Mesenchymal Neuroblastoma Cells Lack Critical Transporters for mIBG Uptake and May Evade Detection and Therapy

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MES neuroblastoma cells are linked to increased motility and chemoresistance, but their capacity to accumulate meta-iodobenzylguanidine (mIBG)—a cornerstone in neuroblastoma imaging and therapy—remains unclear. We hypothesized that MES cells fail to take up mIBG due to reduced expression of the norepinephrine transporter (NET) and vesicular monoamine transporters (VMAT1/2). To investigate this, we analyzed NET, VMAT1, and VMAT2 mRNA expression in 33 neuroblastoma cell lines and correlated results with ADRN and MES transcriptional signatures. Validation was performed by real-time quantitative PCR (RT-qPCR) in 12 lines (3 MES, 9 ADRN). Additionally, two ADRN cell lines (SK-N-BE(2)C-PRRX1 and SH-SY5Y-NOTCH3) were induced to undergo MES transition and monitored by time-series RT-qPCR. Functional uptake and retention of [ 125 I]mIBG were assessed in seven cell lines (3 MES, 4 ADRN), with desipramine and reserpine used to confirm transporter specificity. Results MES cell lines showed markedly lower expression of NET, VMAT1, and VMAT2 compared with ADRN lines. Induction of a MES phenotype in ADRN cells led to a progressive reduction in transporter expression. Functional assays demonstrated that MES cells lacked the ability to accumulate or retain mIBG. Conclusion MES neuroblastoma cells are deficient in critical transporters required for mIBG uptake. These findings suggest that MES-predominant tumors may escape detection by mIBG imaging and resist mIBG-based radionuclide therapy, underscoring the need to account for tumor cell state in clinical imaging and treatment planning. Neuroblastoma meta-iodobenzylguanidine (mIBG) norepinephrine transporter (NET) vesicular monoamine transporter (VMAT1 VMAT2) adrenergic mesenchymal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Neuroblastoma is the most common extracranial solid tumor in children and arises from neural crest cells. [ 1 ] Intertumor heterogeneity is a hallmark of neuroblastoma and underlies its highly variable clinical course, which ranges from spontaneous regression to aggressive metastatic disease and death. [ 2 ] Despite recent advances in survival due to anti-GD2 immunotherapy [ 3 , 4 ], only about 50% of high-risk neuroblastoma patients survive long-term. [ 5 ] Relapsed disease remains a major therapeutic challenge in neuroblastoma, with post-relapse survival being exceedingly rare. [ 6 ] Approximately 90% of neuroblastomas express the norepinephrine transporter (NET) [ 7 ], which allows for the application of meta-iodobenzylguanidine (mIBG), a norepinephrine analog, in both diagnostic imaging and therapy. [ 8 ] mIBG labeled with iodine-123 is a highly NET-selective radiopharmaceutical used for imaging [ 9 ], while iodine-131-labeled mIBG has been employed therapeutically since 1984 to treat neuroblastoma. [ 10 ] Previous studies have linked NET mRNA expression to mIBG avidity [ 11 ], and subsequent data indicate a correlation between NET protein expression and mIBG uptake in tumors. [ 12 ] However, inconsistencies have been observed: certain mIBG-non-avid tumors demonstrate high NET expression, while some mIBG-avid tumors show unexpectedly low levels of NET expression. The vesicular monoamine transporters (VMAT1 and VMAT2) may also contribute to mIBG uptake by enabling vesicular storage of mIBG [ 13 ], yet the mechanisms driving the heterogeneous expression of these transporters remain poorly understood. Interestingly, a recent clinical study by Batra et al. found that lower NET and VMAT2 protein expression were unexpectedly associated with a higher likelihood of response to [ 131 I]mIBG therapy in patients with relapsed or refractory neuroblastoma, challenging traditional