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[Cancer Research 65, 3126-3135, April 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Tumorigenic Heterogeneity in Cancer Stem Cells Evolved from Long-term Cultures of Telomerase-Immortalized Human Mesenchymal Stem Cells

Jorge S. Burns1, Basem M. Abdallah1, Per Guldberg3, Jørgen Rygaard4, Henrik D. Schrøder2 and Moustapha Kassem1

1 Department of Endocrinology and Metabolism and 2 Institute of Pathology, Odense University Hospital, Odense, Denmark; 3 Institute of Cancer Biology, Danish Cancer Society; and 4 Bartholin Instituttet, Kommunehospitalet, Copenhagen, Denmark

Requests for reprints: Jorge S. Burns, Laboratory for Molecular Endocrinology, KMEB, Department of Endocrinology and Metabolism, Odense University Hospital, Medical Biotechnology Center, Winsløwparken 25, DK-5000 Odense C, Denmark. Phone: 45-6550-4081; Fax: 45-6550-3950; E-mail: jburns{at}health.sdu.dk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had {approx}1.2 days doubling time with loss of contact inhibition. All retained 1,25-(OH)2 vitamin D3–induced expression of osteoblastic markers: collagen type I, alkaline phosphatase, and osteocalcin. All shared INK4a/ARF gene locus deletion and epigenetic silencing of the DBCCR1 tumor suppressor gene. Despite in vitro commonality, only four of six clones shared the growth kinetics and 100% tumorigenicity of the parental population. In contrast, one clone consistently formed latent tumors and the other established tumors with only 30% penetrance. Changing the in vitro microenvironment to mimic in vivo growth aspects revealed concordant clonal heterogeneity. Latent tumor growth correlated with extracellular matrix entrapment of multicellular spheroids and high procollagen type III expression. Poor tumorigenicity correlated with in vitro serum dependence and high p27Kip1 expression. Aggressive tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone marrow–derived adult human mesenchymal stem cells (hMSC) can generate specialized mesodermal bone, cartilage, muscle, and adipose tissue, and this multipotency may extend to pluripotency in specific circumstances (1). However, hMSCs lack telomerase activity in vitro (2), with a limited proliferative life span that decreases with age (3). Overcoming this limitation, retroviral vector-mediated telomerase reverse transcriptase expression in primary hMSCs (hMSC-TERT20) greatly extends their in vitro proliferative life span and also improves their osteogenic differentiation in vivo (2, 4). Similarly, endogenous telomerase activity has been considered to be largely responsible for the extended proliferative capacity of cultured embryonic stem cells (5). However, enhanced telomerase expression is often a feature of many tumors, and unlike adult stem cells, embryonic stem cells can spontaneously form teratomas. Initial studies addressing whether telomerase-immortalized human somatic cells developed cancer-associated changes concluded they did not (6, 7). Nonetheless, this has been challenged by spontaneous alterations in c-myc proto-oncogene expression (8) and/or other genetic/epigenetic changes in long-term cultures (9–11) that can lead to in vitro transformation to contact inhibition and anchorage independence (12, 13). That a highly expressed telomerase gene may have "extracurricular" activity beyond maintaining telomere length (14, 15) is consistent with evidence that it enhances cell proliferation (16). However, only recently have spontaneous changes in a telomerase-immortalized human cell strain sufficed for tumorigenesis (17). To clarify whether this merely reflected a rare but dominant subpopulation, the tumorigenic hMSC-TERT20 strain was single cell cloned by limiting dilution. From 110 examples, 6 expanded clones were compared with the parental population with respect to morphology, telomerase expression, previously described genetic and epigenetic changes, osteoblastic differentiation potential, saturation density, contact inhibition, tumorigenicity, and expression of prognostic markers. Initially, we found a discrepancy between common in vitro characteristics and clone-specific differences in tumorigenicity.

However, monolayer culture does not explore all aspects that may govern tumorigenicity. These include (a) host-tissue interactions that nourish and support expansion of the tumor population and (b) growth in three-dimensional clusters. Blood supply is essential for nourishing tumor growth, and rapidly growing solid tumors are often characterized by areas of poor flow and hypoxia with a metabolic environment that compromises growth until there is rescue by vascularization (18). A tumor cell's resistance to nutrient starvation may influence tumorigenicity, and exogenous growth factor dependence can discriminate tumorigenic from nontumorigenic human cancer sublines (19). Thus, we tested growth and survival of the hMSC-TERT20 clones in serum-deprived conditions.

