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. 2019 Jul 26;71(5):893–903. doi: 10.1007/s10616-019-00332-3

Biological characterization of the UW402, UW473, ONS-76 and DAOY pediatric medulloblastoma cell lines

Ricardo Bonfim-Silva 1,✉, Karina Bezerra Salomão 1, Thais Valéria Costa de Andrade Pimentel 2, Camila Cristina Branquinho de Oliveira Menezes 3, Patrícia Vianna Bonini Palma 3, Aparecida Maria Fontes 1
PMCID: PMC6787134  PMID: 31346954

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent advances in molecular technologies allowed to classify MB in 4 major molecular subgroups: WNT, SHH, Group 3 and Group 4. In cancer research, cancer cell lines are important for examining and manipulating molecular and cellular process. However, it is important to know the characteristics of each cancer cell line prior to use, because there are some differences among them, even if they originate from the same cancer type. This study aimed to evaluate the similarities and differences among four human medulloblastoma cell lines, UW402, UW473, DAOY and ONS-76. The medulloblastoma cell lines were analyzed for (1) cell morphology, (2) immunophenotyping by flow cytometry for some specifics surface proteins, (3) expression level of adhesion molecules by RT-qPCR, (4) proliferative potential, (5) cell migration, and (6) in vivo tumorigenic potential. It was observed a relationship between cell growth and CDH1 (E-chaderin) adhesion molecule expression and all MB cell lines showed higher levels of CDH2 (N-chaderin) when compared to other adhesion molecule. ONS-76 showed higher gene expression of CDH5 (VE-chaderin) and higher percentage of CD144/VE-chaderin positive cells when compared to other MB cell lines. All MB cell lines showed low percentage of CD34, CD45, CD31, CD133 positive cells and high percentage of CD44, CD105, CD106 and CD29 positive cells. The DAOY cell line showed the highest migration potential, the ONS-76 cell line showed the highest proliferative potential and only DAOY and ONS-76 cell lines showed tumorigenic potential in vivo. MB cell lines showed functional and molecular differences among them, which it should be considered by the researchers in choosing the most suitable cellular model according to the study proposal.

Keywords: Medulloblastoma cell lines, Migration, Proliferation, Surface proteins, In vivo tumorigenic potential

Introduction

Medulloblastoma (MB) is a tumor derived from neuronal progenitor/stem cells in the external embryonic layer of developing cerebellum (Skowron et al. 2015). It is the most common malignant brain tumor in children and has an incidence of approximately 0.74/100,000 person-year (Pui et al. 2011; Siegel et al. 2012) and 30% of cases show metastatic dissemination (Rutkowski et al. 2010). Currently, MB is classified by the World Health Organization (WHO) based on histology and molecular profile. About histologic subtypes it is classified as classic, large cell/anaplastic (LCA), nodular/desmoplastic and MB with extensive nodularity (Louis et al. 2007, 2016). Patients with nodular/desmoplastic histology have favorable outcomes, while those with large cell and anaplastic histology have the worst prognosis (Eberhart et al. 2002; McManamy et al. 2007). Regarding molecular subtypes, officially, MBs have been divided into 5 major subgroups: WNT, TP53 mutated Sonic Hedgehog (SHH), TP53 wild type SHH, Group 3 and Group 4, and this molecular classification is derived from the recent advances in microarray and genomic sequencing technologies that allowed a deeper molecular understanding of MB (Jones et al. 2012; Northcott et al. 2012; Robinson et al. 2012; Louis et al. 2016). However, recently, a study have identified twelve other molecular subgroups within these 5 molecular subgroups and making association with clinical features (Cavalli et al. 2017).

In cancer research, despite the importance of the analyzes being carried out with tumor tissue samples, tumor-derived cell lines became an important tool to allow the evaluation of some biological properties, such as gene expression modulation, pharmacological inhibitors effects, and so on, which are not possible to be directly assessed in tumor tissues. The cancer cell lines are important models for examining and manipulating the molecular and cellular processes potentially relevant to the tumor initiation and progression, as well as, they have some advantages, such as, in vitro undefined proliferation and easy maintenance. However, it is essential to know the characteristics of cancer cell lines, because, although “represent” the tumor tissue, they have some differences among them that should be considered in the studies.

