave rise to new tumors in animals consistently with the injection of 1,000 ALDHbr cells, and even with as few as 100 ALDHbr cells injected the incidence of tumor formation remained 2 of 3, which reoccurred through serial transplantation. In addition, based upon analysis of reformed tumors arising from the transplantation of isolated ALDHbr cells, both ALDHbr and ALDHlo cells were present concurrently, and distribution of the cells remained closely similar through serial transplantations. These results suggest that only ALDHbr cells, but not ALDHlo cells, have the ability to reinitiate tumors and reconstitute the heterogenicity, reflecting attributes of self-renewal and multipotency.

Further evidence that ALDHbr cells can recapitulate tumor diversity was evident in the immunohistochemistry assay on sections of xenografted tumors. Our results demonstrate that ALDH1-positive cells distributed sparsely over sections of tumors developed by injection of pure ALDHbr cells, in which ALDHlo cells appeared concurrently, indicates a developmental hierarchy was formed in the xenografted human osteosarcoma.

Analysis of stem cell marker genes showed that the tumorigenic population of ALDHbr cells preferentially expressed stem cell markers Oct3/4A, Nanog and Sox-2. It has been suggested that self-renewal and pluripotency of undifferentiated embryonic stem cells are maintained by a crosstalk of these transcription factors.33 Oct3/4A, Nanog and Sox-2 have also been implicated in tumorigenesis, primarily in germ cell tumors.33 However, they are also expressed in several other types of human cancers, including breast cancer and bladder cancer.40 The finding that Oct3/4A, Nanog and Sox-2 are differentially expressed in the tumorigenic population suggests a potential functional role for these transcriptions in human osteosarcoma, a possibility that remains to be investigated.

The capacity for tumor formation between the ALDHbr and ALDHlo populations sorted directly from the cell line did not reach statistical significance, unlike cells sorted from xenografted tumors. It is not clear why the low percentage of ALDHbr cells from the OS99-1 xenografts was more tumorigenic than the high percentage of ALDHbr cells sorted directly from the OS99-1 cell line. Further investigations including genetic analysis and microarray analysis are warranted to define the cause of the difference.

The finding that osteosarcoma-initiating cells can be identified by high ALDH activity offers several potential advantages over other methods. First, ALDEFLUOR assay has been developed for the assessment of ALDH activity in viable cells by flow cytometry and has been made commercially available in a kit format. Second, the detection of ALDH activity is a simpler and more reliable method than surface marker selection-based methods that require examination of clusters of several surface markers. The identification of a specific surface marker typically earns certain credits to target cells of interest, but it is also true that sometimes the surface phenotype of stem cells may remain stable despite a decline in functional activity as observed in the progenitors, so that the existence of qualified stem cells may therefore be overestimated.41 Third, ALDH substrate will not enter the nucleus or bind DNA, and as a consequence, this assay tends to be safer and less toxic compared to other methods that require UV excitation or stain cell dyes that bind to nucleic acids such as in SP analysis.42 Finally, since only viable cells with functional enzymatic activity and an intact membrane can retain the ALDH substrate, apoptotic and necrotic cells with leaky membranes will not be counted in the assessment. The identification of CSCs based on high ALDH activity represents a very useful method for detection and isolation of CSCs.

Of note, however, high ALDH activity might not be as a universal marker for stem cells, so ALDEFLUOR assay is limited for identifying cells with the properties expected of CSCs. It has been demonstrated isolated ALDHbr cells showed lower ability to proliferate and migrate as compared to ALDHlo cells in adipose tissue and endothelial tissue.43, 44 Thus, it is critical to carefully characterize the cells isolated based on high ALDH activity.

Although our data show that a subpopulation of osteosarcoma cells exists within the OS99-1 cell line with markedly enhanced tumorigenic potential and stem cell properties, there are limitations to our study. Specifically, an established cell line and not primary tumor was investigated. Further research using primary tumor is therefore necessary to confirm the findings of this study.

In conclusion, this study provides further support to the CSC hypothesis, with evidence that a subpopulation of cells with high ALDH activity residing in human osteosarcoma cell line OS99-1 is able to initiate and mediate new tumor.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors would also like to thank Dr. Sheila M. Nielsen-Preiss from Montana State University for the generous gift of human osteosarcoma cell line OS99-1. The authors also thank Mrs. Holly Wagner for assistance in the preparation of the manuscript and Mr. Martin J. White for flow cytometry.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References