With respect to cellular growth in three-dimensional clusters, tissue architecture can inhibit tumor growth with build-up of solid stress restricting changes in cell geometry needed for proliferation (20). Tumor-derived matrix metalloproteinases (MMP) can break down and relax the extracellular matrix (ECM) of surrounding tissue, aiding early as well as late stages of tumor growth (21). Recent reviews of this "tumor jailbreak" phenotype (22) highlighted membrane-bound MMP-1 (MT1-MMP) as the principle regulator of pericellular proteolysis, allowing tumor cells to breach host tissue barriers (23, 24). Changing the in vitro microenvironment of tumor cell lines to growth as multicellular spheroids (MCS) may reveal differences in proliferation rate not observed in monolayer culture (25). Thus, we extended our in vitro analysis to include three-dimensional culture of MCS. These altered microenvironments revealed clone-specific differences that correlated with the significant differences in tumorigenic phenotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and derivation of clones. The hMSC-TERT20 strain was initiated by increasing the split ratio of continually passaged hMSC-TERT20 cells to 1:20 (17). hMSC-TERT20 parental cells and derived clones were grown in phenol red–free MEM (Gibco Invitrogen Co., Tastrup, Denmark) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen Co., batch tested) and antibiotics. Cells were maintained in a humidified incubator at 37°C and 5% CO2 and detached when near confluent by incubation with 0.25% trypsin in 0.1% EDTA (Gibco Invitrogen Co.). Seeding density (1:20) was 1,250 cells/cm2 in T80 flasks; vessels were from Nunc (Roskilde, Denmark) unless stated otherwise. For limiting dilution cloning, hMSC-TERT20 population doubling (PD) 440 cells were seeded in 100 µL medium at 0.3 per well in four flat-bottomed 96-well plates. Optical microscopy, within 12 hours of seeding, identified wells with only one cell. Subsequent monitoring confirmed growth of only a single colony per well, and medium was changed weekly. Clones at {approx}60% confluence were passaged into 24-well plates and refed twice weekly with 1 mL medium. Clonogenicity was determined by the ability to grow to at least PD 20 (>106 cells) when subsequently passaged in T25 flasks. Six arbitrarily selected clones showing morphologic uniformity were expanded for detailed characterization. The human fibrosarcoma cell line HT-1080 (26) was grown under the same conditions as hMSC-TERT20 cells. Telomerase-immortalized microvascular endothelial (TIME) cells (27), a gift from Prof. McMahon (University of California at San Francisco, San Francisco, CA), were grown in supplemented ECBM-MV2 medium (PromoCell GmbH, Heidelberg, Germany).

Telomerase activity: real-time quantitative Telomeric Repeat Amplification Protocol assay. The SYBR Green Real-time Quantitative Telomeric Repeat Amplification Protocol (TRAP) assay was a modified version of the method by Wege et al. (28). Briefly, cell pellet extracts were prepared using a 1x CHAPS lysis method (29). Real-time PCR amplification of serial dilutions (1,000, 100, 10, and 1 cell equivalents, including heat-inactivated samples) ensured assay specificity and linearity.

Proliferation rate. Cells from subconfluent cultures were detached by trypsin and counted using a hemocytometer. PD was calculated using the formula: PD = log [(n cells in) / (n cells out)] / log 2. PD times were calculated from the average of three consecutive passages.

Saturation density and contact inhibition. Cells were seeded at 0.53 x 105/cm2 in 12-well plates and grown to confluence. Triplicate wells were harvested using trypsin and cells counted in a hemocytometer to determine saturation density. Parallel cultures were fed every 3 days and counted at 10 days postconfluence. Results represent the average of three separate experiments.

Flow cytometry. Cells harvested using trypsin with 0.1% EDTA were centrifuged at 200 x g for 5 minutes at 4°C. Cell pellets resuspended in ice-cold PBS– were fixed in ice-cold 96% ethanol. The nuclei were released using a 0.04% pepsin solution (Sigma-Aldrich A/S, Brøndby, Denmark) in 0.1 mol/L HCl, and RNA was removed with 10 µg/mL RNase (Qiagen, VWR International A/S) in PBS/TB [PBS + 0.5% (v/v) Tween 20 (Merck, VWR International A/S) + 0.1% (w/v) bovine serum albumin]. The nuclei were spun down and resuspended in 50 µg/mL propidium iodide (Sigma-Aldrich A/S) in PBS/TB and incubated overnight at 4°C. Samples were filtered on a 50 µm nylon mesh before measuring 10,000 to 20,000 nuclei on a FACScan flow cytometer (BD Biosciences, Brøndby, Denmark) using a 488 nm excitation argon laser with >695 fluorescence emission.

Mutation and deletion analysis: denaturing gradient gel electrophoresis. Genomic DNA from 106 cells was isolated using a Purescript DNA Isolation Kit (Gentra Systems Inc., Biotech Line AS, Slangerup, Denmark). TP53 gene mutations were explored using a combination of PCR and denaturing gradient gel electrophoresis. The 12 sets of oligonucleotide primer sequences and conditions for analyzing the entire coding region and all splice junctions were as described (30). For deletion analysis of the INK4a/ARF locus, primers and PCR conditions were as described (31).

Epigenetic analysis: methylation-specific PCR. The methylation status of the DBCCR1 promoter CpG island (Genbank accession no. AF027734) was examined by methylation-specific PCR (32). Genomic DNA was treated with sodium bisulfite (33), and primer sequences and conditions for methylation-specific PCR analysis of the DBCCR1 gene promoter were as described (34). Amplification products were resolved in a 2% agarose gel.