assumptions about the predictive value of transporter expression for therapeutic efficacy. [ 14 ] The epithelial–mesenchymal transition (EMT) is a cellular program that confers motility and invasiveness [ 15 ], playing a key role in tumor progression, metastasis, and therapy resistance. [ 16 ] While EMT and its reverse process, mesenchymal-epithelial transition (MET), are widely studied in various cancers [ 17 , 18 ], these processes are also implicated in the heterogeneity of neuroblastoma. [ 19 – 22 ] Mesenchymal (MES) neuroblastoma cells exhibit increased motility [ 23 ] and resistance to chemotherapy compared to lineage-committed adrenergic (ADRN) cells. [ 21 , 22 ] Moreover, MES cells lack the oncogene ALK , rendering them resistant to ALK inhibitors [ 24 , 25 ], and show reduced expression of GD2, leading to resistance against anti-GD2 antibody therapy [ 26 ], a cornerstone of high-risk neuroblastoma treatment. Our previous work demonstrated that MES neuroblastoma cells can escape detection in minimal residual disease (MRD) assays that rely solely on ADRN-specific markers. To address this, we developed a mesenchymal-specific MRD panel. [ 27 ] In this study, we investigated the expression of NET, VMAT1, and VMAT2, as well as mIBG uptake in MES and ADRN neuroblastoma cells in vitro . We hypothesized that MES cells lack the transporters necessary for mIBG uptake, rendering them invisible to mIBG imaging and resistant to mIBG-based therapies (Fig. 1 ). Material and methods Cell lines and culture conditions Human neuroblastoma cell lines (ATCC®, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO®, Thermo Fisher Scientific, Waltham, USA), supplemented with fetal calf serum, L-glutamine, penicillin-streptavidin, and non-essential amino acids as described previously. [ 23 ] Cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 . The patient-derived 691-MES (AMC691T) and 691-ADRN (AMC691B) neuroblastoma cell lines were cultured under previously reported conditions. [ 28 ] SK-N-BE(2)-C and SH-SY5Y neuroblastoma cells with inducible PRRX1A and NOTCH3 expression, respectively, were used as described in earlier studies. [ 21 , 22 ]. Doxycycline (100 ng/ml) was added to induce PRRX1A or NOTCH3 transgene expression. HEK293 cells (ATCC® CRL-3216™, Manassas, USA) were transiently transfected with a human NET plasmid (addgene plasmid #15475; HEK-NET) or an empty vector (HEK-EV) as controls. [ 29 ] Cell identity was verified by short tandem repeat (STR) analysis, and mycoplasma contamination was routinely checked. Mesenchymal and adrenergic signature scores and monoamine transporter gene expression The MES and ADRN gene signature scores were developed as described by Van Groningen and colleagues. [ 22 ] These scores were based on mRNA expression data from four isogenic MES and ADRN neuroblastoma cell line pairs, identifying 485 MES-specific and 369 ADRN-specific genes. Signature scores for each cell line were calculated by averaging expression levels across the genes in each signature. Signature scores and gene expression data for norepinephrine transporter (NET, SLC6A2 ), vesicular monoamine transporter 1 (VMAT1, SLC18A1 ), and vesicular monoamine transporter 2 (VMAT2, SLC18A2 ) were extracted from a neuroblastoma cell line panel available on the R2 genomics platform ( http://r2.amc.nl/ ; GEO accession number GSE28019). [ 23 ] The data cover 33 neuroblastoma cell lines, which are listed in Supplementary Table 1 . Expression data were log2-transformed for statistical analysis and figure presentation. mRNA expression using real-time quantitative polymerase chain reaction (RT-qPCR) NET, VMAT1, and VMAT2 mRNA expression was analyzed across 12 human neuroblastoma cell lines (691-ADRN, 691-MES, SH-SY5Y, SH-EP2, GI-ME-N, IMR-32, N206, SJNB-1, SJNB-8, NGP, SK-N-SH, and SK-N-BE) using RT-qPCR. RNA was extracted