This study aimed to evaluate the similarities and differences among the human medulloblastoma cell lines UW402, UW473, DAOY and ONS-76 regarding cell morphology, immunophenotyping (surface proteins), gene expression level of adhesion molecules, in vitro migration and proliferative potential and in vivo tumorigenic potential. Some studies have molecularly classified these cell lines, which DAOY cell line is grouped in the TP53 mutant SHH subgroup and the ONS-76 cell line in the TP53 wildtype SHH subgroup (Ivanov et al. 2016). The UW402 and UW473 cell lines were classified by the Pediatric Oncology Laboratory of the Clinics Hospital of Ribeirão Preto Medical School as SHH (Cruzeiro et al. 2019) and TP53 mutant (data not published).

Materials and methods

Cell lines

UW402, UW473, DAOY and ONS-76 human pediatric medulloblastoma cell lines were used in this study. ONS-76, UW402 and UW473 cell lines were kindly provided by the Laboratory of Pediatric Oncology of the Clinics Hospital of the Ribeirão Preto Medical School under authorization of Prof. Dr. Michael Bobola, while DAOY cell line was purchased from the American Type Culture Collection (ATCC, USA). UW402 and UW473 cell lines were grown in DMEM-F12 medium (Gibco, USA) with 10% fetal bovine serum (FBS; Hyclone, USA). The DAOY cell line was cultured in α-MEM (Gibco, USA) 10% FBS and ONS-76 cell line was cultured in RPMI-1640 medium (Gibco, USA) 10% FBS in incubator at 37 °C and 5% CO2. All culture media contained 10,000 units/ml penicillin and 10,000 mg/ml streptomycin.

Cell morphology analysis

Medulloblastoma cell lines were photographed in phase contrast microscopy (Olympus CX31, EUA) and analyzed morphologically. They were classified as polygonal or fibroblastoid morphology, and regarding to the cell growth pattern they were classified as in colony or dispersed.

Immunophenotyping

Cells were incubated with the antibodies peridinin chlorophyll protein complex (PERCP) anti-human CD34, allophycocyanin (APC) anti-human CD45, fluorescein Isothiocyanate (FITC) anti-human CD31, phycoerythrin (PE) anti-human CD133, FITC anti-human CD44, PERCP anti-human CD105, PE anti-human CD166, APC anti-human CD29, FITC anti-human CD144, FITC anti-human CD271, PE anti-human CD24, FITC anti-human CD146 and PE anti-human CD90. Cells were analyzed in the flow cytometry FACSCaliburTM (Becton & Dickson Biosciences, EUA) and the results were shown as mean ± standard deviation.

Real time PCR

The total RNA samples were extracted using the RNeasy mini kit (Qiagen, Germany) and cDNA was synthesized with 1 μg RNA using the High Capacity cDNA Reverse Transcription Kit (Life Technology, USA) according to the manufacturer’s protocol. TaqMan probes (Applied Biosystems, USA) for CDH1 (E-cadherin), CDH5 (VE-cadherin) and CDH2 (N-cadherin) genes and ACTB reference gene (β-actin) were used with Taqman Master Mix (Life Technology, USA) according to the manufacturer’s protocol to perform the Real-Time PCR analysis (qRT-PCR). The results from qRT-PCR were analyzed by the equation 10,000/2ΔCt (level of gene expression relative to the reference gene) and shown as relative expression units and mean ± standard deviation.

In vitro proliferative potential assay

The proliferative potential was assessed by cell counting in Neubauer chamber. For that, 8 × 104 viable cells (triplicate) were cultured in 75 cm2 flask for a period of 120 h. Next, the cells were trypsinized and viable cells were counted in Neubauer chamber with trypan blue. The proliferative potential (PP) was calculated by Doubling time (DT), DT = t × Log2/Log(N/No), in which t = time, N = final number of cells and No = initial number of cells, and the results were showed as mean ± standard deviation.

