Oct-4+/Tenascin C+ neuroblastoma cells serve as progenitors of tumor-derived endothelial cells

Pezzolo, Annalisa; Parodi, Federica; Marimpietri, Danilo; Raffaghello, Lizzia; Cocco, Claudia; Pistorio, Angela; Mosconi, Manuela; Gambini, Claudio; Cilli, Michele; Deaglio, Silvia; Malavasi, Fabio; Pistoia, Vito

doi:10.1038/cr.2011.38

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Original Article
Published: 15 March 2011

Oct-4⁺/Tenascin C⁺ neuroblastoma cells serve as progenitors of tumor-derived endothelial cells

Annalisa Pezzolo¹,
Federica Parodi¹,
Danilo Marimpietri¹,
Lizzia Raffaghello¹,
Claudia Cocco²,
Angela Pistorio³,
Manuela Mosconi⁴,
Claudio Gambini⁴,
Michele Cilli⁵,
Silvia Deaglio⁶,
Fabio Malavasi⁶ &
…
Vito Pistoia¹

Cell Research volume 21, pages 1470–1486 (2011)Cite this article

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Abstract

Neuroblastoma (NB)-associated endothelial microvessels (EMs) may be lined by tumor-derived endothelial cells (TECs), that are genetically unstable and chemoresistant. Here we have addressed the identification of TEC progenitors in NB by focusing on Octamer-binding transcription factor 4 (Oct-4) as a putative marker. Oct-4⁺ cells were detected in primary NB samples (n = 23), metastatic bone marrow aspirates (n = 10), NB cell lines (n = 4), and orthotopic tumors (n = 10) formed by the HTLA-230 NB cell line in immunodeficient mice. Most Oct-4⁺ cells showed a perivascular distribution, with 5% of them homing in perinecrotic areas. All Oct-4⁺ cells were tumor-derived since they shared amplification of MYCN oncogene with malignant cells. Perivascular Oct-4⁺ cells expressed stem cell-related, neural progenitor-related and NB-related markers, including surface Tenascin C (TNC), that was absent from perinecrotic Oct-4⁺ cells and bulk tumor cells. TNC⁺ but not TNC⁻ HTLA-230 cells differentiated in vitro into endothelial-like cells expressing vascular-endothelial-cadherin, prostate-specific membrane antigen and CD31 upon culture in medium containing vascular endothelial growth factor (VEGF). TNC⁺ but not TNC⁻ HTLA-230 cells formed neurospheres when cultured in serum-free medium. Both cell fractions were tumorigenic, but only tumors formed by TNC⁺ cells contained EMs lined by TECs. In conclusion, we have identified in NB tumors two putative niches containing Oct-4⁺ tumor cells. Oct-4⁺/TNC⁺ perivascular NB cells displayed a high degree of plasticity and served as progenitors of TECs. Therapeutic targeting of Oct4⁺/TNC⁺ progenitors may counteract the contribution of NB-derived ECs to tumor relapse and chemoresistance.

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Introduction

Neuroblastoma (NB) is a tumor derived from primitive cells of the sympathetic nervous system and is the second most common solid tumor in childhood ¹. Approximately a half of NB patients present with metastatic disease at diagnosis, and only one-third of them survive at 5 years. About 22% of the NB tumors show amplification of the myelocytomatosis viral related oncogene NB-derived (MYCN) ¹.

Tumor growth is critically dependent on adequate blood supply provided by newly formed endothelial microvessels (EMs). Strong correlations have been established in different malignancies between high degree of tumor vascularization and unfavorable outcome ^{2, 3, 4, 5, 6}.

Endothelial precursors are recruited to form tumor neo-vessels through a phenomenon known as “angiogenic switch” and dependent on the production of pro-angiogenic factors (e.g., VEGF) by tumor cells ^{4, 5, 6}. There is debate as to the respective contribution of bone marrow (BM)-derived vs local endothelial precursors to tumor neo-angiogenesis. An additional pathway of tumor-associated EM generation is represented by the so-called vascular mimicry ⁷, whereby cancer cells are incorporated in vascular structures where they may acquire features typical of “professional” endothelial cells (ECs) ^{8, 9, 10, 11}.

We previously demonstrated that primary NB-associated EM may be lined by TECs and, accordingly, TEC-lined EMs were identified in tumors formed in immunodeficient mice by human MYCN amplified ⁸ and MYCN non-amplified ¹⁰ NB cell lines.

The Octamer-binding transcription factor 4 (Oct-4), together with SOX-2 and NANOG, maintains self-renewal and blocks cell differentiation in embryonic stem cells ^{12, 13, 14} and is expressed by some cancer stem cells ^{15, 16, 17, 18, 19, 20}. Among these factors, Oct-4 and SOX-2 appear to be indispensable for stemness maintenance ²¹. Previous studies have demonstrated that Oct-4 is expressed by side-population cells and tumor-initiating cells from human NB, indicating that Oct-4⁺ NB cells include tumor stem/progenitor cells ^{20, 22}.

In this study we have investigated whether Oct-4 is a marker of NB cells acting as TEC progenitors. Both primary and metastatic NB cells as well as NB cell lines were found to express Oct-4. Tenascin C (TNC), a protein of the extracellular matrix expressed by many tumor cell types and involved in tumor growth, metastasis and angiogenesis ^{23, 24, 25, 26, 27, 28, 29}, was consistently expressed on the surface of most Oct-4⁺ NB cells but not of bulk tumor cells. The availability of TNC as a surface marker allowed one to positively select Oct-4⁺ NB cells to perform functional studies proving that Oct-4⁺/TNC⁺ cells serve as TEC progenitors.