using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. Methods for cDNA synthesis and RT-qPCR were performed as described previously. (van Wezel, Zwijnenburg et al. 2015) Normalization of NET/VMAT expression was done using β-glucuronidase (GUS) as the reference gene, with the formula: normalized cycle threshold (ΔC T ) = C T GUS − C T NET/VMAT. [ 30 ] In PRRX1 -inducible SK-N-BE(2)-C cells, Beta-2-microglobulin (B2M) served as the internal control due to its stable expression. Samples without amplification after 40 cycles were scored as negative. All qPCR reactions were performed in triplicate, and results are presented as ΔC T values (mean ± SD). Primers and probes for VMAT1 and VMAT2 were designed based on previously published gene sequences (ENSEMBL: VMAT1 transcript ENST00000276373.10; VMAT2 transcript ENST00000644641.2). [ 31 ] Oligonucleotides were synthesized by Eurogentec (Liège, Belgium), with detailed sequences provided in Table 1 . Primer/probe sets for β-glucuronidase (GUS), Beta-2-microglobulin (B2M), and the norepinephrine transporter (NET) were adopted from earlier studies. [ 31 , 32 ] Table 1 Primer and probe combinations for VMAT1 and VMAT2 VMAT1 ( SLC18A1 ) Vesicular monoamine transporter 1 Exon Forward primer Reverse primer Probe 5’- GCT TTT TCT GGG ACC TAT ACT CTA CTC T − 3’ 5’- CAA GAC CTG CAA CAG ATG AAA ATG − 3’ 5’FAM- TGT GGC CCG AAC CCT TCA AGG C − 3’TAMRA 5 6 5–6 VMAT2 ( SLC18A2 ) Vesicular monoamine transporter 2 Forward primer Reverse primer Probe 5’- GCT GCT GAA GGA CCC GTA CA -3’ 5’- GCG ATG CCC ATG TTT GC -3’ 5’FAM – CTC ATT GCT GCA GGC TCC ATC TGC TT − 3’TAMRA 9 10 9–10 [ 125 I]mIBG uptake and retention experiments Human neuroblastoma cells were grown in 12-well plates at 80–90% confluence for uptake experiments. Culture medium was replaced with fresh medium containing 1.0 µM of mIBG and a fixed concentration of [ 125 I]mIBG (3.7 kBq/ml; specific activity ≈ 0.65 TBq/mmol; Chelatec, Saint-Herblain, France). For some conditions, NET inhibitor (desipramine) or VMAT inhibitor (reserpine) was added at 1.0 µM (all inhibitors Sigma-Aldrich, Burlington, USA). Cells were incubated for 120 minutes at 37°C with 5% CO2. Following incubation, cells were washed with ice-cold Hank’s Balanced Salt Solution (HBSS) to halt further uptake and preserve intracellular content, then lysed with 0.2 M sodium hydroxide. Cell-associated radioactivity was measured in Ultima Gold® scintillation fluid (PerkinElmer, Waltham, USA) using a PerkinElmer Tri-Carb liquid scintillation counter (4910TR) and normalized to total protein content. Uptake was expressed as nanomoles of mIBG per milligram of protein per hour. All assays were performed in triplicate. For retention studies, neuroblastoma cells were preloaded with radioactive mIBG for 2 hours under the same conditions. After washing, fresh non-radioactive medium was added, with or without inhibitors. Radioactivity retained by cells was measured after 4 and 24 hours of incubation. Control experiments included PC-12 rat pheochromocytoma cells, fibroblasts, and HEK293 cells transfected with NET (HEK-NET) or empty vector (HEK-EV). Data analysis All data are presented as mean ± SD. Group means were compared using Welch’s t-test. Pearson’s correlation test was used to evaluate the relationship between microarray and RT-qPCR expression data. Statistical analyses were performed using GraphPad Prism 9.3.0 (GraphPad Software, San Diego, USA). Statistical significance was indicated by p-values: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001. Results MES neuroblastoma cells lack NET, VMAT1, and VMAT2 mRNA expression To investigate the relationship between adrenergic (ADRN) and mesenchymal (MES) gene signature scores and the expression of NET, VMAT1, and VMAT2 mRNA, we visualized the data using the R2 Genomics Analysis and Visualization platform ( http://r2.amc.nl/ ) (Fig. 2 ). Data from 33 neuroblastoma cell lines were analyzed; raw expression values and signature scores are listed in Supplementary Table 1 . In the bottom-right quadrant of the plots—characterized by high ADRN and low MES scores—NET expression was markedly elevated, with a median of 8.2 (interquartile range [IQR] 7.4–9.0; Fig. 2 a). In contrast, the top-left quadrant, representing high MES and low ADRN scores, showed significantly lower NET expression (median 1.9, IQR 1.3–2.8; p = 0.0006). A similar pattern was observed for VMAT1 (Fig. 2 b), with expression levels of 5.4 (IQR 3.6–7.4) in ADRN-high/MES-low cell lines, compared to 2.7 (IQR 1.3–3.3) in MES-high/ADRN-low cell lines (p = 0.0004). VMAT2 expression followed the same trend (Fig. 2 c), with 5.1 (IQR 4.6–8.4) in ADRN-high/MES-low cells and 3.0 (IQR 2.2–3.5) in MES-high/ADRN-low cells (p = 0.0006). These data indicate that NET, VMAT1, and VMAT2 mRNA expression is significantly lower in MES-type neuroblastoma cell lines, with NET showing the strongest association with the ADRN phenotype. Validation of NET, VMAT1, and VMAT2 mRNA expression To validate the differential expression of NET, VMAT1, and VMAT2 mRNA, we performed RT-qPCR on 12 human neuroblastoma cell lines, including two isogenic pairs (SH-SY5Y with SH-EP2, and 691-ADRN with 691-MES) (Fig. 3 ). RT-qPCR results showed a strong correlation with microarray data for NET (r = 0.9451; Fig. 3 a). Low NET expression (reflected by low ΔC T values) was consistently observed in MES-type cell lines (691-MES, SH-EP2, and GI-ME-N). Among the ADRN-type lines, SJNB-8 stood out as an outlier (ΔC T = − 11.4), lacking a clear ADRN or MES identity (ADRN score − 0.43; MES score 0.20). Similarly, strong correlations were found for VMAT1 (r = 0.8884; Fig. 3 b) and VMAT2 (r = 0.8074; Fig. 3 c) between RT-qPCR and microarray expression data. These findings confirm that MES neuroblastoma cell lines exhibit significantly lower mRNA expression of NET, VMAT1, and VMAT2, and strongly support the reliability of the microarray data. Downregulation of NET, VMAT1, and VMAT2 expression during ADRN-to-MES reprogramming To further investigate whether acquisition of a MES phenotype is associated with downregulation of NET, VMAT1, and VMAT2, we analyzed their mRNA expression during ADRN-to-MES reprogramming in two inducible cell models. In SK-N-BE(2)C cells, reprogramming was triggered by doxycycline-induced expression of PRRX1, with successful MES transition confirmed by upregulation of the mesenchymal marker POSTN (Fig. 4 e). Over a 28-day time course, NET (Fig. 4 a), VMAT1 (Fig. 4 b), and VMAT2 (Fig. 4 c) mRNA levels were significantly reduced in PRRX1-induced cells (+ dox) compared to non-induced controls (–dox), with significant differences already apparent by day 3 (p = 0.0082, p = 0.0013, and p = 0.0362, respectively), and further decreases observed through day 28. Consistent results were obtained in a second model, SH-SY5Y-NOTCH3 cells, in which NOTCH3 induction led to marked downregulation of NET, VMAT1, and VMAT2 mRNA as early as day 3. No such changes were observed in wild-type SH-SY5Y cells treated with doxycycline ( Supplemental Fig. 1 ). A mesenchymal marker was not assessed in this model. Together, these findings show that NET, VMAT1, and VMAT2 are consistently downregulated during the ADRN-to-MES transition, supporting their strong association with the ADRN cell state. MES neuroblastoma cells lack functional mIBG uptake To assess whether MES neuroblastoma cells possess functional transporters required for mIBG uptake, we performed uptake assays using [ 125 I]mIBG (Fig. 5 ). ADRN cell lines (691-ADRN, SK-N-SH, SH-SY5Y, and SK-N-BE) showed robust mIBG uptake, with SK-N-SH exhibiting the highest uptake (0.95 ± 0.24 nmol/mg/h) and SH-SY5Y the lowest (0.42 ± 0.14 nmol/mg/h). Uptake