Carcinogenesis vol.32 no.1 pp.102–109, 2011 doi:10.1093/carcin/bgq214 Advance Access publication October 25, 2010
Hemangioblastomas might derive from neoplastic transformation of neural stem cells/progenitors in the specific niche Dexuan Ma1, Minmin Zhang1,2, Luping Chen1, Qisheng Tang2, Xuqun Tang2, Ying Mao1, and Liangfu Zhou1 1 Department of Neurosurgery and 2Division of Nephrology, Huashan Hospital, Fudan University, Wulumuqi Road 12#, Shanghai 200040, China To whom correspondence should be addressed. Department of Neurosurgery, Shanghai Medical College, Fudan University, Shanghai 200040, China. Tel: þ86 21 52887206; Fax: þ86 21 62492884; Email:
[email protected] Correspondence may also be addressed to Liangfu Zhou. Tel: þ86 21 52887206; Fax: þ86 21 62492884; Email:
[email protected]
Introduction Hemangioblastoma (HB), which represents 1.5–3% of all central nervous system (CNS) tumors (1,2), occurs exclusively in nervous system as a sporadic entity or a component tumor of von Hippel-Lindau (VHL) disease. Patients with sporadic HBs typically seek treatment at age 40– 50, whereas patients with VHL disease-related HBs seek treatment in their 20s or 30s (3). It has demonstrated that HBs in clinicopathology are benign neoplasms with characteristic and well-described histopathological features, including uncontrolled growth of vascular and stromal cells. Hitherto, the histogenesis/cytological origin of HBs still remain uncertain and controversial although World Health Organization classified it in a class of neoplasms related to the meninges in 2007 (4). Tumor-initiating cells (TICs) are characterized as existing in primary tumors and having the ability to reestablish the tumor heterogeneity. In the search for HB TICs, the stromal cells,the underlying neoplastic cells in HBs (5), were correlated with the progeny of neural stem cells (NSCs)/progenitors in some genes as well as protein expression such as neuron-specific enolase (NSE), S100b and glial fibrillary acidic protein (GFAP) etc. and were thought to arise from neuroectodermal differentiation (6). However, in light of that, there exists high vascularity in HBs and that blood vessels are mesodermal origin, investigators speculated that HB may come from the remnants of mesoderm or mesenchyma. Until recently, it has been confirmed that HBs are capable of forming blood islands or tumorlets (2,7). Hemangioblasts, comAbbreviations: CNS, central nervous system; ES, embryonic stem cells; FACS, fluorescence-activated cell sorting; GFAP, glial fibrillary acidic protein; HB, hemangioblastoma; NSC, neural stem cell; NSE, neuron-specific enolase; PBS, phosphate-buffered saline; SSEA1, stage-specific embryonic antigen-1; TIC, tumor-initiating cell; VHL, von Hippel-Lindau.
Material and methods Patients and tissue processing Twenty-eight patients who underwent resection of CNS HBs between November 2008 and March 2010 at the Department of Neurosurgery, Huashan Hospital, Shanghai Medical College, Fudan University (Shanghai, China) were recruited in the study. The patients’ information has been summarized in Table I. All the specimens were ultimately diagnosed by neuropathologists and seven patients met the diagnostic criteria for VHL (1) (Table I). Dissected fresh samples were washed three times in cold phosphate-buffered saline (PBS). One part of the tumor tissue for western blot analysis or immunohistochemistry were rapidly frozen in liquid nitrogen and then stored at 80°C or 4°C, respectively, until use; another part for cell culture was immediately performed within 0.5 h after surgical removal. Research was performed under an institutional review board approved protocol (KY2009-313) and informed consent was obtained from all patients. Western blot analysis The samples were washed twice with PBS and then lysed in lysis buffer (Sigma, St. Louis, MO) including homogenizing process in tissue samples. Cellular proteins were collected and measured using a BCA protein assay kit (Bio-Rad, Hercules, CA). The protein was electrophoresized and then transferred to Hybond polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Japan) using a semidry system. After blocking, blots were, respectively, incubated in goat anti-Nanog and Oct-3/4, mouse anti-alkaline phosphatase and SSEA1 (1:3000; R&D Systems), rabbit anti-human nestin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-b-actin (1:10 000; Sigma) overnight at 4°C. Membranes were rinsed three times, incubated with the horseradish peroxidase-conjugated anti-goat, rabbit or mouse IgG (1:10 000; Sigma) for 1 h at room temperature and developed using ECL western blotting detection reagents (Amersham Pharmacia Biotech). Cell culture and fluorescence-activated cell sorting analysis Dissected fresh tumor tissues were washed with cold PBS, cut into the 0.5 0.5 0.5 mm3 pieces and incubated in ACK Lysis Solution (Invitrogen, Carlsbad, CA) in order to remove red blood cells and subsequently cultured in Dulbecco’s modified Eagle’s medium/F12 medium (Gibco, Gaithersburg, MD), supplemented with penicillin/streptomycin sulfate, N2 and B27 supplements (Invitrogen) as well as human recombinant fibroblast growth factor-2 (20 ng/ml; Chemicon, Germany), epidermal growth factor (25 ng/ ml; Chemicon). The cultures were incubated at 37°C in a humidified 5% CO2 incubator.
