|Molecular Vision 2005;
Received 27 May 2005 | Accepted 3 August 2005 | Published 12 September 2005
Cancer stem cell characteristics in retinoblastoma
Gail M. Seigel,1,2
Lorrie M. Campbell,1,2
1Ross Eye Institute and the Departments of 2Ophthalmology and 3Pathology, University of Buffalo, Buffalo, NY; 4Medical Research Service, Veterans Affairs, Buffalo, NY
Correspondence to: Gail M. Seigel, Ross Eye Institute, Department of
Ophthalmology, University of Buffalo, 3435 Main Street, Sherman 124,
Buffalo, NY, 14214; Phone: (716) 829-2157; FAX: (716) 829-2157; email:
Dr. Malathi Narayan is now a graduate student in the Department of Biochemistry, Medical College of Wisconsin, Madison, WI
Purpose: There is increasing evidence that cancer stem cells contribute to tumor progression and chemoresistance in a variety of malignancies, including brain tumors, leukemias, and breast carcinomas. In this study, we tested the hypothesis that retinoblastomas contain subpopulations of cells that exhibit cancer stem cell properties.
Methods: The following sources of retinoblastoma cells andtissues were examined for the presence of stem cell markers by immunocytochemistry: retinoblastoma tumors from mice transgenic for the SV40 T-antigen (driven by the β-luteinizing hormone promoter), cell pellets of human Y79 and WERI-Rb27 retinoblastoma cell lines, and archival human retinoblastoma pathological specimens. Hoechst dye exclusion, mediated by the stem cell surface marker ABCG2 (ATP-binding cassette transporter, G2 subfamily), was assessed by flow cytometry in mouse tumors and WERI-Rb27 cells.
Results: Small numbers of retinoblastoma cells (less than 1%) exhibited immunoreactivity to stem cell markers ABCG2, aldehyde dehydrogenase 1 (ALDH1), MCM2 (minichromosome maintenance marker 2), SCA-1 (Stem cell antigen-1), and p63. Hoechst dye uptake in mouse tumors and WERI-Rb27 cells was enhanced by addition of 50 μM Verapamil, consistent with activity of the calcium-sensitive ABCG2 protein. Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in mouse retinoblastoma tumors (0.3%) and WERI-Rb27 cells (0.1%) in a verapamil-sensitive manner. ABCG2 and ALDH1 positive cells were Hoechst-dim, as seen by dual labeling in vitro.
Conclusions: Mouse and human retinoblastoma tumor cells contain a small subpopulation of cells that exhibit a cancer stem cell-like phenotype. Especially significant is the expression of ABCG2 in mouse and human tumor cells, a calcium-sensitive cell surface protein that not only acts to exclude Hoechst dye, but also confers resistance to over 20 different chemotherapeutic agents. These findings point to a heterogeneity in retinoblastoma tumors that may have significant impact on future treatment strategies.
Retinoblastomas arise when both alleles of the rb gene are inactivated in the same retinoblast. Rb codes for a nuclear phosphoprotein that regulates retinoblast proliferation and development . As proposed more than two decades ago, retinoblastoma arises from a primitive neuroectodermal cell . The malignant cells typically express a restricted set of markers, particularly IRBP and cone-specific genes, and form various rosette structures representing photoreceptor differentiation. This expression pattern follows the cone photoreceptor default pathway of retinal development. Thus, although the target cell is a pluripotent retinoblast, the cellular phenotype largely resembles that of photoreceptors, in which cones are the dominant cellular phenotype [3,4]. For many years, an elusive goal has been to identify the retinoblastoma cell of origin . There has been a general consensus in the literature that primitive neuro-ectodermal cells are in some way involved in RB tumorigenesis [6,7]. More recently, the theory of a "cancer stem cell" component to retinoblastoma has been put forward .
The hypothesis of cancer "stem cells" originates from previous observations that not every cell within a tumor can maintain tumor growth, and that large numbers of tumor cells are needed to transplant a tumor. Some cancers appear to contain stem-like cells (cancer stem cells) that are slowly dividing, chemoresistant, and are capable of tumor progression [8,9]. The existence of cancer stem cells was first shown in acute myeloid leukemia [10,11] and has since been demonstrated in other cancers, including breast cancer  human brain tumors  and even the C6 glioma cell line, maintained in culture over many years . Therefore, there is precedent for the persistence of cancer stem cells with unique stem cell-like properties. Yet, we still do not know whether all cancers are organized into a hierarchy of cells with different proliferative and differentiation potentials.