in these lines was largely NET-dependent, as the NET inhibitor desipramine significantly reduced mIBG uptake (p < 0.0001). In contrast, the VMAT inhibitor reserpine significantly inhibited mIBG uptake in the pheochromocytoma cell line PC12 (p = 0.00073) but had no significant effect on uptake in ADRN neuroblastoma cells, suggesting minimal VMAT involvement in these lines. MES cell lines (691-MES, SH-EP2, GI-ME-N) demonstrated minimal mIBG uptake, comparable to negative controls lacking monoamine transporters (HEK-EV and fibroblasts), and this uptake was not affected by NET or VMAT inhibition. Extending incubation times did not increase mIBG uptake in MES cells. These findings indicate that MES neuroblastoma cells lack functional NET-mediated mIBG uptake, highlighting a key functional difference between MES and ADRN phenotypes. MES neuroblastoma cells show no mIBG retention To assess mIBG retention, cells were pre-loaded with [ 125 I]mIBG, washed, and then incubated with fresh medium for either 4 or 24 hours (Fig. 6 ). ADRN cell lines retained significant amounts of mIBG (> 59% after 4 hours and > 51% after 24 hours), as long as NET function was preserved (no desipramine). Inhibition of VMAT with reserpine did not affect retention. In contrast, MES cell lines (691-MES, SH-EP2, GI-ME-N) showed no mIBG retention, mirroring the behavior of control cells lacking monoamine transporters (HEK-EV, fibroblasts). No retention was observed under any inhibitory conditions. These findings confirm that MES neuroblastoma cells lack the ability to retain mIBG, and that VMAT does not significantly contribute to mIBG retention in ADRN neuroblastoma cells. Discussion In this study, we investigated the impact of mesenchymal differentiation in neuroblastoma on the uptake of meta-iodobenzylguanidine (mIBG) through the norepinephrine transporter (NET) and vesicular monoamine transporter (VMAT). Our findings indicate that MES neuroblastoma cell lines exhibit either undetectable or low mRNA expression levels of NET, VMAT1, and VMAT2 compared to cell lines that exhibit a lineage-committed adrenergic (ADRN) phenotype. Additionally, the reprogramming of ADRN cell lines into a mesenchymal phenotype via PRRX1 and NOTCH3 induction resulted in a dramatic decrease in mRNA expression of NET, VMAT1, and VMAT2 over time. Crucially, MES neuroblastoma cells are unable to concentrate or retain mIBG in vitro due to the absence of these essential monoamine neurotransmitter transporters. Neuroblastoma cell lines can adopt either a lineage-committed ADRN phenotype or an immature MES phenotype based on distinct lineage-specific core regulatory networks. [ 19 , 21 , 22 ] These findings align with previous studies highlighting the morphological and biochemical heterogeneity of neuroblastoma cells in vitro . [ 33 ] Biedler and colleagues originally classified neuroblastoma cells into two phenotypes: N-type (neuroblast-like cells exhibiting catecholamine activity) and S-type (large, flattened, adhesive cells). Subsequent research revealed spontaneous bidirectional interconversion between these cell types [ 34 ] and described an intermediate type (I-type) representing a transitional state in the trans-differentiation process. [ 35 ] More recently, studies have shown that MES neuroblastoma cells are not detectable by current minimal residual disease mRNA marker panels. [ 27 ] Notably, MES cells exhibit relative resistance to chemotherapy [ 22 ], and those harboring ALK mutations show resistance to ALK inhibitor treatment. [ 24 , 25 ] Therefore, MES cells are prime candidates for therapeutic escape, underscoring the need to evaluate and optimize detection methods for their sensitivity to MES cells. While our findings demonstrate that MES neuroblastoma cells are unable to concentrate or retain mIBG in vitro, the clinical implications of this