Results

Identification of Oct-4⁺ cells in primary NB samples and NB cell lines

Primary NB tissue sections from 23 patients at different disease stages were tested for Oct-4A isoform expression by immunofluorescence. Oct-4A is specifically involved in stemness maintenance, is highly expressed in human embryonic stem cells and downregulated following differentiation ³⁰. Oct-4A, together with SOX-2 and NANOG, governs the transcriptional regulatory network by activating expression of pluripotency-related genes and repressing expression of differentiation-related genes ³⁰. Oct-4A protein is usually localized in the nucleus as a transcription factor. However, Wang et al. ³¹ found that Oct-4A protein was diffusely localized in both cytoplasm and nucleus.

In all, 21 out of 23 tumor samples were found to contain 2-30% Oct-4A (henceforth referred to as Oct-4) positive cells (Table 1). Most Oct-4⁺ cells were detected in perivascular spaces (Figure 1A, 1), consistent with the selective homing of normal stem cells ^{32, 33} and some cancer stem cells to perivascular niches ^{34, 35, 36}. A small minority of Oct-4⁺ cells (approximately 5%) were identified in perinecrotic areas (Figure 1A, 2). All Oct-4⁺ cells displayed granular cytoplasmic (Golgi-like) and punctate nuclear staining patterns (Figure 1A-1D), in accordance with other studies in bladder, prostate and lung cancers ^{37, 38, 39}.

Table 1 Demographic characteristics of NB patients and percentage of Oct-4⁺ cells in primary tumor samples and in bone marrow (BM) aspirate samples

Full size table

In order to demonstrate the neoplastic origin of Oct-4⁺ cells, seven MYCN-amplified NB tumors were tested by combining immunofluorescence with FISH (Table 1, cases #4, 18-23). Oct-4⁺ cells showed MYCN amplification as double minutes, indicating that these cells were tumor-derived (Figure 1A, 3 and 4). No correlation was found between the proportion of Oct-4⁺ cells and tumor stage, patient age or MYCN amplification.

Cytospins from metastatic BM aspirates of 10 patients with stage 4 NB (range 10-80% NB84⁺ cells) were stained for Oct-4 by immunofluorescence (Figure 1B). Oct-4⁺ cells were detected in all samples (Table 1). Next, these aspirates were double stained with anti-Oct-4 and anti-NB84 (an NB-specific marker) mAbs ^{40, 41}. All BM Oct-4⁺ cells co-expressed NB84 (range 0.2–1.5%), thus proving their tumor origin (Table 1 and Figure 1B).

Three MYCN amplified (HTLA-230, GI-LI-N and LAN-5) and two MYCN non-amplified (SHSY-5Y and ACN) human NB cell lines were also tested for Oct-4 expression. HTLA-230, SHSY-5Y, LAN-5 and ACN cell lines, but not GI-LI-N cells, expressed Oct-4 (range 2-15%) with a predominant nuclear staining pattern (Figure 1C).

Identification and immunophenotypic characterization of Oct-4⁺ tumor cells in an orthotopic mouse model of human NB

In subsequent experiments, HTLA-230 cells were injected in the adrenal gland capsule of nude mice to generate orthotopic tumors recapitulating the natural history of primary human NB ⁴². The choice of the HTLA-230 cell model was based on previous studies from our group showing that these MYCN-amplified cells formed metastatic tumors containing TEC-lining EM ⁸. In analogy to that observed with primary NB, 10 orthotopic tumors were found to contain Oct-4⁺ cells predominantly in perivascular spaces (16.8% ± 2%) (Figure 1D, 1 and 2), with a small subset of them homing in perinecrotic areas (4% ± 2%) (Figure 1D, 3). All Oct-4⁺ cells harbored MYCN amplification, indicating that they were tumor-derived (Figure 1D, 4).

Oct-4⁺ cells did not express the human EC marker CD31 (hCD31) (Figure 1D, 1) or the pericyte marker α-SMA (Figure 1D, 2) and adhered or homed in close proximity to α-SMA⁺ pericytes coating EM (Figure 1D, 2). Notably in this respect, FISH experiments with human MYCN-specific and mouse Cot-1 DNA probes showed that pericytes were always of murine origin (data not shown), similarly to that previously reported in primary NB and in the HTLA-230 pseudometastatic model ⁸. As in primary NB, the granular cytoplasmic pattern of Oct-4 staining in orthotopic HTLA-230 tumors prevailed over the nuclear punctate pattern (Figure 1A-1D) ³⁰.

In order to investigate the immunophenotypic profile of perivascular Oct-4⁺ NB cells from orthotopic tumors, these cells were tested by immunofluorescence for the expression of panels of stem cell-related, neural progenitor-related and NB-related markers. Figure 2A summarizes the differential expression of all the markers investigated in Oct-4⁺ vs Oct-4⁻ NB cells from 10 different orthotopic tumors.

Most Oct-4⁺ cells expressed surface TNC, a marker of neural stem/progenitor cells ²⁸ (Figure 2B, 1-3), and HIF-2α (Figure 2B, 5-7), that controls Oct-4 expression in embryonic stem cells ⁴⁰ (Figure 2A), but not HIF-1α ³⁵. HIF-2α, but not TNC, was expressed also by approximately 65% Oct-4⁻ tumor cells (Figure 2B, 5-7 and Figure 2A). Most Oct-4⁺ cells co-expressed SOX-2 (Figure 2B, 9-11), another key transcription factor for stemness maintenance ^{13, 14}, which was detected also in a small minority of Oct-4⁻ NB cells (Figure 2A).