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The cytological origin of central nervous system hemangioblastoma (HB) remains unclear and controversial, largely owing to a lack of in-depth characterization of tumorigenic cells and their progeny tracking. We have now detected a cell subpopulation by stage-specific embryonic antigen-1 expression, which were defined as tumor-initiating cells (TICs) in both sporadic and familial HBs. These TICs subpopulations had universal neural stem cell characteristics. Nevertheless, the freshly sorted TICs endowed with potential of multi-progeny derivatives, including HB components and non-HB ingredients, depended on environmental induction in vitro. Importantly, the freshly harvested TICs formed malignant tumors by injection into conventional mice model, while did redevelop the characteristic HB-like structures within a special mice model with HB-microenvironment, indicating HB niche dependency for the TICs derivative specification. Taken together, the data of the present study suggested that HBs might derive from neoplastic transformation of neural stem cells/progenitors in the specific niche.
mon precursors of hematopoietic and endothelial cells, have been detected and characterized as neoplastic cells in HBs (8,9); further, they were committed to initiate in the period of the primitive streak in the mouse embryo (10). Thus, they were deemed to derive from ‘embryologically arrested stem cells’. However, these assumptions or inference either are restricted in the stromal cells (not explain vasculogenesis in HBs) or built on normal developmental mechanisms of vascular system, which lacked the support of direct evidence. Stage-specific embryonic antigen-1 (SSEA1) is a carbohydrate adhesion molecule distributed in adult human CNS (11,12). Although SSEA1 is a well-established marker of mouse embryonic stem (ES) cells, it is not expressed in human ES. Further, there is evidence that human embryonic tumors (13,14) and mesenchymal tumors, both benign and malignant, were unreactive for SSEA1 (15). The expression of SSEA1 exists in the differentiated stage of human ES (15–17) and follows a timely ordered pattern during the development of neural cells of certain brain areas (11,12), thus SSEA1 can act as a marker for both neural stem cells/progenitors and brain TICs (18,19). In this study, we have identified in HB tissues a subpopulation of SSEA1-positive (SSEA1þ) cells, which have the ability of differentiating into the stromal-like cells and vascular cells, these potential of multi-progeny derivatives, however, preserved only in the presence of HB niche. Based on the existing literatures, we propose to use reconstructing HB niche experiments to verify our hypothesis that SSEA1þ cells could be the TICs of HBs.
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Table I. Summary of patients’ information, tumor characteristics and number of TICs
17 11 37.6 15–69 21 4 3 20 8 21a 7a
28.3 ± 8.7; 37 ± 9.5 (5) 21.5 ± 7.6; 41.6 ± 10.2 (3) 11.7 ± 6.3; 33 ± 7.6 (4) 12.4; 25.9 (1)
a
Five cases had the family history; two cases were recurrent tumors; three cases had two or more multiple lesions (including extracranial lesions in two cases). b The other 15 samples, particular in cystic specimens, were too small and were applied in immunohistochemistry, western blotting, primary culture or primary tissue implantation in animal only.
Non-adherent spherical clusters of cells (tumorspheres) in culture were selected and dissociated into single-cell suspensions after enzymatic dissociation. The supernatant containing liberated cells was collected and washed with PBS, and the cells at a density of 1 106 cells/ml were isolated by the fluorescence-activated cell sorting (FACS) Vantage SE flow cytometer (BD Biosciences, Japan) with the following antibodies: anti-CD133-PE (1:500; eBioscience, San Diego, California), anti-SSEA1-fluorescein isothiocyanate (1:800; eBioscience). The sorted cells were cultured under the identical culture conditions and also passaged by trituration through a fire-polished pipette and reseeded into fresh proliferative medium. As control, the human ES line (HuES-14) gifted by the Harvard Office of Technology Development of Harvard University (20) was cultured according to the Instruction Manual. Induction of differentiation and Matrigel-based capillary formation assay For induction of differentiation, cells were cultured in Neurobasal media without growth factors or Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal bovine serum. Uncoated plastic dishes were used for tumorsphere culture assays. For adherent culture, the plates were precoated with polyornithine/laminin mixture. Capillary-like formation was analyzed as described in ref. 21. Briefly, 48well plates were coated with 100 ll of Matrigel (BD Biosciences) and incubated 30 min at 37°C. Serum-starved prospective cells (1 105) were plated on solidified Matrigel in 300 ll of serum-free medium. Capillary-like structures were recorded for a period of 48 h and representative images taken 24 h after plating were shown. Reconstruction of HB niche in vitro and in vivo Primary culture for HBs in vitro was described as above. Cocultures for both the labeled putative TICs and HB niche were performed in order to ensure the interaction between the TICs and the other cells in tumor, as well as the tumor receptor-mediated recognition. Primary tumor specimen implantation was applied for establishment of HB tumorigenicity. Simply, samples of human HBs were obtained within 30 min after surgical resection. Tumors were suspended in sterile Dulbecco’s modified Eagle’s medium/F12 and mechanically dissociated using scissors and then minced with a sterile scalpel blade over ice to yield 0.5 0.5 0.5 cm3 pieces. After anesthetizes, A 1 cm incision was made in the skin at the right armpit of