Anchorage-independent growth (soft agar colony assay). Soft agar assays were as described (6) with minor modifications. Briefly, 2 x 104 cells were plated in triplicate 35 mm wells of six-well plates in 2 mL medium with 0.36% low gelling temperature agarose (Sigma-Aldrich A/S) plus 10% FBS on a 4 mL base layer of 0.9% agarose in medium. Cells were fed additional medium, 10% FBS (200 µL) weekly. After 2 to 6 weeks, colonies >40 cells were counted under an optical microscope. HT-1080 cells were a positive control.

Osteoblastic differentiation studies. Cells seeded at 4.2 x 104/cm2 in six-well plates were grown in standard medium for 24 hours. At 50% to 60% confluence, medium was replaced with control medium (MEM with 10% FBS) or medium with osteoblastic inductors, 1,25-(OH)2 vitamin D3, L-ascorbic acid and ß-glycerophosphate as described (35) for 4 days. Total RNA was isolated with TRIzol (Invitrogen A/S, Tastrup, Denmark) according to manufacturer's instructions. Expression of collagen type 1{alpha}, alkaline phosphatase, and osteocalcin relative to ß-actin mRNA expression was analyzed from two confluent wells per sample by real-time PCR. Methods determining the integrity and purity of total RNA, conditions for first-strand cDNA synthesis and real-time PCR, and software for generating relative expression values were as described (35).

Xenograft tumorigenicity. Immunodeficient mice (NMRI nu/nu) were maintained in pathogen-free conditions. Cells (5 x 106) at passage 6 were mixed with Matrigel 1:1 before implantation (100 µL) to facilitate establishment (6). Perpendicular diameters of emerging tumors were measured with digital calipers every 2 days to estimate tumor volume until the xenografts reached 1 to 1.5 cm3 or termination of the study. HT-1080 cells were a positive control. Tissue samples fixed in 4% formaldehyde-0.075 mol/L NaPO4 (pH 7), dehydrated, embedded in paraffin, and sectioned at 4 µm were processed for histologic and immunohistochemical evaluation. H&E staining determined histologic structure.

Serum dependence. Cells were seeded at 2 x 105 per well in 24-well plates in medium (10% FBS). After overnight attachment, the wells were washed two times with PBS– before applying low-serum (0.1% FBS) or serum-free medium. Proliferation and viability was monitored for 10 days by harvesting triplicate wells and counting with a hemocytometer using trypan blue dye exclusion to define viable cells.

Spheroid culture and immunocytochemistry. MCS cultures formed after seeding 106 cells on ultralow adhesion tissue culture dishes (Corning, Biotech Line A/S, Slangerup, Denmark). DNA synthesis and proliferative index were determined by 5-bromo-2'-deoxyuridine (BrdUrd) uptake and Ki-67 expression, respectively. Cell spheroid cultures labeled with 100 µmol/L BrdUrd for 90 minutes were fixed overnight in 0.5% w/v paraformaldehyde, PBS (pH 7.2; for BrdUrd staining), or 4% formaldehyde (for Ki-67 staining), washed three times in 9 mg/mL isotonic saline, and pelleted (280 x g, 8 minutes) in polystyrene conical tubes. The final pellet was aspirated dry before preparing a cell block using Shandon Cytoblock (Thermo Electron Corporation, Pittsburgh, PA) according to manufacturer's instructions. Cell blocks were embedded in paraffin and sectioned (4 µm) using conventional histopathologic methods. Immunocytochemistry for BrdUrd used the chromogen diaminobenzidine according to manufacturer's instructions (Roche A/S, Hvidovre, Denmark). pKi-67 antigen was detected using MIB-1 primary antibody (DAKOCytomation, Glostrup, Denmark). Procollagen type III (PIIINP) was detected using a polyclonal rabbit anti-human PIIINP antibody (36), a gift from J. Risteli (University of Oulu, Oulu, Finland). Cyclin D1 in MCS sections was detected using a murine monoclonal antibody (clone DCS-6, Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) and in tumor sections using a rabbit monoclonal antibody (ref. 37; clone SP4, Lab Vision Corp., AH Diagnostics, Aarhus, Denmark). p16INK4a expression in MCS was examined using a murine monoclonal antibody (clone E6H4, DAKOCytomation). p27Kip1 in MCS was detected using a murine monoclonal antibody (ref. 38; clone SX53G8, a gift from X. Lu, Ludwig Institute for Cancer Research UCL, London, United Kingdom). In tumor sections, p27Kip1 was detected using a rabbit polyclonal IgG targeting the amino terminus (clone sc-527, Santa Cruz Biotechnology, Inc., AH Diagnostics, Aarhus, Denmark). pRB1 was detected using a mouse monoclonal antibody (NCL-RB1, Novocastra Laboratories Ltd, Newcastle upon Tyne, United Kingdom). Detection employed Immunoperoxidase and Envision Plus according to manufacturer's instructions (DAKOCytomation, Glostrup Denmark).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hMSC-TERT20 clones share common in vitro monolayer characteristics. The hMSC-TERT20 population was single cell cloned 186 PDs after first showing tumorigenic potential (Fig. 1A). Eight 96-well plates seeded at limiting dilution yielded 117 wells containing single cells that showed high clonogenicity (94%); 110 clones expanded by 20 PDs to over a million cells. Most expandable clones (82%) matched the parental population morphologically; cuboidal cells formed a dense cobblestone pattern when confluent. Some (12%) had a flattened squamous morphology, a minority (4%) contained cells with a fusiform morphology, and two clones grew exceptionally slowly [doubling time (DT), ~15 days]. From the majority of clones showing uniform and consistent morphology, six were arbitrarily selected for comparison.