Most Oct-4⁺ cells expressed the catalytic subunit of human telomerase (hTERT) ⁴³ (Supplementary information, Figure S1, 1-3) and the cancer stem cell-related markers CD133 ¹⁵ (Supplementary information, Figure S1, 5-7), CD24 ⁴⁴ (Figure 2B, 13-15), CD271 (nerve growth factor receptor p75) ⁴⁵, as well as βIII tubulin ⁴⁶, c-kit and nestin ^{47, 48} (Figure 2A). hTERT, CD133, CD24, nestin and CD271 were expressed in different proportions also by Oct-4⁻ tumor cells (81% ± 4%, 85% ± 2%, 71% ± 4%, 33% ± 2% and 66% ± 5%, respectively, Figure 2A). βIII tubulin and c-kit were expressed by most Oct-4⁻ NB cells (Figure 2A).

Most Oct-4⁺ and Oct-4⁻ cells expressed VEGF-R2 (Supplementary information, Figure S1, 13-15) ⁸, and the neuroblastic markers GD2 (Supplementary information, Figure S1, 9-11), CD56 (Supplementary information, Figure S1, 17-19) ^{8, 49, 50} and NB84 (Figure 2A). All isotypic controls used for immunofluorescence tested negative (Figure 2B, 4, 8, 12, 16; Supplementary information, Figure S1, 4, 8, 12, 16, 20).

In summary, TNC was the only surface marker that was able to discriminate Oct-4⁺ and Oct-4⁻ cells in orthotopic NB tumors (Figure 2A). Notably in this respect, the few perinecrotic Oct-4⁺ cells did not express TNC.

Identification of perivascular Oct-4⁺/TNC⁺ cells in primary tumors and NB cell lines

We next asked whether perivascular Oct-4⁺ cell co-expressing TNCs that had been detected in HTLA-230 orthotopic tumors were present also in primary NB samples. Therefore, we stained by double immunofluorescence tissue sections from 10 primary tumors (out of the 23 previously tested, see Table 1) with anti-Oct-4 and anti-TNC mAbs. Perivascular but not perinecrotic Oct-4⁺ cells were found to express surface TNC (range from 3% to 29%) (Figure 3A), similarly to that observed in the HTLA-230 orthotopic model (see Figure 2B, 1-3). In contrast, TNC was never expressed by Oct-4⁻ cells from primary tumors.

Oct-4⁺ cells from in vitro cultured HTLA-230, SHSY-5Y, ACN, and LAN-5 NB cell lines also co-expressed surface TNC that was undetectable on Oct-4⁻ cells from these cell lines as well as on the Oct-4⁻ GI-LI-N cells (Figure 3A). One representative experiment showing co-expression of Oct-4 and TNC in cytospins from the HTLA-230 cell line is shown in Figure 3B, 4–6.

Expression of TNC mRNA in human HTLA-230 cell line

In order to exclude the possibility that detection of surface TNC on NB cells was due to release of soluble TNC from the tumor extracellular matrix and subsequent binding to receptors expressed on malignant cells, we next performed real-time PCR experiments with specific primers detecting all TNC isoforms ²⁸ using mRNA from the TNC⁺ HTLA-230 and TNC⁻ GI-LI-N NB cell lines. As shown in Supplementary information, Figure 2S, TNC mRNA was detected in HTLA-230 but not GI-LI-N cells, indicating that the former cells produced TNC endogenously.

Orthotopic NB tumors formed by TNC⁺ but not TNC⁻ HTLA-230 cells contain EMs lined by TECs

Next, we asked whether TNC⁺/Oct-4⁺ NB cells could serve as TEC progenitors and therefore contribute to tumor vasculogenesis. Tumor-derived TNC promotes or enhances the process of neovascularization in different tumor models ^{23, 24, 25, 26, 27, 28, 51}.

In preliminary experiments, we investigated whether orthotopic tumors formed by HTLA-230 cells in nude mice contained EM lined by TEC. Indeed, tumors harvested after 3 weeks from malignant cell inoculation contained approximately a half of mouse EM and the other half of human EM (data not shown), as previously demonstrated in the HTLA-230 pseudo-metastatic model ⁸.

We next isolated TNC⁺ and TNC⁻ HTLA-230 cells with high degree of purity by a two-round immunomagnetic procedure. Either cell fraction was inoculated orthotopically in two groups of six nude mice each. Tumors formed by TNC⁺ and TNC⁻ HTLA-230 cells were harvested after 3 weeks. The average volume of tumors formed by TNC⁺ cells was larger than that formed by TNC⁻ cells (P = 0.039) (Figure 4D). Tumors formed by both TNC⁺ and TNC⁻ cells were subjected to EM count by immunofluorescence with hCD31 and mCD34 mAbs that proved to be rigorously species-specific in a previous study from our group ⁸. EM lined by hCD31⁺ TEC represented approximately a half of the EM present in tumors formed by TNC⁺ HTLA-230 cells (49% ± 1.35%), the remaining EM being lined by mCD34⁺ EC (51% ± 1.35%) (Figure 4A and 4B). In contrast, all EM present in tumors formed by TNC⁻ HTLA-230 cells were lined by mCD34⁺ EC (Figure 4A and 4C). Finally, orthotopic tumors formed by the TNC⁻/Oct-4⁻ GI-LI-N NB cell line contained mouse EM only (data not shown).