Lineage tracking and derivative specification of the putative TICs within HB microenvironment The sorted SSEA1þ cells were harvested and labeled with PKH26 (Sigma) according to the manufacturer’s instructions and were subsequently applied for lineage tracking in vitro and in vivo. Briefly, the SSEA1þ cells were suspended in diluent C at a density of 2 106 cells/ml and immediately mixed with an equal volume of a 2 PKH26 stock solution in diluent C and then further incubated at room temperature. After 10 min, an equal volume of fetal bovine serum was added to stop the staining reaction. Labeled cells were diluted with culture medium containing serum and washed three times. Cell viability was .95%, as determined by Trypan blue exclusion. As control, the unlabeled SSEAþ cells, SSEAcells or human ES at a density of 1 105 cells/ml were subcutaneously injected into nude mice without HB tissue tumorigenicity, respectively. Similarly, the labeled SSEA1þ cells (1 105) also injected into the established model with the implanted human HB specimens. Five cases were performed this way, and the labeled cells were found in the sections of all five cases. The pathological results of tumors generated in animals were also analyzed. There was no implantation or injection procedure-related animal lethality. The animals were killed at given time points (primary tumor xenografts 6 weeks after subcutaneous transplantation and 4 weeks after the labeled SSEA1 cell injection) for the analysis of tumor histology and immunohistochemistry. Cell and tissue staining Cells were plated in poly-D-lysine-coated chamber slides for 6 h prior to fixation in 4% paraformaldehyde and blocked with appropriate normal serum. We used the following primary antibodies: mouse anti-SSEA1 (1:500; R&D Systems), rabbit anti-nestin (1:200; Santa Cruz), rabbit anti-Ki67 (1:200; Abcam, Cambridge, MA), rabbit anti-GFAP (1:1000; sigma), mouse anti-S-100b (1:800; sigma) or NSE (1:200; Abcam). For staining of frozen tissue, the samples were fixed in 4% paraformaldehyde, cryoprotected in 25% sucrose, embedded in optimum cutting temperature and sectioned at a thickness of 10 mm. Sections were permeabilized with Tris-buffered saline containing 0.05% Tween 20, blocked with Tris-buffered saline containing 0.05% Tween 20 þ 10% normal serum. The following primary antibodies were used: mouse anti-SSEA1 (1:500; R&D Systems), rabbit anti-inhibin-a (1:200; Santa Cruz), goat anti-CD31 (1:200; Santa Cruz) or mouse anti-smooth muscle antibody (1:500; Sigma). Primary antibodies were visualized by appropriate secondary antibodies conjugated to Alexa Fluor 488, 546 (Molecular probes, Eugene, OR). All sections were stained 40, 60-diamidino-2-phenylindole (Sigma). For double staining, the respective primary antibodies were from different species and were applied sequentially to minimize the probability of cross-reactivity. Negative control experiments were done by omitting the primary antibody, and these revealed no positive staining (data not shown). For staining of paraffin sections, tissue was fixed overnight in 10% formalin, paraffin embedded and sectioned at a thickness of 5 lm. Sections were deparaffinized and rehydrated and stained with hematoxylin and eosin (H&E) as well as mouse anti-SSEA1 (1:500; R&D Systems). The staining was visualized using an avidin–biotin–peroxidase complex (ABC-Elite; Vector Labs, Burlingame, CA). Peroxidase activity was revealed by diaminobenzidine tetrahydrochloride.
Results Expression of SSEA1 in primary human HB tissue and their putative TICs To ascertain whether there is a small subpopulation of distinct TICsthat are responsible for tumor formation, we detected the expression of well-known markers. In this study, immunohistochemistry showed that there was expression of SSEA1 in both sporadic (A) and inherited HBs (B) of all tested 18 samples. To determine whether SSEA1 expression is specific for both sporadic and inherited HBs, we performed western blot using HB tissues, human ES and normal brain tissues. Western blot analysis demonstrated that ES expressed Nanog, Oct-3/4 as well as alkaline phosphatase and some markers of human ES; whereas HB tissues (five samples in sporadic and inherited HBs, respectively) expressed SSEA1, nestin only (Figure 1D). Meanwhile, In agreement with previous results (11,12), SSEA1 was also detected in human cerebellar tissue (two samples) (Figure 1D). These data indicated that SSEA1þ cells in both sporadic and inherited HBs might
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Patient and tumor characteristics of HBs Gender Male Female Age (years) Mean Range Location Cerebellum Brainstem Spinal cord Cystic Solid Sporadic Hereditary 13 tested samples for cell sortingb SSEA1þ %; CD133þ % (number of cases) Solid tumors Sporadic HBs Hereditary HBs Cystic tumors Sporadic HBs Hereditary HBs
nude mice, and three pieces of xenografts were implanted. The skin incision was closed with absorbable suture. The tumor growth in the right armpit was pathologically examined weekly at the maximum duration of 12 weeks.