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  		Figure 1. hMSC-TERT20-derived clones retain common genetic and epigenetic changes. A, growth curve for hTERT infected human mesenchymal cells passaged at 1:20 (hMSC-TERT20). Days in culture refer to the cumulative period since infection with the hTERT retroviral vector. Time points when tumorigenicity was first detected in nude mice and single-cell clones were initiated by limiting dilution. B, detection of INK4a/ARF deletion in hMSC-TERT20 clones. C, detection of DBCCR1 hypermethylation in hMSC-TERT20 clones by methylation-specific PCR. DNA treated with sodium bisulfite was PCR amplified with primer pairs specific for unmethylated (U) and methylated (M) DBCCR1 alleles. SssI-methylated DNA provided a positive control for methylated DBCCR1 alleles.

 
Telomerase activity was normalized to the parental hMSC-TERT20 population and all six clones showed levels of activity equivalent to that found in the HT-1080 positive control (Table 1).


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  		Table 1. Phenotype of hMSC-TERT20 and clones

 
The DT of each clone in monolayer culture was very similar to that of the parental hMSC-TERT20 population (DT, 1.2 days) as was the corresponding proliferation-associated Ki-67 labeling index (LI; Table 1).

The clone with highest saturation density, hMSC-TERT20-BD6, had tightly packed cuboidal cells with a density of 36.8 x 104 cells/cm2 at confluence with continued growth forming a multilayered culture. All the clones lacked contact inhibition (Table 1).

The DNA content profile for all the clones indicated a diploid phenotype (Table 1) in contrast to the tetraploid fibrosarcoma cell line HT-1080. This agreed with previous chromosomal analysis of the hMSC-TERT20 parental population, consistently showing a normal 46 chromosome XY phenotype (17).

In contrast to early-passage hMSC-TERT20 cells (PD 85), all clones showed genetic deletion at the INK4a/ARF locus and hypermethylation of the DBCCR1 gene promoter (Fig. 1B) like the parental population, hMSC-TERT20 PD 417 (17). Despite exploring the entire coding region and all splice junctions, no TP53 gene alteration was detected in the parental hMSC-TERT20 population or its clones (data not shown).

Growth in soft agar revealed heterogeneity among the clones. Four clones grew poorly in soft agar or not at all, whereas hMSC-TERT20-BD11 and -DB9 resembled the positive hMSC-TERT20 parental cells and HT-1080 control (Table 1).

All clones stimulated by osteogenic medium expressed osteoblastic differentiation markers, although the basal levels and degree of induction varied between the clones (Fig. 2A). Clones hMSC-TERT20-BB3 and -DB9 had the lowest Col1A1 expression under basal conditions, but in osteogenic medium it was induced 10- to 25-fold. Low basal alkaline phosphatase expression meant that hMSC-TERT20-DB9 also had a relatively high induction of this marker (Fig. 2B). However, when normalized to ß-actin levels, the absolute level of alkaline phosphatase expression (2 arbitrary units) was much lower than that of clones expressing the highest levels: hMSC-TERT20-BC8 (20 arbitrary units) and hMSC-TERT20-BD11 (40 arbitrary units). Osteocalcin was only detected in osteogenic medium (Fig. 2C).



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  		Figure 2. Real-time PCR gene expression analysis of induced osteoblastic differentiation markers and relative expression of genes encoding ECM proteins and MMP. A-C, expression of osteoblastic markers collagen 1{alpha}, alkaline phosphatase, and osteocalcin comparing cell clones with the parental population grown in basal medium (white columns) or basal medium + osteogenic growth factors (black columns). D, relative expression of MT1-MMP. E, relative expression of collagen type III. Expression of target genes was normalized for ß-actin. Columns, mean of at least two independent experiments. *, P < 0.002 (paired t test).

 
Clonal heterogeneity in tumor formation. Despite common DTs and genetic/epigenetic changes, the clones had heterogeneous tumor phenotypes (Fig. 3A) that did not correlate with soft agar colony formation assays. Four clones (hMSC-TERT20-BB3, -BD6, -DB9, and -BD11) readily formed tumors, and hMSC-TERT20-BB3 and -BD11 formed tumors first. Histomorphologically, hMSC-TERT20-BD11 tumors had more dispersed cells with a spindle-shaped morphology, whereas hMSC-TERT20-BB3, -BB6, -CE8, and -DB9 tumors had densely packed cuboidal cells. Although hMSC-TERT20-BB3 and -BD11 had the fastest tumor volume growth (Fig. 3A), this reflected different phenotypes. hMSC-TERT20-BB3 had a cell density of 45 nuclei/100 µm2 and a Ki-67 LI of 61.3%, whereas hMSC-TERT20-BD11 had a cell density of only 26 nuclei/100 µm2 but a Ki-67 LI of 42.7%. Microscopic analysis of the lung, liver, kidney, spleen, or lymph nodes did not show any metastases.