In the orthotopic tumors formed by TNC⁺HTLA-230 cells, mean overall EM density was 29.2 microvessels per 40× field visually (range 12.8–94.3). In the orthotopic tumors formed by TNC⁻ HTLA-230 cells mean overall EM density was 7.4 microvessels per 40× field visually (range 0.0-24). Therefore, in tumors formed by TNC⁺HTLA-230 cells microvessel density was significantly higher than in those formed by TNC⁻ HTLA-230 cells (P = 0.022).

These experiments demonstrate unambiguously that TNC⁺NB cells contained TEC progenitors.

Kinetics of human and mouse EM formation in orthotopic tumors formed by TNC⁺ cells in immunodeficient mice

In order to investigate the kinetics of development of tumor microvessels lined by human or mouse EC, nude mice were inoculated orthotopically with the TNC⁺ HTLA-230 cells ⁴² and groups of six mice each were sacrificed at various times. ECs lining EM were detected by staining with anti-mCD34 and anti-hCD31 mAbs ⁸. After an initial predominance of mouse over human EM, the two types of EM reached an equilibrium. Both the increment in the proportion of human EM and the decrease of mouse EM on day 35 compared to day 7 were statistically significant (P = 0.038 for both comparisons) (Figure 5A). Human EM contained intra-luminal red blood cells (Figure 5B, 3), indicative of their contribution to tumor vascularization. Individual EMs lined by juxtaposed mouse ECs and human TECs were never detected.

We next investigated expression of additional EC-related markers and of NB-related markers in human TECs from orthotopic tumors harvested 3 weeks after inoculation of TNC⁺ HTLA-230 cells (Figure 6A). Most TECs were found to express the EC-related markers PSMA ⁵² (Figure 6B, 1) and hvWF (Figure 6B, 2). These cells were tumor-derived since they carried MYCN amplification as homogeneously staining regions (Figure 6B, 1 and 2). Most human TECs expressed hCD105, a marker of EC activation ⁵³ (Figure 6B, 3), HLA-class I (Figure 6B, 4) but not HLA-class II (data not shown) antigens, as well as HLA-G, a non-polymorphic HLA-class Ib molecule expressed by embryonic cytotrophoblast cells and endowed with potent immunosuppressive activities ⁵⁴ (Figure 6B, 5). At variance with TEC, bulk tumor cells did not express PSMA, vWF, CD105, HLA-class I or HLA-G (Figure 6A). Most TECs were found to express the NB-associated markers GD2 ⁴⁹ NB84 ⁴¹, and CD56 ⁵⁰ (Figure 6B, 6-8), which were shared by most tumor cells (Figure 6A).

Plasticity of TNC⁺ cells isolated from the HTLA-230 cell line

In subsequent experiments, we investigated the plasticity of TNC⁺ cells from the HTLA-230 cell line in three different in vitro models, i.e., the ability to form neurospheres or tubes and the potential to differentiate into endothelial-like cells.

When cultured in serum-free medium promoting neurosphere formation, TNC⁺ cells grew in suspension and generated neurospheres (Figure 7A, 1), whereas TNC⁻ cells did not (Figure 7A, 2). Notably, the latter cells showed morphological evidence of differentiation represented by neurite formation (Figure 7A, 2) as did the parental cell line (data not shown).

To evaluate whether the ability to form neurospheres was maintained, primary neurospheres, enzymatically digested after 7-10 days and re-plated as single-cell suspension, generated second passage spheres. This feature was retained for 10 passages. Neurospheres expressed βIII tubulin, nestin, HIF-2α, CD24, Oct-4, and Notch-2 ⁵⁵ (Figure 7B, 1-4), but not CD34, PSMA, CD31 or VE-cadherin.

We next used a 3D in vitro system with high-density Matrigel to investigate the ability of TNC⁺ and TNC⁻ HTLA-230 cells to form tubes resembling vascular structures. Normal ECs isolated from human umbilical vein (HUVECs) were tested as control.

In four different experiments, both TNC⁺ and TNC⁻ cells formed a network of tubes (Figure 7A, 3 and 4), similar to those observed with HUVECs. However, the kinetics of formation of tubes was definitely faster with TNC⁺ than with TNC⁻ cells. Thus, the former cells gave rise to a network of tubes 2 h after plating and complete tube formation was detected after 6 h. In contrast, TNC⁻ cells started forming the network of tube structures after 12 h and reached a plateau after 20 h in culture. Tubes formed by TNC⁺ cells persisted for up to 5 days, while tubular structures generated by TNC⁻ cells rapidly dissolved in a few hours. Furthermore, the network of tubes formed by TNC⁺ cells was thicker and more branched than that generated by TNC⁻ cells, which displayed a less-organized morphology (Figure 7A, 3 and 4). The number of branches of the tube network formed by TNC⁺ cells was higher than that formed by TNC⁻ cells (TNC⁺ cells, 70 ± 27 vs TNC⁻ cells, 23 ± 12 branches per field; P = 0.004).

GI-LI-N NB cells, which did not give rise to TECs in orthotopic tumors, were also tested in the same high-density Matrigel system mentioned above. GI-LI-N cells formed tubes already after 2 h that waned after 4-6 h, suggesting that these tubular structures were unstable (data not shown).