In vitro migration potential assay

The migration potential was evaluated by Scratch assay. Cells were cultured in six-well plates (in triplicate for each cell line), and when they reached approximately 95% confluence a scratch was made. The wells were photo-documented by phase contrast microscopy (Olympus IX71 and PD controller software, USA) in two different points along the scratch in each well, and identified as time 0. After 24 h, the wells were again photo-documented at the same time 0 positions and using the TScratch software (software developed by Tobias Gebäck and Schulz, ETH Zurich) the percentage of scratch area was measured in the initial and final time images. The migration potential was calculated as percentage of migrated area (%MA), %MA = 1 − (N/No), in which N = % of the final area and No = % of the initial area, and the results were showed as mean ± standard deviation.

In vivo tumorigenic potential assay

For the evaluation of the tumorigenic potential, 3 × 106 cells of medulloblastoma cell lines in 100 μl matrigel (Matrigel® matrix, Corning, USA) were infused subcutaneously into the back of Nude mice (n = 3 of each cell line), previously anesthetized with a mixture of 2% isoflurane and oxygen. Mice were monitored at 7, 15, 30 and 60 days after cell infusion and nodular volume was measured using a digital caliper. Nodular volume (NV) was calculated as NV = L × W × H × 4/3 π, which L = length, W = width and H = height, and the results were showed as mean ± standard deviation. The experiments with mice have been approved by the Ethics Committee on Animal Experiments of the Medical School of Ribeirão Preto under the protocol number 137/2011.

Statistical analysis

One-way ANOVA test was used to evaluate whether differences among the samples were statistically significant at p < 0.05. All statistical tests were performed in the statistical program GraphPad Prism 5 for Mac OS X.

Results

Cell morphology, cell growth pattern and adhesion molecules expression level of human medulloblastoma cell lines

UW402, UW473, ONS-76 and DAOY human medulloblastoma cell lines are a morphologically mixed cellular population, containing from polygonal cells to fibroblastic cells. UW402 and UW473 cell lines are similar in morphology, with most of cells with a fibroblastic shape (Fig. 1a, b), while DAOY and ONS-76 cell lines have a majority of cells with a polygonal shape (Fig. 1c, d). Regarding the cell growth pattern, UW402 and UW473 cell lines proliferate more dispersed, while DAOY and ONS-76 cell lines proliferate as aggregated cells (colony).

Fig. 1.

Fig. 1

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Cell morphology and cell growth pattern of the medulloblastoma cell lines. a UW402. b UW473. c DAOY. d ONS-76

Due to this difference in cell growth pattern (dispersed and colony) and also to investigate the origin of the cells, the gene expression level of some adhesion molecules, such as N-cadherin (CDH2, neural), E-cadherin (CDH1, epithelial) and VE-cadherin (CDH5, vascular-endothelial), was analyzed in the medulloblastoma cell lines. ONS-76 and DAOY cell lines (cell lines with a growth pattern in colony) showed the highest gene expression level of E-cadherin (p < 0.0001) (Fig. 2a) and ONS-76 cell line showed the highest gene expression level of VE-cadherin (p < 0.0001) (Fig. 2b). All medulloblastoma cell lines showed higher gene expression level of N-cadherin when compared to other cadherins, with UW402 and ONS-76 cell lines showing the highest gene expression level of N-cadherin (p < 0.0001) (Fig. 2c).

Fig. 2.

Fig. 2

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Cadherins gene expression levels in the medulloblastoma cell lines. a E-cadherin (CDH1), b VE-cadherin (CDH5) and c N-cadherin (CDH2). The results are shown as relative expression units and mean ± standard deviation. One-way ANOVA test was used to evaluate whether differences among the samples were statistically significant at p < 0.05

Cell surface proteins in human medulloblastoma cell lines

UW402, UW473, ONS-76 and DAOY human medulloblastoma cell lines were evaluated by flow cytometry for the expression of cell surface proteins related to hematopoietic cell (CD34 and CD45), endothelial cells (CD31 and CD144), medulloblastoma stem cells (CD133) and other cell surface proteins such as CD105, CD90, CD44, CD146, CD271, CD29 and CD166 in order to assess the differences and similarities among the medulloblastoma cell lines creating a immunophenotypic profile. Similarities regarding the percentage of positive cells for CD34, CD45, CD31, CD133, CD44, CD105, CD166 and CD29, and somewhat difference for CD144, CD271, CD24, CD146 and CD90 were observed among the medulloblastoma cell lines (Table 1). In all cell lines, there was a low percentage of positive cells for CD34, CD45, CD31 and CD133 and a higher percentage for CD44, CD105, CD166 and CD29. For CD144 and CD271, there was a higher percentage of positive cells from ONS-76 cell line when compared to the other cell lines (p = 0.0069, p = 0.0009, respectively) and the DAOY and ONS-76 cell lines showed a higher percentage of CD24-positive cells when compared to UW402 and UW473 cell lines (p = 0.0033). UW473 and ONS-76 cell lines showed a higher percentage of CD146-positive cells when compared to UW402 and DAOY cell lines (p = 0.0035) and the UW402 cell line showed a lower percentage of CD90-positive cells when compared to the other cell lines (p < 0.0001).