ABCG2 (or BCRP), an ATP-binding cassette transporter in the G2 subfamily, is a cell surface, drug-resistance marker that has been utilized to identify stem cells from a variety of tissues, including tumors and leukemias. Specifically, ABCG2 expression confers upon cells the ability to exclude Hoechst dye 33342 [15-17], and confer resistance to at least 20 different chemotherapeutic agents, including methotrexate, doxorubicin, indolcarbazole, and others . This dye-excluding "side population" (SP) phenotype has been used in a variety of tissues to sort out presumptive stem cells, including stem cells from hematopoetic populations , bone marrow, skeletal muscle , mammary gland , lung , and developing retina . The side population has been identified as a group of cells able to exclude the Hoechst 33342 dye, a characteristic abolished with 50 μM verapamil treatment . The SP phenotype, coupled with expression of stem cell markers are two of the hallmarks of stem cells, both cancer stem cells and otherwise. In the present study, we tested the hypothesis that retinoblastoma contains subpopulations of cells that express these unique stem cell characteristics.
Animal experiments were performed in accordance with NIH guidelines (DHEW publication NIH80-23) and with approval by the Institutional Animal Care and Use Committee of the Veterans Affairs Medical Research Service. Fresh retinoblastoma (RB) tumors were harvested from adult SV40 β-LH-T-Ag mice. Breeding pairs of mice for the initiation of our colony were generously provided by Dr. Daniel Albert . The mouse model was created by expression of a viral oncogene (simian virus 40 T-Antigen), in the retina of transgenic mice, resulting in heritable ocular tumors with histological, ultrastructural and immunohistochemical characteristics comparable to those of human RB . Animals were killed by CO2 inhalation and their eyes placed in 4% paraformaldehyde buffered with PBS. The fixed eyes were incubated in 30% sucrose and OCT compound. Frozen sections were cut for immunohistochemistry.
For cell culture studies, we used the well-established cell lines Y79  and WERI-Rb27 , derived from genetically related donors . The WERI-Rb27 cell line was derived from a 24-month-old boy with bilateral retinoblastoma, with abnormal mRNA transcripts corresponding to the retinoblastoma gene. Y79 cells originated from the intraocular tumor of a 2 1/2 year-old female. WERI-Rb27 cells were maintained in DMEM with 10% calf serum, 1X MEM nonessential amino acids (GIBCO, Grand Island, NY), 1X MEM vitamins (GIBCO), 0.37% sodium bicarbonate, 0.058% l-glutamine and 100 μg/ml gentamicin. Y79 cells were maintained in RPMI 1640 with 20% fetal calf serum, 0.37% sodium bicarbonate, 0.058% l-glutamine, 10 mM HEPES, and 100 μg/ml gentamicin.
Human archival RB tumor specimens
Serial paraffin sections of three human RB tumors were examined for the presence of stem cell markers. Three human RB cases were used for comparison with the human cell lines and mouse tumors. However, three human cases were insufficient to make statistically significant correlations between expression of stem cell markers and tumor stage, treatment or prognosis. This will be the topic of a future study.
Table 1 lists the primary antibodies, their sources, and concentrations for all staining techniques used in this report. For negative controls, we tested these antibodies on mouse fibroblasts (NIH 3T3 cells) and human cervical carcinoma cells (HeLa cells), with no staining observed (data not shown). Paraffin-embedded retinal tissue sections of archival human specimens were rehydrated through xylene and graded alcohols and underwent antigen retrieval. Antigen retrieval was accomplished by placing slides in 10 mM sodium citrate buffer (pH 6.0), heated at 95 °C for 10 min. The slides and buffer were then cooled to room temperature over an interval of 20 min, followed by three rinses in water for 2 min each. The water was blotted off and the primary antibody was added to the slides. Frozen sections skipped the previous steps and were rinsed directly in PBS, then carried through the rest of the protocol. Goat serum (5%) was used for blocking of nonspecific staining on slides. All tissue sections were incubated in 0.25% Triton X-100 for 5 min. After a rinse in phosphate-buffered saline (PBS), sections were incubated for 1 h with primary antibody. After rinsing three times for 5 min each in PBS, sections were incubated with 1 μg/ml of biotinylated goat anti-rabbit or anti-mouse immunoglobulin (Zymed/Invitrogen, Carlsbad, CA) for 60 min. Tissue sections were incubated for 20 min with horseradish peroxidase-conjugated avidin (Elite kit, Vector Laboratories, Burlingame, CA). The sections were rinsed in 0.05 M Tris (pH 7.4) and antigens were detected with diaminobenzidine (Vector). The brown to black reaction product was visualized by light microscopy. Negative controls that consisted of incubations in 5% isotype control serum without primary antibody did not generate reaction product.