Finally, TNC⁺ cells were grown in differentiating EndoGRO-MV-VEGF medium supplemented with VEGF. In this condition, TNC⁺ cells lost TNC expression after 3 days in culture (data not shown) and acquired expression of the endothelial-specific markers PSMA, VE-cadherin, and CD31 after 10–15-day culture (Figure 7C, 1-3). In three different experiments, PSMA and VE-cadherin were upregulated on higher than 90% TNC⁺ cells, while CD31 was detected on 30% of them. PSMA, VE-cadherin and CD31 were undetectable in TNC⁻ cells cultured for 10-15 days in EndoGRO-MV-VEGF medium supplemented with VEGF (Figure 7C, 4-6).

Discussion

The concept of vascular mimicry, originally referred to the ability of highly aggressive human melanoma cells to form vascular channels in vivo and in vitro ⁷, has been extended to indicate the differentiation of tumor cells into ECs witnessed by de novo expression of EC-specific genes ⁷.

Vascular mimicry has been reported in different tumors ^{7, 8, 9, 10, 11}. Although the mechanisms underlying differentiation of human tumor cells into EM are largely unknown, a few studies provide insights into the nature of the malignant cells involved in the phenomenon. Thus, Bussolati et al. ⁵⁶ have shown that CD105⁺ tumor-initiating cells from renal carcinoma can differentiate into TECs in vitro and generate human microvessels in immunodeficient mice. The same group has demonstrated that breast cancer stem/progenitor cells are capable of differentiating into TECs ⁹. Likewise, Alvero et al. ⁵⁷ have shown that TECs may originate from a subset of ovarian cancer cells with a highly angiogenic phenotype and the features of cancer stem cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis ⁵⁸. It has been proposed that genetically abnormal TECs display an embryonic/immature phenotype associated with increased angiogenic potential, leading to embryonic-like vasculogenesis in the tumor microenvironment ⁵³.

We have previously demonstrated in a pseudo-metastatic tumor model that both primary NB cells and NB cell lines generate TECs lining the EM coated by host-derived pericytes ^{8, 10}.

Previous studies ^{20, 22} have demonstrated that side-population cells of the SK-NB-BE(2) NB cell line and tumor-initiating human NB cells express the embryonic stem cell ^{12, 13, 14} and cancer stem cell marker Oct-4 ^{15, 16, 17, 18, 19, 20}. The human Oct-4 gene can generate at least three transcripts (Oct-4A, Oct-44B and Oct-4B1) and four protein isoforms (OCT4A, OCT4B-190, OCT4B-265 and OCT4B-164) by alternative splicing and alternative translation initiation ³¹. The Oct-4A isoform confers pluripotency on ES cells.

In this study, we first investigated Oct-4A expression in primary NB at different stages and detected variable amounts of Oct-4⁺ cells in most of them. Lower proportions of Oct-4⁺ cells were found in metastatic BM samples. This latter finding may be explained by the fact that BM aspiration does not allow to recover the Oct-4⁺ tumor cells attached to the stroma. Oct-4⁺ cells from primary tumors and metastatic BM aspirates carried amplification of the MYCN oncogene or expressed the NB84 neuroblastic marker, respectively, and were located predominantly in perivascular areas and at a lower extent in perinecrotic areas, similarly to that reported recently for primary glioma ⁵⁹.

To investigate the functional relationships between Oct-4⁺ NB cells and TECs, we set up an orthotopic NB model in immunodeficient mice using the HTLA-230 cell line. This model recapitulates the onset and progression of NB observed in patients. Orthotopic tumors contained a half of the EMs lined by MYCN-amplified TECs. Oct-4⁺ cells showed a predominant perivascular distribution, with few of them homing in perinecrotic areas.

Hypoxia plays a critical role in the establishment of niches where cancer stem cells home and maintain stemness and self-renewal ⁶⁰. It has been recently shown that the perinecrotic niche in gliomas is strongly hypoxic (approximately 1% oxygen tension) and this microenvironment may promote HIF-2α-dependent reprogramming of non-stem tumor cells towards a cancer stem cell-like phenotype ⁶¹. Previously it was shown that in NB biopsy specimens HIF-2α marks a perivascular niche containing cells that express neural crest markers ³⁵, and that HIF-2α maintains an undifferentiated state in NB tumor-initiating cells ²². HIF-2α regulates Oct-4 gene expression ⁴⁰ and we identified Oct-4⁺ NB cells co-expressing HIF-2α in perivascular areas, consistent with the results of Pietras et al ³⁵. In addition, we have reported for the first time the existence of a perinecrotic putative niche in human NB where scanty Oct-4⁺ cells home. The relations between Oct-4⁺ cells present at the latter site and those found in perivascular areas are so far unknown, but it is tempting to speculate that the perinecrotic Oct-4⁺ NB cells are more stem-like cells than the perivascular ones, which have moved to a less hypoxic area and may be prone to differentiation. On the other hand, perivascular Oct-4⁺ cells can migrate back to the perinecrotic putative niche and revert to a cancer-stem cell-like phenotype likely through epigenetic mechanisms ⁶². In this respect, the possibility that TECs can de-differentiate back to progenitor state under the influence of stimuli like hypoxia cannot be excluded and warrants further investigation.

TNC is involved in CNS embryogenesis, especially in the formation of neural stem cell niches ^{27, 28, 29}. TNC expression, which is under the control of Oct-4 ⁶², is low in normal brain tissue and upregulated in several malignant brain tumors in association with increased vascularity, short time to relapse, and poor prognosis ^{26, 63}. Little information was so far available on TNC expression in human NB.