Table 1.

Analysis of the cell surface protein in the UW473, UW402, DAOY and ONS-76 medulloblastoma cell lines

Cell surface protein UW473 (%) UW402 (%) DAOY (%) ONS-76 (%)
CD34 1 ± 0.8 0.3 ± 0.1 1.1 ± 1.5 0.9 ± 1.3
CD45 0.7 ± 0.4 1.6 ± 1.6 0.7 ± 0.6 2.7 ± 3.2
CD31 0.4 ± 0.1 0.2 ± 0.3 0.6 ± 0.1 1 ± 0.5
CD133 3.1 ± 3.6 4.3 ± 0.8 5.4 ± 4.1 2 ± 2.4
CD44 90.5 ± 4.2 85.4 ± 2.4 84.3 ± 1.2 94.6 ± 0.7
CD105 96.4 ± 1.8 73 ± 0.4 81 ± 3.3 93.1 ± 1.2
CD166 75.4 ± 5.5 77.1 ± 2.8 69.7 ± 13.1 91 ± 1.1
CD29 99.5 ± 0.6 98.6 ± 0.4 95.2 ± 0.3 99.3 ± 0.1
CD144* 0.6 ± 0.5 0.4 ± 0.1 0.1 ± 0.1 14.8 ± 2.6
CD271* 27 ± 16.2 1.1 ± 0.2 0.6 ± 0.8 55.8 ± 2.3
CD24* 51.6 ± 0.8 64.6 ± 6.4 20.6 ± 6.4 25.1 ± 6
CD146* 90.7 ± 3.8 55.8 ± 4.9 34.6 ± 12 86.2 ± 2.7
CD90* 99.3 ± 0.9 4.4 ± 0.1 88.5 ± 3.3 41 ± 4.6
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* Statistically significant, p < 0.05

Migration and proliferative potential of human medulloblastoma cell lines

The migration potential of UW402, UW473, DAOY and ONS-76 human medulloblastoma cell lines was evaluated by Scratch assay. DAOY cell line showed the highest migration potential with a migrated area 83 ± 25%, 67 ± 10% e 56 ± 22% higher than the UW402, UW473 and ONS-76 cell lines, respectively (p < 0.0001) (Fig. 3a, b). The proliferative potential of UW402, UW473, DAOY and ONS-76 human medulloblastoma cell lines was measured by viable-cell counting after 120 h. ONS-76 cell line showed a proliferative potential 3.5 ± 0.1, 3.4 ± 0.4 and 1.5 ± 0.1-fold increase when compared to UW402, DAOY and UW473, respectively (p > 0.0001) (Fig. 3c). Cell doubling time was also calculated for those medulloblastoma cell lines, with the ONS-76 cell line showing a cell doubling time of 22 ± 0.3 h, UW473 of 25 ± 0.7 h, UW402 of 32 ± 0.5 h and DAOY of 32 ± 1.5 h.

Fig. 3.

Fig. 3

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In vitro migration and proliferative potential of the medulloblastoma cell lines. a Photodocumentation of the Scratch assay at 0 h and 24 h. b Quantification of Scratch assay by percentage of migrated area. c Proliferation assay by cell doubling time. The results are shown as mean ± standard deviation. One-way ANOVA test was used to evaluate whether differences among the samples were statistically significant at p < 0.05

In vivo tumorigenic potential of human medulloblastoma cell lines

UW402, UW473, DAOY and ONS-76 human medulloblastoma cell lines were evaluated for in vivo tumorigenic potential in Nude mice. Mice were monitored by measuring tumor volume at 7, 15, 30 and 60 days after cell injection. Only DAOY and ONS-76 gave rise to tumor nodules in Nude mice after 60 days of cell injection (Fig. 4a, b). From this result, it can be suggested that DAOY and ONS-76 are tumorigenic cell lines while UW402 and UW473 are non-tumorigenic cell lines according to the approach used.