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represent the neuroectodermal cells similar to those of normal human brain. Interestingly, we found that inhibin-a, a marker of the non-glial tumor (22–24), was expressed in the stromal cells of CNS HBs and appeared in a small subset of SSEA1þ cells (,6%) in both types of HBs (Figure 1C). Primary culture from human HBs exhibits the characteristic phenotypes To investigate whether there are any TICs and their evolving process within human HBs, we primarily cultured tumor cells from post-surgery specimens of both sporadic and inherited HBs and plated the cells in serum-free medium, containing epidermal growth factor and fibroblast growth factor-2.
Characterization and isolation of the putative TICs from human HBs In order to determine the characterization of the tumorspheres, we performed immunohistochemical analysis. We intriguingly found that there were many SSEA1þ cells and they expressed nestin and Ki67 (Figure 3A and B), a proliferation marker expressed in all phases of the active cell cycle. The Ki67 was primarily restricted to SSEA1þ cells within the tumorspheres (Figure 3B). The Ki67 index, which is thought to correlate with tumor proliferation, was 46–73% and 3–5% in the tumorspheres and patient paraffin sections, respectively (data not shown). These data showed that SSEA1 might represent a potential TIC enrichment marker applicable to both sporadic and inherited HBs. To evaluate the presence of the putative TICs within human HBs, we first determined the expression of SSEA1 and/or CD133 by FACS analysis in acutely isolated HB tumor cells in vitro. Acutely isolated cells from 13 tested HBs contained distinct CD133þ and/or SSEA1þ populations. The percentage of CD133þ cells ranged 17.5–48.3%, whereas those of SSEA1þ cells run 8.9–33.6%. Interestingly, there is significant overlap between two subpopulation cells in both sporadic and familial HBs (Figure 3C and D), and the percentage of
Fig. 2. Primary culture from human HBs exhibits the characteristic phenotypes. Primary culture for HBs displayed non-adherent and adherent forms (A). Non-adherent tumorspheres revealed appearance of differentiated morphology along with expansion and scattering outside (B and C). Moreover, at the earlier phase, these scattering and differentiated cells revealed different phenotypes, including cytoplasmic vacuolization (lipid-laden cytoplasm) (D), a typical feature of underlying neoplastic stromal cells, as well as atypical tube structure (E). However, all cells ultimately lost these characteristics after long culture or several passages in vitro (F); scale bar 5 50 lm. Magnification 100 (A); 400 (B and C); 200 (D–F).
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Fig. 1. Expression of SSEA1 in primary human HB tissue and their putative TICs. Positive immunohistochemistry for SSEA1 was observed in the section of both sporadic (A, 400) and familial hemangioblastomas (B, 400). Double labeling of SSEA1 (C1), DAPI (C2) and inhibitn-a (C3) showed that inhibitn-a immunoreactivity was observed in a small subset SSEA1þ cells (C). Western blot analysis (D) demonstrated that embryonic stem cell line expressed Nanog, Oct-3/4 as well as alkaline phosphatase, not SSEA1, whereas HB tissue did in contrast. Meanwhile, SSEA1 was also detected in human normal cerebral tissue; scale bar 5 10 lm.
A number of non-adherent spherical clusters of cells named tumorspheres were observed in all 13 tested tumor samples in 7–10 days and the cell number in spheres increased with increased incubation. The tumorspheres displayed a free-floating structure in culture (Figure 2A). However, after 15–20 days, the peripheral cells of the tumorspheres became adherent with appearance of differentiated morphology (Figure 2B and C). Moreover, these adherent cells revealed different phenotypes at the earlier phase along with cells expansion and scattering, these phenotypes include cytoplasmic vacuolization (lipid-laden cytoplasm), a typical feature of underlying neoplastic stromal cells (Figure 2D), as well as atypical tube structure (Figure 2E). The data suggested that the underlying neoplastic cells, stromal cells and vasculars in HBs may come from the tumorsphere-like cells. Interestingly, the two types of the stromal-like cells and the atypical tube structure were discrete and not miscellaneous in the culture dishes. Although, we were able to maintain the state of these tumorspheres with their proliferation for a long period of time (,2–3 months) under the medium, it is unnecessary for the differentiation of the stromallike cells and the atypical tube structures. The data suggested that the committed differentiation might have nothing to do with the conventional medium. Finally, all cells lose these characteristics when longterm culture in vitro (3–5 months) under identical culture conditions (Figure 2F).
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SSEA1þ and CD133þ cells were widely variable among different individuals (Table I). Differentiation potential of the putative TICs from human HBs It has been demonstrated that cocultured stem cells sometimes take on the characteristics of the cell type with which they are cultured. Because TICs interact with other cells within the microenvironments and each profoundly influences the behavior of the other, we reproduced this proximity in vitro by coculturing labeling putative TICs with HB niche, to determine whether it could duplicate the conversion of TICs to tumor-like cells of HBs. SSEA1 and/or CD133-dependent cells sorting from the tumorspheres were performed using FACS, followed by labeling PKH26. All putative TICs (except for cell sorting) or primary HBs used in these experiments were not passaged in vitro, minimizing the possibility that alteration in nature of TICs or microenvironment disruption would occur and affect the outcome. Coculturing PKH26-labeled SSEA1þ cells and HB niche showed that the labeled SSEA1þ cells could be induced to adopt a stromal-like cell phenotype by displaying a typical feature of cytoplasmic vacuolization (Figure 4A). In contrast, the similar result could not be observed in CD133þ/SSEA1 cells (data not shown). These data suggested that the underlying neoplastic stromal cells may derive from SSEA1þ subpopulation. Consistent with the previous results, both SSEAþ cells and CD133þ cells could not form typical tube structures. To further investigate whether the putative TICs have potential of forming capillary-like structures, we performed Matrigel-based capillary formation assay. Strikingly, CD133þ/SSEA1 (Figure 4B), CD133þ/SSEA1þ (Figure 4C) and CD133/SSEA1þ cells (Figure 4D) could form tube structures though there are some differences in morphology among three structures, especially for SSEA1þ/ CD133þ population (Figure 4C). These data indicated that the putative TICs from the tumorspheres had the ability of forming vasculars and they might depend on environmental induction.