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  		Figure 3. Growth kinetics for hMSC-TERT20 clone tumors. A, growth curves for first round implantations: hMSC-TERT20-BB3 ({square}), hMSC-TERT20-BC8 ({blacksquare}), hMSC-TERT20-BD6 ({bullet}), hMSC-TERT20-BD11 ({blacktriangleup}), hMSC-TERT20-CE8 ({circ}), hMSC-TERT20-DB9 ({triangleup}). B, growth curves for individual hMSC-TERT20-BC8 tumors from second round implantations: BC8-NMT1 ({circ}), BC8-NMT2 ({blacktriangleup}), BC8-NMT3 ({square}).

 
Consistently, hMSC-TERT20-CE8 only formed tumors after a dormant period of 24 days; then, the latent tumors grew with kinetics similar to the other clones (Fig. 3A).

In first round analysis, hMSC-TERT20-BC8 (passage 6) formed small nodules at 10 days, in three of five sites, but they receded with no macroscopically visible tumors 18 days postimplantation. Different tumorigenic clones on the opposite flank of the host forced termination of the hMSC-TERT20-BC8 experiment at 21 days. Testing nontumorigenicity more stringently, five further hMSC-TERT20-BC8 (passage 9) implantations were analyzed for an extended period. The three tumors from this second round had atypical growth kinetics (Fig. 3B). Histologic analysis of the tumor that appeared at 1 week with a volume plateau at 0.5 cm3 revealed a multilobed tumor with interlobular heterogeneity, suggesting selective expansion of only a subset of implanted cells. Cells cultured from the tumor latent until day 97 morphologically resembled those of the original hMSC-TERT20-BC8 clone (data not shown). Ultimately, the hMSC-TERT20-BC8 clone might best be described as poorly tumorigenic.

Poor tumor formation correlated with serum dependence in vitro. When grown in low-serum (0.1% FBS), hMSC-TERT20-BC8 cells were the most susceptible to death (Table 1). By day 9, viability declined to only 16% with the majority dead floating cells (Fig. 4B). Similar serum dependency was seen in 18% of the 110 expandable clones. In contrast, hMSC-TERT20-BD11 cells not only resisted death (90% viable on day 9) but, compared with the parental population (Fig. 4A), showed enhanced morphologic change to a reticular network of cords of cells termed capillary morphogenesis, a phenotype typical of endothelial cells (refs. 27, 39; Fig. 4C). Similar capillary morphogenesis on serum withdrawal was seen in 16% of the 110 expandable clones. Within 4 days, the capillary morphogenesis of hMSC-TERT20-BC8 had deteriorated, whereas that of hMSC-TERT20-BD11 was reinforced with more cells forming thicker cords. hMSC-TERT20-BD11 cells also showed capillary morphogenesis and high viability under serum-free conditions; changes in cell shape were visible within 2 hours; and intercellular organization to a reticular network initiated within 6 hours and was uniform across the whole culture by 12 hours. Confluence was not necessary for morphologic change, but cord-like structures formed more quickly and distinctly in regions of higher cell density. Immunocytochemical analysis at 3 days showed 14% of the hMSC-TERT20-BD11 cells expressed the marker CD31 with equivalent intensity to control endothelial cells (Fig. 4F). hMSC-TERT20-BD11 cells in 10% FBS medium were predominantly negative for CD31 (Fig. 4E). As expected for mesenchymal stromal cells and in common with endothelial cells, hMSC-TERT20-BD11 cells stained strongly for CD105 (endoglin; Fig. 4H and I).



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  		Figure 4. Contrasting phenotypes of the parental population and poorly tumorigenic versus highly tumorigenic clones. A, phase-contrast photomicrograph showing capillary morphogenesis of hMSC-TERT20 cells after 9 days in 0.1% FBS medium. B, clone hMSC-TERT20-BC8 under same conditions showed mostly floating dead cells. C, clone hMSC-TERT20-BD11 in 0.1% FBS showed enhanced capillary morphogenesis. D-F, peroxidase immunocytochemical staining (brown) for endothelial marker CD31 in (D) TIME endothelial cells (positive control), (E) clone hMSC-TERT20-BD11 cells in 10% FBS medium, and (F) clone hMSC-TERT20-BD11 cells in 0.1% FBS medium. G-I, peroxidase immunocytochemical staining (brown) for CD105 in (G) TIME cells (positive control), (H) clone hMSC-TERT20-BD11 cells in 10% FBS, and (I) clone hMSC-TERT20-BD11 cells in 0.1%FBS. J-R, peroxidase immunocytochemical staining (brown) for prognostic markers (J-L) pRB1, (M-O) cyclin D1, and (P-R) p27Kip1 in tumor sections derived from (J, M, and P) hMSC-TERT20, (K, N, and Q) clone hMSC-TERT20-BC8, and (L, O, and R) clone hMSC-TERT20-BD11. Insets, immunocytochemistry for corresponding MCS sections, peroxidase-immunostained (brown, positive) nuclei contrasted with counterstained (blue, negative) nuclei. (A-R) Bar, 100 µm.