Fluorescent immunostaining in suspension
WERI-Rb27 cells and dissociated mouse tumor cells were double labeled in suspension with Hoechst 33342 and either ABCG2 or ALDH1. Cells (2x106) were centrifuged at 800 rpm (149x g) for 5 min. The supernatant was decanted and cells resuspended in 4 ml of DMEM with 10% calf serum. Hoechst 33342 dye (Molecular Probes, Eugene, OR) was added at 5 μg/ml, and cells were incubated at 37 °C for 90 min. Cells were washed twice in PBS and centriguged at 149x g for 5 min. Cells were resuspended in 200 μl PBS and divided into two tubes. No blocking serum was used. One tube received 6.25 μg/ml ABCG2 antibody and was incubated 1 h at room temperature, while the other tube received 5% isotype control antibody. Cells were washed two times with PBS and resuspended in 100 μl PBS. TRITC conjugated anti-mouse IgG (Sigma Chemical, St. Louis, MO) was added at 1:100 dilution to both tubes. Cells were incubated for 1 h at room temperature. Cells were washed two times with PBS and resuspended in residual supernatant after decanting, then pipetted onto a slide and coverslipped for microscopic viewing. Fluorescent cells were visualized with a Nikon ES600 microscope with epifluorescent filters for Hoechst and TRITC. Images were captured with a SONY ICX 285AL SPOT camera (Diagnostic Instruments, Sterling Heights, MI).
Identification of side populations
Methods for identification of side populations were adapted from previous reports [26,27]. Briefly, cells were incubated with 5 μg/ml Hoechst 33342 dye (with or without verapamil (MP Biomedicals, Eschwege, Germany)) for 90 min, rinsed in PBS and centrifuged at 149x g for 5 min. Cells were resuspended in PBS with 2 μg/ml propidium iodide (Sigma). Cell samples (mouse tumor cells and WERI-Rb27 cells) were analyzed at the Roswell Park UV cell sorter facility using a FACSVantage cell sorter with Turbosort with UV capabilities and CellQuest software for data acquisition and analysis. An Argon laser (333-363 nm) was used to excite the Hoechst dye. Fluorescence emission was collected with a 405/30 nm band pass filter for Hoechst blue and a 660 nm ALP (long pass filter) for Hoechst red. Dead cells were excluded by propidium iodide fluorescence at 564-606 nm. The side population "SP" was identified as a group of cells able to exclude the Hoechst dye, a characteristic abolished with 50 μM verapamil treatment .
RB side populations (SPs) detected by flow cytometry
In previous studies, using a novel microtiter assay, we have shown verapamil-sensitive Hoechst 33342 dye uptake in WERI-Rb27 cells, Y79 cells, and mouse tumor cells, but not NIH 3T3 mouse fibroblast cells . In this study, we examined cells from mouse tumors, and WERI-Rb27 cells for the presence of side populations by flow cytometry. As seen in Figure 1, a small, but persistent number of cells (0.3% for the mouse tumor, 0.1% for WERI-Rb27) displayed uptake of Hoechst 3342 dye that was abolished by the addition of 50 μM verapamil, the definition of a side population . As seen in Figure 1, boxes are drawn that bound the area of the side population.