We found that all perivascular Oct-4⁺ NB cells in primary and ortothopic tumors co-expressed surface TNC that was absent in either perinecrotic Oct-4⁺ cells or bulk tumor cells. TNC⁺/Oct-4⁺ NB cells were detected consistently in close proximity or adherent to the EM wall, suggesting that ECs regulate TNC⁺/Oct-4⁺ cell viability and self-renewal ^{28, 64}. TNC⁺/Oct-4⁺ perivascular tumor cells from orthotopic tumors were found to express SOX-2, CD133, HIF-2α, CD24, VEGF-R2, βIII tubulin, c-kit and nestin, as well as GD2, CD56 and NB84. SOX-2, as Oct-4, maintains self-renewal of undifferentiated embryonic stem cells ^{12, 13, 14}. CD133, βIII tubulin, c-kit and nestin are expressed by NB progenitor cells ^{15, 46, 47, 48}. CD24 identifies a tumor-initiating cell population in BM metastatic NB, and NB84 has been detected in neurospheres thereof ⁶⁵. Besides being neuroblastic markers, CD56 ⁶⁶ is expressed by immature and TECs, and GD2 by mesenchymal stem cells ⁶⁷.

The overall immunophenotypic profile of TNC⁺/Oct-4⁺ perivascular NB cells was consistent with their progenitor cell nature. Accordingly, these cells showed high plasticity both in vitro and in vivo. Thus, TNC⁺/Oct-4⁺ cells grew as neurospheres in vitro for many passages, a feature typical of tumor stem/progenitor cells ^{35, 68}. These neurospheres expressed stem cell-related markers such as βIII-tubulin, HIF-2α, nestin and Notch-2, which controls TNC expression and angiogenesis ⁶⁹. Three lines of evidence demonstrated unambiguously that TNC⁺/Oct-4⁺ cells were the progenitors of NB-derived ECs, and namely (i) in vitro these cells, but not their TNC⁻ counterparts, differentiated into endothelial-like cells expressing VE-cadherin, PSMA and to a lesser extent CD31 after culture with VEGF, and (ii) in vivo TNC⁺, but not TNC⁻ HTLA-230 cells, formed ortothopic tumors in which a half of the EMs were lined by NB-derived ECs. However, both TNC⁺ and TNC⁻ cell fractions were tumorigenic, pointing to the progenitor rather than cancer stem cell nature of TNC⁺/Oct-4⁺ cells. (iii) In vivo the TNC⁻/Oct-4⁻ GI-LI-N NB cell line formed orthotopic tumors completely devoid of TECs lined by TECs.

The virtually ubiquitous detection of TNC⁺/Oct-4⁺ TEC progenitor cells in primary tumors is in apparent disagreement with the previous demonstration that only one-third of primary NB samples contained TECs ⁸. It is conceivable that unknown micro-environmental factors influence in the individual cases the ability of TNC⁺/Oct-4⁺ NB cells to generate tumor-derived EM. A possible factor is the scanty distribution of hypoxic areas in tumors where Oct-4⁺ side-population NB cells have been shown to migrate in vivo ^{70, 71}. Notably, in this respect, we observed that TECs lining the EM tended to concentrate in the tumor core where oxygen tension is very low.

In conclusion, we have identified in primary and ortothopic NB tumors two putative niches containing Oct-4⁺ tumor cells and shown that surface TNC allows to dissect these Oct-4⁺ cell subsets. Oct-4⁺/TNC⁺ perivascular NB cells displayed a high degree of plasticity and served as progenitors of TECs. Therapeutic targeting of Oct4⁺/TNC⁺ progenitors may counteract the contribution of NB-derived ECs to tumor relapse and chemoresistance.

Materials and Methods

Patients

Tumors from 23 NB patients as well as 10 metastatic BM aspirates were studied. Six patients had stage 1 disease, 5 stage 2, 3 stage 4s and 19 stage 4 according to the International Neuroblastoma Staging System classification ⁷² (Table 1). All patients were tested at disease onset in the absence of any treatment. The study was approved by the local Ethics Committee.

Immunofluorescence study

Indirect immunofluorescence was performed on 4-μm-thick formalin-fixed, paraffin-embedded or cryopreserved tissue sections or cytospins as previously described ⁸. The following antibodies were used: anti-human(h)CD31 (diluted 1:50; Dako Cytomation, Hamburg, Germany), anti-mouse(m)CD34 (diluted 1:50; Novus Biologicals, Littleton, CO, USA), anti-human endoglin (CD105) (1:50; Dako), anti-alpha-smooth muscle actin (α-SMA; 1:50; Dako), anti-von Willebrand factor (vWF) (1:50; Dako), anti-VE-Cadherin (1:50; Chemicon International, Millipore, Billerica, MA, USA), anti-GD2 (1:500; secreted from the hybridoma cell line 14.G2, a generous gift of RA Reisfeld, The Scripps Institute, La Jolla, CA, USA), anti-NB84 (1:100; Dako), anti-prostate-specific membrane antigen (1:50; anti-CB-PSMA) that detects specifically an epitope expressed by newly formed microvessels ⁷³, anti-HIF1α (1:100; Novus Biologicals), anti-HIF2α (1:100; Novus Biologicals), anti-βIII tubulin (1:50; Novus Biologicals), anti-CD24 (1:50; Novus Biologicals), anti-Oct-4 (1:50, Clone 10H11.2 that detects the Oct-4A isoform; Chemicon International, Millipore), anti-SOX-2 (1:50; Chemicon International), anti-Tenascin C (1:50; Clone CB-TNX; MediaPharma, Chieti, Italy), anti-nestin (1:50; Chemicon International), anti-CD117 (c-Kit) (1:100; Chemicon International), anti-CD271 (nerve growth factor receptor p75) (1:50; Chemicon International), anti-CD133 (1:50; Miltenyi Biotec Inc, Auburn CA, USA), anti-VEGF R2 (1:100; R&D Systems Inc, Minneapolis, USA), anti-Notch2 (1:50; Abcam Inc), anti-CD56 (1:50; Invitrogen Corp, Camarillo, CA, USA), anti-CD57 (1:50; Invitrogen Corp), and anti-Telomerase (1:100; LabVision, Fremont, CA, USA). Antigen retrieval slides were incubated with primary antibodies overnight at 4 °C, followed by 30-min incubation with secondary antibodies of the appropriate species conjugated with different fluorochromes (1:50; Dako). After washing, the slides were counterstained with DAPI and coverslipped. Results were photographically documented using a digital photo-device (Nikon Instruments, see below). Isotype-matched non-binding mAbs were used in all antibody-staining experiments to exclude non-specific reactivity.