 
Clonal heterogeneity in three-dimensional spheroid cultures correlated with in vivo phenotype. We explored whether tumor-specific heterogeneity (e.g., hMSC-TERT20-CE8 latency) might be attributable to three-dimensional growth. Cells grown on ultralow adhesion plastic soon formed multicellular aggregates. Most clones formed overnight aggregates 100 to 200 µm in diameter consisting of a few hundred cells, but hMSC-TERT20-CE8 formed smaller spheroids <50 µm in diameter with <50 cells. Spheroid cultures were grown in suspension for 2 weeks before analysis. The cellular density of the solid cell spheroids mimicked that observed in the tumor sections (e.g., hMSC-TERT20-BD11 had the widest internuclear distances; Fig. 5D). Moreover, hMSC-TERT20-CE8 had a unique three-dimensional culture phenotype of minimal DNA synthesis (BrdUrd labeling virtually undetectable) confirmed by Ki-67 immunocytochemistry showing low cell cycle activity (Table 1; Fig. 5E, inset). Unlike the widely distributed pKi-67-positive cells seen in most clones, the staining pattern of hMSC-TERT20-CE8 sections showed preferential staining of cells in the spheroid periphery (Fig. 5A-F, inset).



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  		Figure 5. Peroxidase immunocytochemical staining (brown) for PIIINP and pKi-67 expression in 4 µm sections of (A-F) three-dimensional MCS and (G and H) tumors: (A) clone hMSC-TERT20-BB3, (B) clone hMSC-TERT20-BC8, (C) clone hMSC-TERT20-BD6, (D) clone hMSC-TERT20-BD11, (E) clone hMSC-TERT20-CE8, and (F) clone hMSC-TERT20-DB9. Insets, corresponding MCS sections stained for Ki-67, darkly stained positive nuclei indicating cell cycle activity. G, tumor derived from clone CE8; H, tumor derived from clone hMSC-TERT20-BD6. Insets, corresponding tumor sections stained for Ki-67. (A-H) Bar, 100 µm.

 
The fibroproliferative response adjacent to malignant tumors is often characterized by increased expression of type I and III procollagens. Thus, we explored whether the hMSC-TERT20 clones shared these tumor-stroma characteristics. Coincident with reduced growth, hMSC-TERT20-CE8 spheroids had intense and organized staining for PIIINP (Fig. 5E). Staining the tumors for PIIINP and Ki-67 showed that the in vivo phenotype corresponded with the spheroid cultures. In hMSC-TERT20-CE8 tumors, regions of stronger PIIINP staining had reciprocally less pKi-67 staining that was preferentially distributed to the tumor periphery (Fig. 5G). This staining pattern was not observed in tumors from the other clones (e.g., hMSC-TERT20-BD6; Fig. 5H). These results were consistent with previous reports that inhibition of three-dimensional cell growth could be generated by solid stress imposed by a meshwork of ECM molecules (23). MT1-MMP was highlighted as the primary metalloproteinase conferring a growth advantage to tumors in three-dimensional cultures by pericellular proteolysis of the ECM. Thus, MT1-MMP expression was analyzed by semiquantitative real-time PCR. However, all clones expressed similar MT1-MMP levels, with hMSC-TERT20-CE8 having the second highest level (Fig. 2D). Nonetheless, real-time PCR analysis of monolayer cultures showed that collagen type III mRNA expression correlated with the spheroid immunocytochemistry (Fig. 2E).

Tumorigenicity correlated with expression of prognostic markers. We wished to determine whether the parental cell strain showed heterogeneity for markers correlated previously to cancer in other tumor systems and whether these markers then associated with the tumorigenic potential of its clones. Given INK4a/ARF locus alteration, we analyzed members of its complex signaling network in the tumors and MCS of hMSC-TERT20 cells compared with the most aggressive (hMSC-TERT20-BD11) and least tumorigenic (hMSC-TERT20-BC8) clones. As anticipated from genetic observations, p16INK4a was not detected (data not shown). However, the retinoblastoma protein (pRB1) was highly expressed in all cases (Fig. 4J-L; LI >90% positive nuclei). In hMSC-TERT20 tumors, cyclin D1 (LI 61%) and p27Kip1 (LI 49%) showed heterogeneous expression and this was also seen in sections from the cultured MCS (Fig. 4M and P). In contrast, clone hMSC-TERT20-BD11 tumors showed high cyclin D1 expression (LI 84%), and despite a similar LI, the p27Kip1 nuclear staining intensity was often less than that seen in the parental population (Fig. 4R). Clone hMSC-TERT20-BC8 showed a markedly elevated p27Kip1 expression (LI 83%; Fig. 4Q).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Telomerase may be considered an immortalizing agent of choice, with enhanced chromosome stability compared with other popular immortalizing agents (e.g., SV40 large T antigen; ref. 40). Many laboratories have "telomerized" a wide range of different human cell types (41, 42). Nonetheless, varying consequences of telomerase expression may reflect different technical approaches. For example, recent reports of similar genetic alterations in long-term cultures of telomerase-immortalized cells (11, 13) used a coselectable marker. Neomycin selection can influence gene expression and metabolism and may have a role in generating genetic alterations (43, 44). However, our use of a nonselectable vector confirms that genetic alterations in long-term cultures of telomerase-immortalized cells are not simply artifacts from use of selectable markers. Other contributory factors, including vector integration site, levels of ectopic gene expression, culture conditions, including plating density, and inherent qualities of different cell types, will inevitably vary (45). Therefore, the outcome of ectopic telomerase expression in long-term cultures is likely to remain unpredictable.