ABCG2 staining co-localizes with Hoechst-dim cells in RB
The presence of side populations in RB cell cultures, as seen in Figure 1, led us to examine the co-localization of Hoechst-dim cells with ABCG2 immunoreactive cells in SV40 β-LH-T-Ag mouse tumors, WERI-Rb27, and Y79 cells. If, in fact, ABCG2 confers the ability to exclude Hoechst dye, one would predict that cells immunoreactive for ABCG2 would necessarily be Hoechst-dim. This result is shown in Figure 2. In each case, cells that were ABCG2 bright (less than 1% of the population, Figure 2A,E,I), were Hoechst-dim (Figure 2B,F,J), as seen definitively in the merged image (Figure 2C,G,K). Brightfield images were captured (Figure 2D,H,L) to ensure that the labeled cells appeared healthy and intact. It was important to determine cell morphology, as less intact cells occasionally took up the TRITC-conjugated secondary antibody in a nonspecific manner.
ALDH1 staining co-localizes with Hoechst-dim cells in RB
Aldehyde dehydrogenases (ALDHs) are intracellular enzymes that oxidize aldehydes to carboxylic acids . Measures of ALDH1 activity have been used successfully to enrich for hematopoetic progenitor populations . We stained WERI-Rb27 and Y79 cells to detect the presence of ALDH1-immunopositive/Hoechst dim cells that would further indicate a potential cancer stem cell phenotype. In each case, cells that were ALDH1 bright (less than 1% of the population, Figure 3A,E), were Hoechst-dim (Figure 3B,F), as seen more definitively in the merged image (Figure 3C,G). Brightfield images were captured (Figure 3D,H) to ensure that the labeled cells appeared healthy and intact.
Subpopulations of mouse RB cells are immunoreactive to stem cell markers
We examined immunoreactivity of stem cell and progenitor markers in frozen sections of SV40 βLH-T-Ag mouse tumors (Figure 4). A negative control panel (isotype control serum) is shown in Figure 4A. Stem cell/progenitor cell markers were detected immunohistochemically in small numbers of mouse RB tumor cells (less than 1%). These markers included: ALDH1 (Aldehyde dehydrogenase-1, Figure 4B), SCA-1 (mouse-specific stem cell antigen, Figure 4C) , the chemoresistance marker ABCG2 (Figure 4D), and p63 (Figure 4E), a marker expressed by corneal limbal epithelial stem cells . It is important to note that there are six known isoforms of p63 with distinct subcellular localization patterns (nuclear versus cytoplasmic) and different antibody specificities. The particular antibody that we used from BD Biosciences is clone 4A4, a mouse IgG2A made against the N-terminal portion (amino acids 1-65) of a p63-GST fusion protein that cross reacts with both human and mouse p63. This antibody detects both nuclear and cytoplasmic portions of the protein. In all cases, these markers were observed in frozen sections of mouse RB tumors, with less than 1% of the cells immunoreactive.
Subpopulations of cells in human RB tissues are immunoreactive to stem cell markers
We examined immunoreactivity of stem cell and progenitor markers in archival human retinoblastoma specimens (Figure 5). These markers included the stem cell/chemoresistance marker ABCG2 (Figure 5A,B) and ALDH1 (Aldehyde dehydrogenase-1, Figure 5C). Interestingly, ABCG2 immunoreactive cells were seen sporadically in the retina (Figure 5B). In both cases, the number of immunoreactive cells was less than 1% of the total number of cells.
Expression of MCM2 in RB
MCM2 (minichromosome maintenance protein 2) exhibits G1 phase-specific expression and is a useful marker for detecting slowly cycling putative neural stem cells in situ . MCM2 is correlated with poor prognosis in lung carcinoma . We examined archival specimens of human RB and detected an interesting pattern of MCM2 expression (Figure 6). MCM2 immunoreactive cells were seen in tumor tissues surrounding blood vessels (Figure 6A), adjacent to the optic nerve (Figure 6B), and sporadically within the retina itself, both in the inner nuclear layer and photoreceptor layer (Figure 6C-E).
One present challenge in the treatment of retinoblastoma is the incidence of metastatic or secondary tumors that reduce life span and quality of survival. Patients with germline retinoblastoma have a markedly increased frequency of secondary malignant neoplasms . Those who survive are at increased risk for developing additional malignancies at a rate of about 2% per year . Additional malignancies that occur after primary RB are believed to result from the Rb mutation itself, radiation-induced neoplasms, or seeding and invasion of metastatic tumor cells. We propose yet another possible, nonmutually exclusive, mechanism: The persistence of subpopulations of RB cancer stem cells that escape chemotherapy could cause metastasis. As evidence, we present a small subpopulation of RB cells that exhibit some characteristics of a cancer stem cell phenotype including the ABCG2 chemoresistance marker.