Next, we wondered whether the putative TICs have other differentiation potential or what is the difference between the putative TICs and NSCs/progenitors. To address the question, we cultured the putative TICs within the absence of fibroblast growth factor-2/epidermal growth factor or in the presence of serum. Freshly isolated SSEA1þ cells underwent significant morphological changes, including extension of processes and spread-out cell shapes. In order to ascertain the fate of the putative TICs under inducing differentiation, we performed these cells for immunocytochemical analysis. The differentiation conditions induced significantly increased expression of the astroglial differentiation markers GFAP/S100b and/or the neuronal marker NSE (Figure 4E–G). The data suggested that SSEAþ cells from human HBs had a differentiation potential for neural progeny similar to normal neural stem cells. Tumorigenic potential of SSEA1þ cells in mice transplantation model A most effective tool in TICs research would be tumorigenic experiments in animal model. To assess whether the subset of SSEA1þ cells is enriched for tumorigenic potential in vivo, we injected acutely isolated SSEA1þ cells and SSEA1 from fresh HBs into nude mice. Cells from eight different HBs were evaluated for tumorigenic potential of SSEA1þ and SSEA1 cells subpopulations. Unexpectedly, acutely isolated SSEA1þ cells generated three malignant tumors with the similar features in pathomorphology (3/8) (Figure 5A), whereas SSEA1 cells failed to generate tumors (0/8) (data not shown), suggesting that tumorigenicity of SSEA1þ cells were more efficient than SSEA1 cells under this situation. In addition, as control, human ES formed malignant teratomas (3/5) (Figure 5B). Taken together, the results indicated that the hostile microenvironment of animal could play a vital role for malignant neoplasm formation. To obtain HB niche, primary tumor specimen implantation was applied for this study because of the difficulty in routinely obtaining the tumor in nude mice by injecting single-cell suspensions from HBs.
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Fig. 3. Characterization and isolation of the putative TICs from human HBs. Immunochemical staining showed that SSEA1þ cells were predominant in numbers within the tumorspheres (A and B) and were immunoreactive for nestin, a neural stem/progenitor cell marker (A). Meanwhile, Ki67, a proliferation protein marker, was positive for most SSEA1þ cells (B). Flow cytometry analysis of SSEA1 (fluorescein isothiocyanate labeled, y-axis) and CD133 (phycoerythrin labeled, x-axis) in the putative TICs of both sporadic (C) and familial (D) hemangioblastomas. Numbers in each quadrate represent the percentage of cells.
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A total number of 10 human HB xenografts was established, seven from sporadic HBs and three from familial HBs. These xenografts retained many basic features of the primary HB in animal model (Figure 5C). To evaluate the fate of the putative TICs or sorted SSEA1þ cells from human HBs, we performed in vivo lineage-tracing experiments by using PKH26-labeled SSEA1þ cells. The labeled SSEA1þ cells from human HBs were injected into the HB niche of adult nude mice; the resultant tumors were demonstrated to sustain many of the features of the primary HB (Figure 5D). Importantly, immunohistochemical analysis using a human-specific antibody showed that the labeled SSEA1þ cells could not only differentiate into a-inhibinþ cells (Figure 5E) but also generate vascular endothelial cells (CD31þ) (Figure 5F) and vascular smooth muscle cells (SMAþ) (Figure 5G), which took part in vascular formation.