Fig. 4.

Fig. 4

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In vivo tumorigenic potential of the medulloblastoma cell lines. a Photo documentation of nodule growth and b quantification of tumor volume follow up at 7, 15, 30 and 60 days after cell injection of UW402, UW473, ONS-76 and DAOY. The results are shown as mean ± standard deviation. One-way ANOVA test was used to evaluate whether differences among the samples were statistically significant at p < 0.05

Discussion

Cancer cell lines have become an essential tool for studying the tumor cell properties and to understand the molecular and cellular mechanisms underlying the tumor development and progression. Knowledge of cell lines characteristics is of great importance before studying any type of cancer because, depending on the study purpose and the type of experiment to be perform, a specific cell line should be used. In this article, we focused on characterizing four commonly used human pediatric medulloblastoma cell lines, UW402, UW473, DAOY and ONS-76, as regards cell morphology and expression of adhesion molecules, cell surface proteins, in vitro migration and proliferative potential, and in vivo tumorigenic potential.

We observed that there was a certain relationship between the cell growth pattern and E-cadherin adhesion molecule expression level. DAOY and ONS-76 cell lines showed a colony growth pattern and a higher E-cadherin expression level while UW402 and UW473 cell lines showed a dispersed growth pattern and a lower E-cadherin expression level. Neutralizing antibodies against E-cadherin inhibited colony formation in embryonic germ cells (Kanatsu-Shinohara et al. 2008), while the rescue of E-cadherin expression, re-established the cell-to-cell contact (Hawkins et al. 2012). In human embryonic stem (ES) cells, immunochemical analysis showed that the cells at the periphery of the colonies did not express the E-cadherin, suggesting an epithelial-mesenchymal transition (EMT) (Ullmann et al. 2007).

During EMT, the expression of E-cadherin is downregulated (Lee et al. 2006) and this process is important to embryogenesis, as well as to tumorigenesis (Barrallo-Gimeno 2005; Lee et al. 2006). In embryonic carcinoma cells, E-cadherin is critical for SC1-induced colony growth, and contributes to maintain undifferentiated ES cells (Du et al. 2014). In fact, a decrease in E-cadherin expression is related to advanced tumor stages, invasion, migration, metastasis and poorly differentiated tumors (He et al. 2017; Pantazi et al. 2017). In MB, overexpression of microRNA-21 or Hand-1 that inhibited cell migration, increased E-cadherin expression (Grunder et al. 2011; Asuthkar et al. 2016). However, a relationship between E-cadherin expression level and migration potential was not observed in MB cell lines studied, perhaps due to low overall E-cadherin expression level.

N-Cadherin and VE-cadherin are associated with stimulation of proliferation, invasion and metastasis, and a positive association between N-cadherin immunoreactivity and dissemination levels in primary MB samples was described (Li et al. 2009; Aragon-Sanabria et al. 2017; Rochefort et al. 2017). In the present study, MB cell lines showed higher levels of N-cadherin when compared to other cadherins, meanwhile, VE-cadherin was most expressed in the ONS-76 cell line; this cell line showed an increase in the proliferative potential when compared to other MB cell lines, and showed tumorigenic potential. ONS-76 has been shown to have a primitive and immature profile, being able to differentiate and originate a neuronal phenotype, and a high expression of neuronal markers (Sun et al. 2013; Zanini et al. 2013). In accordance with CDH5 gene expression level (VE-cadherin), ONS-76 showed a higher percentage of CD144/VE-cadherin positive cells when compared to other MB cell lines. In melanoma, VE-cadherin was exclusively expressed by highly aggressive cells and down regulation of VE-cadherin expression in the aggressive cells abrogated their ability to form vasculogenic networks, suggesting a role of VE-cadherin+ cells in tumor vasculogenesis independent of endothelial cells (Hendrix et al. 2001). With this, we can suggest that VE-cadherin+ ONS-76 cells may have the same ability to form vasculogenic networks without the presence of endothelial cells.