We found that expression of MCM2, a neural stem cell marker, is evident not only within the RB tumor itself, but sporadically within histologically unaffected areas of the retina in RB patients. Tumorigenic cells with characteristics similar to neural stem cells have been isolated from pediatric brain tumors . These cells were multipotent, self-renewing and able to produce proliferating neurospheres with the capacity to differentiate into neurons and glia. In a separate study , a cell derived from human brain tumors expressed the surface marker ABCG2, self-renewed, and could differentiate in culture to form tumor cells that resembled the original tumor. In addition, there was a positive correlation between self-renewal capacity and increasing aggressiveness of the original tumor. This may explain the poor prognosis of patients having MCM2 immunoreactivity in lung carcinomas , and MCM2 could be a similarly important clinical marker in RB, as well. Tumors containing a higher percentage of cancer stem cells may metastasize and spread more readily. As evidence for cell hierarchy within solid tumors, Al-Hajj et al.  injected human breast cancer cells into mice and identified a distinct subpopulation capable of generating heterogeneous primary and secondary tumors similar to the original pathology specimens. Their results suggested that the tumorigenic cells can both self-renew and form nontumorigenic cancer cells .
Despite the many years since they were first propagated in culture, Y79 and WERI-Rb27 cells appear to have retained their heterogeneity, with regard to cancer stem cell characteristics. As precedent, Kondo et al.  analyzed the regenerative and tumor formation potential of six well-known cell lines: C6 (rat glioma), MCF-7 (breast cancer), U-20 and SaOS-2 (osteosarcoma), B104 (rat neuroblastoma), and HeLa (adenocarcinoma, cultured over 50 years). They found that all but the U-20 and SaOS-2 cells contained side population cells. In the C6 glioma line, sorted SP cells could produce both new SP and non-SP cells in culture, produce neurons and glia in culture, and form tumors containing both glia and neurons, when injected into nude mice . It is unknown how long-term propagation may affect RB heterogeneity. Therefore, we included fresh RB mouse tumors, along with preserved human pathological specimens in our analysis.
In summary, mouse and human RB tumor cells contain a small subpopulation of cells that exhibit some cancer stem cell characteristics. Especially significant is the expression of ABCG2, a calcium-sensitive cell surface protein that not only acts to exclude Hoechst dye, but also confers resistance to over 20 different chemotherapeutic agents. Previous studies showed heterogeneous drug resistance marker immunoreactivity in pathology specimens of retinoblastoma, namely the multidrug resistance proteins (MDR)-P-glycoprotein (P-gp) and vault protein lung resistance protein (LRP) . The presence of these resistance markers, including now ABCG2, suggests the possibility of subpopulations of RB cells that can escape standard chemotherapeutic treatments. In this study, we describe specific stem cell characteristics in retinoblastoma, but we cannot yet assert that these are conclusively cancer stem cells. More studies are needed. Self-renewal experiments, using sorted cells, could be used to show that the isolated side population can eventually generate a population that resembles the original unsorted population. In addition, we are in the process of determining resistance to a panel of chemotherapy drugs, another hallmark of cancer stem cells. Future studies involving transplantation of sorted cells (with and without chemotherapy treatments) will address the issue of metastatic potential.
Major challenges have to be overcome before our understanding of cancer stem cells can be harnessed and translated to meaningful treatments for patients. These challenges include identifying the stem cell subpopulation, characterizing differentiation and tumor-forming potential, and deciding the best therapeutic strategy based upon these findings. The existence of even a small subpopulation of chemoresistant cancer stem cells in retinoblastoma would have a significant impact on therapeutic strategies necessary for tumor eradication.
The authors thank: David Sheedy of Roswell Park Cancer Institute for technical assistance; Dr. Daniel M. Albert for providing breeding pairs of the SV40 βLH-T-Ag mice. This work was supported by Fight for Sight (GMS) and NEI R01EY009412 (FGF), and an unrestricted departmental grant from Research to Prevent Blindness. Dr. Seigel is also supported by the Sybil Harrington Special Scholar Award from Research to Prevent Blindness. Dr. Gonzalez-Fernandez is the Elizabeth and Ira P. Ross Chair of Ophthalmic Pathology.
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