Discussion We have successfully isolated and identified a unique cell population from human HBs that, on the basis of a number of different criteria, demonstrated characteristics of neural stem-like cells. With PKH26 labeling-tracking assay that has been used to identify the putative TICs in both sporadic and familial HBs, we showed that TICs harbor tumorigenic potential within an HB niche that was well-established advance, indicative of that the specific environment may play a critical and major role in regulating and controlling TICs fate. In contrast, the malignant tumors generated when these TICs were injected into a non-HB niche in nude mice. Therefore, we provided evidence that
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detailed understanding of association between TICs and its niche in HBs, might be an important step to clarify the mechanisms of the initiation, propagation and recurrence in HBs. The origin of the cells that initiate and propagate HBs has been a subject of debate for many years. Little is known about the tumorigenic cells and the crucial factors that control HB formation and maintenance, largely because tumorigenic cells have been poorly isolated, characterized and identified. One purpose of our study was to identify such markers; for this, we used a combination of western blotting, immunocytochemistry and FACS analysis. Western blotting demonstrated that some markers of ES, such as Nanog, Oct-3/4 and alkaline phosphatase, were not expressed in the cells of HBs. Although CD133, a universal TICs enrichment marker for brain tumors, is also present in both sporadic and inherited HBs, however, its expression patterns were not suitable for identifying the cytological source because it is also expressed in hematopoietic stem cells, endothelial progenitor cells, neural stem cells and some other cell types, which represent multi-germinal layer origin (25,26). Intriguingly, we found and isolated a unique cell population by expression of the cell surface markers SSEA1. These cells not only displayed universal stem cell characteristics, such as subclonogenicity, multipotency and self-renewal capacity, but also harbored a potential of stromal cells and vasculogenesis (neovascularization not angiogenesis), the typical features of HB, after extended expansion in vitro and transplantation in vivo. HB tissues expressed neural ectodermrelated factors, such as SSEA1 and nestin. In addition, there are evidence that SSEA1 is absent in human ES and embryonic carcinomas (13,14), as well as mesenchymal tumors, both benign and
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Fig. 4. Differentiation potential of SSEA1þ cells form human HBs. Coculturing PKH26-labeled SSEA1þ cells and HB niche showed that the labeling putative TICs displayed cytoplasmic vacuolization, a typical characteristic of stromal cells, similar to unlabeling cells (A). Matrigel formation assay of SSEA1/CD133þ (B), SSEA1þ/CD133þ (C) and SSEA1þ/CD133 cells (D) revealed that they could generate a type of tube-structure, especially for SSEA1þ/CD133þ population (C). Under inducing differentiation condition, SSEA1þ cells could differentiate into the cells expressing GFAP (E) and S100b (F), as well as NSE (G) by immunofluorescence. Scale bar 5 50 lm (A–D); 20 lm (E–G).
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malignant neoplasm (15). These data suggested that HBs were closely related to neoplastic transformation of neural stem-like cells, but not identical to polyembryonic origin. There are innumerable similarities between brain TICs and adult NSCs/progenitors (27). Two kinds of cells shared common multiple cell surface markers, are able to form neurospheres when cultured in vitro, and have multipotent progeny. In this study, we also found that SSEA1 is expressed in normal brain as control. Moreover, in both types of HBs, a small subset of SSEA1þ cells was positive for inhibin-a. Significantly, SSEA1þ cells could be induced into SSEA1 cells including vascular cells and the stromal-like cells; this indicated that SSEA1 antigenic determinants may be lost during neoplastic transformation (14). Taken together, the data suggested that neural epithelial marker SSEA1 is lost and stromal cell marker inhibin-a is gained in the cells, indicative of dynamic evolutional process. The results support the prevalent theory on the origin of brain tumor and assert that they are a result of transformation from NSCs/progenitors (28,29). Additionally, inconsistent with the previous hypothesis that brain TICs may derive from mutation of adult stem cells, we showed that the sorted SSEA1þ cells from HBs in vitro could abduct into neural cells by expression of GFAP, S100b and NSE, whereas lack of cytoplasmic vacuolization (lipid-laden cytoplasm), a typical feature of stromal cells of HB. Furthermore, injection of SSEA1þ cells into the nude mice resulted in malignant tumor formation. Taken together, these results in fact raised the possibility that the fates of the putative TICs reflect an environmental response rather than intrinsic property only, at least in HBs.
The existence of high vascularity in HB has bewildered its cytological origin. Hitherto, scientific discovery in HB almost never provided unequivocal answer regarding this issue. The previous assumptions or inference on the ground of normal vascular development also lacked support of direct evidence. Moreover, the vascular architecture in brain tumor also lose normal characteristics of the brain vasculature, such as the blood-brain barrier because brain tumor growth generates hypoxic signals and vascular endothelial growth factor production (30), these information indicated that there existed different latent mechanism for between neovascularization in adult brain tumor and vasculogenesis in embryologic development or adult angiogenesis and not yet identical. With the discovery of transdifferentiation phenomenon defined as an irreversible switch in postnatal life of one differentiated cell to another, it has broken down the conventional framework of the committed differentiation process. Transdifferentiation, which occurs during certain physiological processes and leads to transition of tumor cell phenotypes in the development of neoplasia, is a type of metaplasia, and sometimes refer to the origination of the tumor initiative cells from differentiated cells (31). There is gradual accumulative evidence that involves the ability of neural precursor cells of ectodermal origin to transdifferentiate into mesodermal cell types (32–34). This phenomenon has been termed transformation or epithelial–mesenchymal transition in tumors (35). Accordingly, it becomes so interesting and important to isolate TICs and track their progeny fate. With advent of dilemma during putative tumor stem cells advancement, the tumor niche that harbors TICs and provides the necessary factors to maintain its stemness and specify cell-fate determination is
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Fig. 5. Tumor formation in nude mice injected with sorted SSEA1þ cells from human HBs. Malignant tumor formation (A) was found in a mouse at the injection site of SSEA1þ cell suspensions, whereas malignant teratomas (B) were seen in human ES cells as control. Primary tumor specimen implantation revealed similar histologic features to the corresponding patient’s HBs (C). The same results were present in PKH26-labeled SSEA1þ cells injection after primary tumor specimen implantation (D). Immunostaining showed that PKH26-labeled SSEA1þ cells expressed inhibin-a (E), CD31 (F) and SMA (G). Magnification 400 (A–D); scale bar 5 50 lm.