Cell surface proteins can be useful as biomarker. These proteins showed roles in many biological functions, including cell-to-cell communication and responses to external environment; and could be useful to distinguish different medulloblastoma cell lines (Arcinas et al. 2010; Gedye et al. 2014; Liang et al. 2015). UW402, UW473, DAOY and ONS-76 showed a low percentage of CD34, CD45 and CD31 positive cells, as expected, because CD34 and CD45 are expressed mostly in hematopoietic cells and CD31 in mature endothelial cells (Woodford-Thomas and Thomas 1993; Krause et al. 1998; Woodfin et al. 2007). These cell lines showed a high percentage of CD44, CD105, CD106 and CD29 positive cells, which are markers expressed in mostly tumor cell lines (Fonsatti et al. 2010; Geng et al. 2013; Liang et al. 2015; Sneha et al. 2017).

CD133 is considered the most important cancer stem cells (CSC) marker in nervous system tumors, including medulloblastoma (Singh et al. 2003, 2004). As expected for a CSC subpopulation, the percentage of CD133 was lower in all analyzed medulloblastoma cell lines. CSC showed to be more resistant to treatments and an increased presence of these cells is related to an increased tumor recurrence and makes the tumor more aggressive (Blazek et al. 2007; Raso et al. 2008). Sphere assay is a validated method to study CSC in vitro (Read et al. 2009) and Zanini et al. (2013) showed that DAOY and ONS-76 medulloblastoma cell lines are able to form spheres in vitro (Zanini et al. 2013).

We observed a higher percentage of CD271+ ONS-76 cells when compared to other cell lines. CD271 is known as p75 neurotrophin receptor (p75NTR) or nerve growth factor receptor (NGFR). This marker was expressed in primary SHH MB samples, and selected for lower self-renewing progenitors or CSC (Liang et al. 2015). CD271 overexpression resulted in a decrease of in vitro tumorsphere number but an increase in tumorsphere size (Liang et al. 2015), and it was demonstrated that ONS-76 formed larger tumorspheres when compared to other MB cell lines (Zanini et al. 2013).

CD24 glycoprotein controls neuronal differentiation, proliferation and immune response. This marker is overexpressed in several cancers (Calaora et al. 1996; Chou et al. 2007; Cao et al. 2012) and comparing to other brain tumor, MB showed a higher expression of CD24 (Sandén et al. 2015). CD44+/CD24−/low cells are described to characterize breast stem cells and more aggressive cells (Ricardo et al. 2011), and ONS-76 and DAOY showed a higher percentage of CD44+ cells and CD24− cells, and a higher in vitro migration potential and in vivo tumorigenic potential when compared to UW402 and UW473 cell lines. This result can suggest that CD44+/CD24− profile also could be useful to distinguish more aggressive cell in medulloblastoma. CD146 was demonstrated as a target for CD44 signaling, and this protein could inhibit breast tumor progression (Ouhtit et al. 2017). DAOY showed a lower expression of CD146, and this cell line showed a higher proliferation, migration and tumorigenic potential.

CD90 (Thy-1) is a GPI-anchored cell surface protein with role in cell–cell and cell–matrix interaction. This protein is expressed on neurons, endothelial cells, mesenchymal stem cells, and could be overexpressed in the tumor microenvironment (Kumar et al. 2016). CD90 overexpression has been shown to promote cell migration and sphere formation in hepatocellular carcinoma cells (Zhang et al. 2018). In the present study, CD90 could be useful to distinguish UW402 cell line, that showed a lower percentage of positive cells for this marker when compared to other MB cell lines.

Briefly, although being derived from the same tumor type, MB cell lines showed functional and molecular differences among them, which should be considered by the researcher to choose the ideal cellular model according to the hypothesis of the study proposal, and, therefore, the results from the present study are useful in this stage of experimental design.

Acknowledgments

We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Process Numbers: 2011/18664-7 and 2011/20829-4 for the financial support for this study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

All the animal procedures of this study were done in agreement with the ETHICAL PRINCIPLES IN ANIMAL RESEARCH adopted by the Brazilian College of Animal Experimentation (COBEA) and this study was approved by the Ethics Committee on Animal Experiments of the Ribeirão Preto Medical School under Protocol Number 137/2011.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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