EM counts

EM density was assessed by a combination of anti-hCD31 or anti-mCD34 staining and morphological analysis and examination of 20-50 microscopic fields (0.5 mm²) per tumor for a minimum of 500 cells counted. Tumor sections were examined under low magnification (40×) to detect areas with high EM density and microvessels were counted in a representative high magnification (100×) field in each area. The most intense vascular areas (hotspots) were selected subjectively from each tumor section as described by Weidner ⁷⁴. Sclerotic and necrotic areas were avoided. EM with a clearly defined lumen or well-defined linear vessel shape, but not single EC, was taken into account for EM counting.

Fluorescent in situ hybridization (FISH)

FISH was performed as described ⁸ using rhodamine-labeled MYCN-specific DNA probe (QBIOgene Inc, Hamburg, Germany) and mouse Cot-1 DNA probe (Life Technologies Gibco BRL, Paisley, Scotland).

Image analysis

Digital images were acquired using a Nikon E-1000 fluorescence microscope (Nikon Instruments, Tokyo, Japan) equipped with appropriate filter sets and the Genikon imaging system software (Nikon Instruments).

In vivo experiments

All procedures involving animals were performed in respect of the National and International current regulations (D.lvo 27/01/1992, no. 116, European Economic Community Council Directive 86/609, OJL 358, December 1, 1987). All procedures were reviewed and approved by the licensing and Ethical Committee of the National Cancer Research Institute, Genoa, Italy, and by the Italian Ministry of Health. Female Hsd athymic nude (nude/nude) mice, 7-8 weeks old, were purchased from Harlan Laboratories (Harlan Italy, S Pietro al Natisone, Italy) and housed in sterile enclosures under specific pathogen-free conditions. In all experiments, mice were anesthetized with ketamine (Imalgene 1000, Merial Italia SpA., Milan, Italy), subjected to laparotomy and injected with HTLA-230 or GI-LI-N cells (1.5 × 10⁶ cells in 20 μl of saline solution) in the capsule of the left adrenal gland, as previously described ⁴². No mice died as a result of this treatment. Mice were sacrificed when signs of poor health became evident.

Finally, TNC⁺ and TNC⁻ cells (1 × 10⁵ cells) isolated from the HTLA-230 cell line (see below) were implanted in the capsule of the left adrenal gland of nude/nude mice (Harlan Laboratories, Indianapolis, Indiana, USA). In some experiments, groups of six mice each were injected with TNC⁺ and TNC⁻ cells and sacrificed at various times following cell inoculation (7, 14, 21, 28 and 35 days) by cervical dislocation after being anesthetized with xilezine.

Isolation and culture of TNC⁺ and TNC⁻ HTLA-230 cells

TNC⁺ cells were isolated from the human HTLA-230 NB cell line by magnetic cell sorting using the anti-TNC CB-TNX mAb ⁷⁵ and the MACS system (Miltenyi Biotec, Auburn, CA, USA). Briefly, cells were labeled with the anti-TNC mAb for 20 min, then washed twice and suspended in MACS buffer (PBS supplemented with 1% BSA and 5 mM EDTA). After washings, cells were separated into TNC⁺ and TNC⁻ fractions on a magnetic stainless steel wool column (Miltenyi Biotec), according to the manufacturer's recommendations. TNC⁻ cells were subjected to a further round of depletion of residual TNC⁺ cells using the same immunomagnetic procedure. At the end of the separation process, TNC⁺ and TNC⁻ cell fractions were cytocentrifuged and stained by immunofluorescence with anti-TNC mAb. Higher than 98% of the TNC⁺ cell fraction expressed TNC, whereas the TNC⁻ cell fraction contained less than 1% of TNC⁺ cells.

To evaluate the ability of these cell fractions to grow in non-adhesive condition as floating spheres, cells were plated in serum-free DMEM-F12 (Invitrogen), supplemented with 2% B27 (Invitrogen), 10 ng/ml basic fibroblast growth factor (bFGF) and 20 ng/ml epidermal growth factor (EGF) (all from Sigma-Aldrich Corp., St Louis, MO, USA) ⁷⁶.