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Funding National Natural Science Foundation of China (30973104 to L.Z., 30973091 to D.M.); China Postdoctoral Science Foundation funded project (200902205 to D.M.).
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Acknowledgements The authors thank Harvard Office of Technology Development of Harvard University for human ES line (HuES-14) and Prof. Lei Xiao of Chinese Academy of Sciences as well as Dr Hongying Sha of fudan University for technical assistance. Conflict of Interest Statement: None declared.
References 1. Lonser,R.R. et al. (2003) von Hippel-Lindau disease. Lancet, 361, 2059–2067. 2. Hussein,M.R. (2007) Central nervous system capillary haemangioblastoma: the pathologist’s viewpoint. Int. J. Exp. Pathol., 88, 311–324. 3. Vater,G.E. (2005) Hemangioblastoma. In Berger,M.S. and Prados,M.D. (eds.) Neuro-oncology. Elsevier Saunders, Philadelphia, PA, pp. 294–300. 4. Louis,D.N. et al. (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol., 114, 97–109. 5. Vortmeyer,A.O. et al. (1997) von Hippel-Lindau gene deletion detected in the stromal cell component of a cerebellar hemangioblastoma associated with von Hippel-Lindau disease. Hum. Pathol., 28, 540–543. 6. Ishizawa,K. et al. (2005) Stromal cells in hemangioblastoma: neuroectodermal differentiation and morphological similarities to ependymoma. Pathol. Int., 55, 377–385. 7. Vortmeyer,A.O. et al. (2003) Developmental arrest of angioblastic lineage initiates tumorigenesis in von Hippel-Lindau disease. Cancer Res., 63, 7051–7055. 8. Glasker,S. et al. (2006) Hemangioblastomas share protein expression with embryonal hemangioblast progenitor cell. Cancer Res., 66, 4167–4172. 9. Park,D.M. et al. (2007) von Hippel-Lindau disease-associated hemangioblastomas are derived from embryologic multipotent cells. PLoS Med., 4, e60. 10. Huber,T.L. et al. (2004) Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature, 432, 625–630. 11. Gocht,A. et al. (1996) CD15-containing glycoconjugates in the central nervous system. Histol. Histopathol., 11, 1007–1028. 12. Gocht,A. et al. (1992) The carbohydrate epitope 3-fucosyl-Nacetyllactosamine is developmentally regulated in the human cerebellum. Anat. Embryol. (Berl.), 186, 543–556. 13. Draper,J.S. et al. (2002) Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J. Anat., 200, 249–258. 14. Henderson,J.K. et al. (2002) Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells, 20, 329–337. 15. Fox,N. et al. (1983) Immunohistochemical localization of the mouse stagespecific embryonic antigen 1 in human tissues and tumors. Cancer Res., 43, 669–678. 16. Shamblott,M.J. et al. (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc. Natl Acad. Sci. USA, 95, 13726–13731. 17. Thomson,J.A. et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145–1147. 18. Panchision,D.M. et al. (2007) Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. Stem Cells, 25, 1560–1570. 19. Son,M.J. et al. (2009) SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell, 4, 440–452. 20. Cowan,C.A. et al. (2004) Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med., 350, 1353–1356. 21. Zhou,Z. et al. (1999) Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice. Angiogenesis, 3, 181–188. 22. Takei,H. et al. (2010) Novel immunohistochemical markers in the diagnosis of nonglial tumors of nervous system. Adv. Anat. Pathol., 17, 150–153. 23. Takei,H. et al. (2007) New immunohistochemical markers in the evaluation of central nervous system tumors: a review of 7 selected adult and pediatric brain tumors. Arch. Pathol. Lab. Med., 131, 234–241. 24. Hoang,M.P. et al. (2003) Inhibin alpha distinguishes hemangioblastoma from clear cell renal cell carcinoma. Am. J. Surg. Pathol., 27, 1152–1156. 25. Corbeil,D. et al. (2000) The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J. Biol. Chem., 275, 5512–5520. 26. Shmelkov,S.V. et al. (2005) AC133/CD133/Prominin-1. Int. J. Biochem. Cell Biol., 37, 715–719.