The tumorigenic phenotype of hMSC-TERT20 cells arose from the fundamental force of natural selection for cells that grow more rapidly in vitro. The spontaneous genetic and epigenetic changes we observed, deletion at the INK4a/ARF locus and epigenetic silencing by methylation of the tumor suppressor gene DBCCR1, correspond to similar changes frequently found in human tumors and osteosarcoma-derived cell lines (46–48). The bmi-1 pathway, important for stem cell self-renewal, targets dual inactivation of p16INK4a and p14ARF (49). Thus, p16INK4a and p14ARF pathways are likely to govern key fundamental aspects of cell growth regulation common to tumors and stem cells, with susceptibility for spontaneous alteration in both in vitro and in vivo microenvironments. Our finding no TP53 gene alteration supports data that this is no longer necessary after inactivation of the p53 pathway via ARF loss (50, 51). Although its role may be permissive rather than directly oncogenic (52), telomerase is likely to be very influential by being an initiating event in our tumorigenesis model. The tumorigenic hMSC-TERT20 population and its clones had a diploid karyotype and DNA content. When escape from a senescence-induced G1 arrest is mediated by p53 loss in a telomerase-null background (53) or p16INK4a inactivation is an early event (54), aberrant growth is often accompanied by telomere attrition, genomic instability, and development of aneuploid karyotypes. Studies exploring the prognostic significance of telomerase, p16INK4a, and/or p53 alterations will need to consider the order of events.

Tumor interactions with surrounding host tissue were not components of the in vitro selection pressure that led to evolution of the hMSC-TERT20 population. Moreover, induction of tumor vasculature can be a nonautonomous event, also favoring growth of neighboring cells. Large differences in the tumorigenic potential of individual cells within a tumor has led to the classification of a rare but dominant subpopulation, known as cancer stem cells (55). Thus, it was highly relevant to determine whether tumorigenicity was the outcome for (a) most cells in the hMSC-TERT20 population, (b) a dominant subset of autonomous cells, or (c) interdependent interactions between different subsets of nonautonomous cells. Consistent with the hMSC-TERT20 population representing a cancer stem cell model, all of the single-cell clones tested were tumorigenic to a greater or lesser extent. Although some cells did not survive the single-cell cloning procedure (6%), cloning per se did not necessarily introduce a bias favoring the outgrowth of more aggressive cells. The parental population was highly tumorigenic and clonogenic from the outset, and two of the six clones formed less aggressive tumors. Therefore, in mesenchymal stem cells, telomerase activation, subsequent immortalization, and long-term culture can induce a cellular autonomy capable of forming rapidly growing tumors.

The original observation that late-passage hMSC-TERT20 cells formed tumors was unexpected, contrasting with the bone formation hitherto characteristic of hydroxyapatite/tricalcium phosphate implantation experiments with early-passage hMSC-TERT20 cells (2). However, tumorigenicity did not totally prevent induction of osteoblast differentiation markers when cells were treated with osteogenic medium. This need not be surprising because human osteosarcoma cell lines show induction of differentiation markers (56) and hTERT expression plus p16INK4a inactivation did not abrogate differentiation pathways in keratinocytes (57). Nonetheless, no single clone had optimal induction of all differentiation markers. The relationship between osteoblast proliferation and differentiation involves complex hormone modulation of gene expression dependent on the maturation state of the osteoblast (58). Although some differentiation pathways were clearly retained in the clones, complete differentiation to bone was compromised.

It may be more than coincidental that adult mesenchymal stem cells provided the first example of spontaneous tumorigenicity in telomerized human cells (17). Parallels between tumor cells and stem cells fuel hypotheses that tumors may originate from the transformation of stem cells, and cancer stem cells may ultimately represent the most dangerous subpopulation that drives tumorigenesis (55, 59). Depending on the class of tumor, there is a minor or major contribution of bone marrow–derived cells to the stroma surrounding a tumor (60). Reyes et al. (39) reported that a tumor microenvironment could recruit mesenchymal multipotent adult progenitor cells (MAPC) and induce endothelial differentiation contributing to the tumor vasculature. Thus, hMSC-TERT20 cells may be more effective at forming tumors than other telomerized somatic cells by virtue of an innate ability to also form a supportive stroma.

Mathematical modeling of cellular interactions predicted that production of an angiogenic growth factor and prevention of programmed cell death are major factors for enhanced tumor growth (61). The initial suspicion that the poor tumorigenicity of clone hMSC-TERT20-BC8 reflected differences in angiogenic potential was not supported by evidence for vascular endothelial growth factor (VEGF) expression and murine vessels seen in the implanted region (data not shown). However, this clone was the most sensitive to death from serum deprivation, suggesting a relatively high requirement for one or more exogenous growth factors. The other clones showed the corollary that cells surviving serum-deprived conditions might be more tumorigenic. To a good approximation, tumor growth rate was proportional to percentage viability after 9 days in low-serum culture.