In vitro formation of capillary-like structures was studied on cells seeded onto Matrigel-coated wells in culture medium (Tube Formation Kit, Trevigen Inc., Gaithersburg, MD, USA). TNC⁺ and TNC⁻ HTLA-230 cells (1-3 × 10⁴ cells/well) were seeded onto Matrigel-coated wells in Endothelial Basal Medium without serum in the presence or absence of the angiogenesis mediators VEGF (5-15 ng/ml) and bFGF (20-50 ng/ml). Sulforaphane at 1-10 μM was added as a control inhibitor of tube formation. As positive control for this system, we used normal ECs isolated from human umbilical vein cells (HUVECs). Each experiment was repeated at least three times. Cell organization onto Matrigel was observed over a 2-24-h period with an inverted microscope with a 10× phase-contrast objective and experimental results were recorded.

To evaluate cell differentiation, TNC⁺ and TNC⁻ HTLA-230 cells were incubated in EndoGRO-MV-VEGF complete media kit (Millipore) with VEGF (5 ng/ml) on EC attachment factor (Roche) coated plates. The medium was changed twice a week for 3 weeks. Thereafter, cells were cytocentrifuged and stained by immunofluorescence for PSMA, VE-cadherin and CD31.

Real-time qPCR

mRNA was extracted from human NB cell lines HTLA-230 and GI-LI-N and human melanoma cell line MZ2-MEL ⁷⁷ as positive control ⁵¹ using RNeasy Mini Kit from Qiagen (Qiagen GmbH, Hilden, Germany) and subjected to RT-PCR. Briefly, 1 μg of RNA was reverse transcribed using the RevertAid^TM First Strand cDNA Synthesis kit (Fermentas Inc., Burlington, ON, Canada) following the instructions of the manufacturer. In all, 5 μl of cDNA was used in 25 μl PCR reaction, using the 2× PCR Master Mix (Fermentas Inc.). The following oligonucleotide primer pairs were used at the indicated annealing temperature: TNC, forward 5′-GCAGCCACAGAGTTGGAC-3′ and reverse 5′-GAGATCCTCTGTGACGACCT-3′ at 58 °C ²⁸; GAPDH, forward 5′-ACATCGCTCAGAACACCTATGG-3′ and reverse 5′-GGGTCTACATGGCAACTGTGAG-3′ at 60 °C. All reactions were carried out in epgradient S Mastercycler (Eppendorf, Hamburg, Germany) starting with a 2-min denaturation step at 94 °C followed by 35 cycles of 15 s at 94 °C, 30 s at the respective annealing temperature, and 70-s elongation at 72 °C. The reaction was followed by a 7-min final elongation step at 72 °C and cooled down to 4 °C. The PCR products were separated on a 1% agarose gel.

Statistical analysis

Analysis of quantitative data was performed by non-parametric repeated measurement analysis of variance (Friedman's test). Angular transformation was applied and regression analysis of transformed data was performed in some experiments ⁷⁸. To compare quantitative variables between two groups of observations, the non-parametric Mann-Whitney U test was used. Statistical significance of differences between experimental and control groups was determined by ANOVA with Tukey's multiple comparison test using GraphPad Prism 3.0 software (GraphPad Software, Inc.).

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Acknowledgements

We thank the late Dr Giorgio Corte for reading the manuscript. Special thanks go to Dr Paolo Bianco for discussion and suggestions. This work was supported by Fondazione Italiana Neuroblastoma, Fondazione “Guido Berlucchi”, Fondazione “Bianca e Wilma Querci”, Progetto di Ricerca Regione Liguria “Endotelio di derivazione tumorale: caratterizzazione e targeting immunologico a fini terapeutici” and Ministero della Salute Progetto Strategico Oncologico 2006 “Microambiente tumorale: ruolo nella progressione neoplastica sulle difese dell'ospite. Identificazione di nuovi bersagli per lo sviluppo di terapie innovative”. CC was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC).

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Laboratory of Oncology,
Annalisa Pezzolo, Federica Parodi, Danilo Marimpietri, Lizzia Raffaghello & Vito Pistoia
AIRC Tumor Immunology Unit,
Claudia Cocco
Epidemiology and Biostatistics Unit, Istituto Giannina Gaslini, Largo G. Gaslini 5, 16147, Genova-Quarto, Italy
Angela Pistorio
Department of Experimental and Laboratory Medicine, Laboratory of Pathology, IRCCS G. Gaslini Hospital, Genova, Italy
Manuela Mosconi & Claudio Gambini
Animal Research Facility, National Cancer Institute, Genova, Italy
Michele Cilli
Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy
Silvia Deaglio & Fabio Malavasi

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( Supplementary information is linked to the online version of the paper on the Cell Research website.)

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Supplementary information, Figure S1

Further immunophenotypic characterization of perivascular Oct-4⁺ NB cells present in orthotopic tumors formed by HTLA-230 NB cells in immunodeficient mice. (PDF 96 kb)

Supplementary information, Figure S2

RT-qPCR analysis of TNC expression in HTLA-230 cell line. (PDF 87 kb)

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Pezzolo, A., Parodi, F., Marimpietri, D. et al. Oct-4⁺/Tenascin C⁺ neuroblastoma cells serve as progenitors of tumor-derived endothelial cells. Cell Res 21, 1470–1486 (2011). https://doi.org/10.1038/cr.2011.38

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Received: 29 August 2010
Revised: 07 December 2010
Accepted: 10 January 2011
Published: 15 March 2011
Issue Date: October 2011
DOI: https://doi.org/10.1038/cr.2011.38

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