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beginning to be recognized and highlighted (36,37). Similar to the role of stem cells niche, increasing evidence has emerged that factors derived from the tumor niche serve to regulate and maintain tumor cells (38,39). In present study, primary culture for human HBs eventually lost its typical cytological characteristics and subclonogenicity of the putative TICs through several passages. We reasoned that longterm culture and passages in vitro may lead to disruption and impairment of the tumor niche. In addition, the utilization of in vivo transplantation, a previous gold standard for the unambiguous identification of TICs, especially in benign tumors, such as HBs, may confound with bewilderment because interspecies differences between mouse and human cells during tumor progression in the biology of oncogenic or cytological transformation are substantial (40) and the results of this assay might owe to high heterogeneity of the hostile microenvironment (41). Although VHL gene/protein among multifactors has already been fully researched and suggested association with inherited HB, epidemiological survey has demonstrated that the lack of pVHL protein is neither a sufficient condition nor an essential factor in the forming and development of HB. It would be reasonable to believe that VHL may be a predisposing factor only. Given HBs are the result of multi-genes or factors, it is of interest and necessity to reconstruct an HB niche in tracing the putative TICs fates because the pivotal factor(s) for HB initiation and progression still remain unknown until now. To address a potential role for tumor niche at the initiation of TICs transition, we cocultured the labeled putative TICs and HB niche in vitro because this method has been shown to retain receptor-mediated recognition and association of these cell types (34). Intriguingly, we found that labeled candidates can differentiate into the stromal-like cells, displaying the typical features of primary tumor. In the subsequent experiments, we injected the labeled prospective TICs into the established HB model. The validity of using primary tumor specimen implantation is supported by previous work, which showed that neoplasm xenografts in both benign angioblastomas and malignant cancers retain many of the features of the primary tumor (42,43). As a result, the labeled candidates not only differentiated into the stromal-like cells but also took part in vascular formation. These data indicated that HB niche, a three dimensional structure composed of cells, was important for the progeny fates determination of the putative TICs and that vascular formation may depend on this three dimensional structure of tumor niche instead of in vitro-expanded cells. In addition, we also found somewhat difference of cell morphology and proliferative rate among several niche. Taken together, these observations revealed new insight and pivotal roles for the tumor microenvironment in TICs initiation, propagation and recurrence. With establishment of conception that tumor is system biology disease (44,45), tumor is appreciated as not only a heterogenous pathology with respect to cell type and tissue origin but also as a disease involving dysregulation of multiple pathways governing fundamental cell processes (45). The previous effective method by sorting putative TICs required for malignant tumor taken in immunodeficient mouse might not be suitable for benign tumor, at least for HBs. This is because that the benign tumor is a low heterogeneity in pathology and that it has never been found metastasis of HBs. Furthermore, there is no report of HBs in animal models. The present study could provide a possibility by solving a key limitation in reconstructing HB niche to reproduce the process of HB’s formation in vitro and in vivo. However, this method only generates an approximate simulation because it is impossible to copy primary niche in animal models except for human. In this sense, almost all tumors are not clonal (as Suzanne Kadereit pointed out). Therefore, whether our study proximally imitates the process of HB initiation and progression and whether other cell(s) also take part in its formation remain to be further investigated in future.
The origin of hemangioblastomas
27. Zaidi,H.A. et al. (2009) Origins and clinical implications of the brain tumor stem cell hypothesis. J. Neurooncol., 93, 49–60. 28. Singh,S.K. et al. (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res., 63, 5821–5828. 29. Hemmati,H.D. et al. (2003) Cancerous stem cells can arise from pediatric brain tumors. Proc. Natl Acad. Sci. USA, 100, 15178–15183. 30. Kim,W.Y. et al. (2009) Brain angiogenesis in developmental and pathological processes: mechanism and therapeutic intervention in brain tumors. FEBS J., 276, 4653–4664. 31. Thowfeequ,S. et al. (2007) Transdifferentiation in developmental biology, disease, and in therapy. Dev. Dyn., 236, 3208–3217. 32. Stocum,D.L. (2002) Development. A tail of transdifferentiation. Science, 298, 1901–1903. 33. Wurmser,A.E. et al. (2004) Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature, 430, 350–356. 34. Vescovi,A. et al. (2002) Neural stem cells: plasticity and their transdifferentiation potential. Cells Tissues Organs, 171, 64–76. 35. Micalizzi,D.S. et al. (2009) Epithelial-mesenchymal transition in development and cancer. Future Oncol., 5, 1129–1143. 36. McGovern,M. et al. (2009) A ‘‘latent niche’’ mechanism for tumor initiation. Proc. Natl Acad. Sci. USA, 106, 11617–11622.
37. Witz,I.P. (2009) The tumor microenvironment: the making of a paradigm. Cancer Microenviron., 2 1 (suppl. 1), 9–17. 38. Kortylewski,M. et al. (2009) Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment. Cancer Cell, 15, 114–123. 39. Milsom,C. et al. (2009) Tissue factor and cancer stem cells: is there a linkage? Arterioscler. Thromb. Vasc. Biol., 29, 2005–2014. 40. Rangarajan,A. et al. (2004) Species- and cell type-specific requirements for cellular transformation. Cancer Cell, 6, 171–183. 41. Hill,R.P. (2006) Identifying cancer stem cells in solid tumors: case not proven. Cancer Res., 66, 1891–1895discussion 1890. 42. Tang,Y. et al. (2007) A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice. Plast. Reconstr. Surg., 120, 869–878. 43. Li,C. et al. (2007) Identification of pancreatic cancer stem cells. Cancer Res., 67, 1030–1037. 44. Hornberg,J.J. et al. (2006) Cancer: a systems biology disease. Biosystems, 83, 81–90. 45. Kreeger,P.K. et al. (2010) Cancer systems biology: a network modeling perspective. Carcinogenesis, 31, 2–8. Received July 23, 2010; revised October 7, 2010; accepted October 13, 2010
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