The most serum-independent clones, hMSC-TERT20-DB9 and -BD11, were also the best examples of low-serum inducible capillary morphogenesis, a characteristic of endothelial cells (27) and also the bone marrow–derived MAPC (39). Like MAPC, hMSC-TERT20-BD11 cells could express endothelial cell markers, but there were significant differences. MAPC required a 9-day low-serum conditioning period before VEGF-induced endothelial differentiation in confluent cultures on fibronectin (39). In contrast, hMSC-TERT20-BD11 cells maintained in 10% FBS showed capillary morphogenesis within 12 hours of serum withdrawal even at subconfluent densities. Moreover, hMSC-TERT20-BD11 cells did not require exogenous VEGF or provision of ECM (e.g., Matrigel) for cord formation. This resembled an in vitro angiogenesis model of murine vascular endothelial cells (62). Thus, hMSC-TERT20-BD11 cells represent a simplified serum-free human model system for determining the regulation of capillary morphogenesis in vitro without the undefined components of serum or Matrigel. Although we found no evidence that cords of hMSC-TERT20-BD11 cells were directly forming blood-conductive vessels in hMSC-TERT20-BD11 tumors, they may have an indirect vascular contribution by enhancing the ingrowth of murine vasculature (60).

Tumor dormancy or latency poses a significant problem when treating malignancy with cytotoxic agents that require an active cell cycle (63). Growth restraint of dormant but viable cells is imperfectly understood, but proposed mechanisms include angiogenic failure, host immune surveillance, absence of growth mitogen, or induction of differentiation. These mechanisms need not be mutually exclusive but have been extended by a growing awareness that ECM interactions may inhibit tumor progression (64). Latent tumor growth of clone hMSC-TERT20-CE8 correlated with reduced cell cycle activity in three-dimensional culture and high PIIINP expression. The PIIINP staining pattern in hMSC-TERT20-CE8 MCS showed well-organized collagen bundles, suggesting an additional qualitative clonal difference in intermolecular cross-linking of the fibrillar collagen. Although these differences did not simply reflect differences in MT1-MMP expression, others have shown that mutated collagen resistant to collagenolytic attack can suppress growth of MT1-MMP-expressing cells (23). Collagen type III was 4-fold more resistant to MT1-MMP catalytic activity than type I collagen (65); thus, it may qualify as a cellular entrapment molecule. In the clinic, regular organization and intensive staining of PIIINP correlated with benign as opposed to malignant ovarian tumors (36), and reduced cross-linking was associated with increased malignancy (66). Nonetheless, collagen type III need not be the only ECM molecule responsible for the hMSC-TERT20-CE8 phenotype. The ECM protein fibulin-1D could markedly extend the tumor latency of a human fibrosarcoma cell line (67). It remains possible that PIIINP overexpression is an indirect indicator of altered matrix deposition rather than the principle matrix molecule governing tumor dormancy.

Further proteins clinically correlated to tumorigenicity were heterogeneously expressed in the parental hMSC-TERT20 strain and more specifically associated with the tumorigenic potential of the least and most aggressive clones. The uniformly high pRB1 expression most likely reflected absence of p16INK4a expression (68). Patients with tumors that have low p27Kip1 expression usually have an inferior prognosis to those with high expression (69), and we found highest p27Kip1 expression in the poorly tumorigenic clone. Overexpression of cyclin D1 is known to correlate with the early onset of cancer and tumor progression, and we observed highest cyclin D1 expression in the most aggressive clone hMSC-TERT20-BD11. Notably, increased cyclin D1 expression need not necessarily correlate with increased DNA synthesis, and additional roles for cyclin D1 include regulation of cell adhesion and motility (70).

The hope for a much wider application of stem cells in therapeutic intervention, possibly enhanced with gene therapy, is modulated by caution regarding tumorigenic potential. The quality control of therapeutic stem cells will be improved with a more comprehensive understanding of possible risk factors. hMSC-TERT20 cells represent a useful cell system for defining spontaneous tumorigenic events along the path from stem cell to cancer stem cell.


   Acknowledgments
 
Grant support: Danish Medical Research Council, Danish Center for Stem Cell Research, Karen Elise Jensen's Fond, Novo Nordisk Foundation, Gross M. Brogaard og Hustru Foundation, Jacob Madsen og Hustru Foundation, and Danish Lægeforening Research Foundation; Alfred Benzon's Foundation World Laboratory fellowship and research fellowship (B.M. Abdallah).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Inger Andersen, Lone Christiansen, and Tina K.L. Nielsen for excellent technical assistance; Birgitte Elsnab and Graham Leslie for help with fluorescence-activated cell sorting analysis (Odense University Hospital); and Margit Baeksted and Morten Schou (Bartholin Instituttet) for expertise in animal husbandry.

Received 7/22/04. Revised 11/30/04. Accepted 1/12/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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