observation remain uncertain. The timing and localization of mesenchymal phenotype acquisition by neuroblastoma cells in vivo have yet to be clearly established. Notably, single-cell RNA sequencing studies have not identified MES-like cells in primary neuroblastoma tumors. [ 36 , 37 ] Nonetheless, key questions persist regarding the role of MES neuroblastoma cells in treatment-induced selection, metastatic dissemination, and drug-resistant disease recurrence following an initial complete response. It remains speculative whether MES neuroblastoma cells can evade detection by mIBG imaging, potentially contributing to mIBG non-avid disease and reduced sensitivity to targeted radionuclide therapy. If MES cells exist as a minor subpopulation within primary tumors, their resistance to radiation may be mitigated by cross-fire effects from high-energy β-emissions of [ 131 I]mIBG absorbed by neighboring ADRN cells. [ 38 ] However, if MES cells occur as solitary, migratory entities with metastatic potential—possibly driving relapse after apparent remission, as some data suggest [ 27 ] – their lack of mIBG uptake becomes a more pressing concern. Understanding the in vivo behavior and therapeutic susceptibility of MES neuroblastoma cells is therefore critical for improving the efficacy of mIBG-based treatment strategies. Our results indicate that mIBG retention in ADRN cell lines relies solely on the efficient re-uptake of accumulated mIBG by NET, rather than vesicular sequestration by VMAT. This finding aligns with earlier studies demonstrating that mIBG loading in neuroblastoma cells is predominantly governed by cytoplasmic retention and efficient recycling by NET located on the plasma membrane, rather than VMAT-controlled vesicular sequestration. [ 39 – 43 ] While NET has long been regarded as the primary transporter responsible for mIBG uptake in neuroblastoma, recent research has identified a positive correlation between VMAT2 protein expression and mIBG tumor avidity, suggesting a potential role for VMAT2-mediated vesicular mIBG sequestration. [ 13 ] However, this study primarily provides statistical correlation between VMAT2 expression and mIBG avidity without elucidating the underlying mechanisms governing mIBG transport in neuroblastoma cells or the specific role of VMAT in this process. Therefore, while VMAT's role in mIBG uptake and retention is well established in pheochromocytomas and carcinoid tumors [ 44 ], our study suggests a minor role for VMAT in neuroblastoma tumors, indicating that low VMAT expression in MES neuroblastoma cells may be of limited significance. We recognize several limitations to our study. Primarily, our findings are based on in vitro experiments using neuroblastoma cell lines, which may not fully capture the complexity of tumor behavior in vivo . Further research is needed to determine whether, when, and where neuroblastoma cells adopt a mesenchymal phenotype in patients. Functional studies using mesenchymal tumor organoids or in vivo xenograft models may offer valuable insight into the biological role of MES cells and their relevance for mIBG avidity. In addition, mIBG uptake and retention experiments were not performed in the two transgenic adrenergic cell lines containing inducible regulators of mesenchymal transformation. Including these models in future studies could provide important mechanistic insights into the regulation of NET function and mIBG uptake during the ADRN-to-MES transition. Conclusions In conclusion, our findings indicate that MES neuroblastoma cells lack mIBG uptake in vitro due to the absence of NET expression. This has significant clinical implications, as MES neuroblastoma cells may evade detection through mIBG imaging and potentially escape targeted radionuclide mIBG therapy. Abbreviations ADRN adrenergic MES mesenchymal mIBG meta-iodobenzylguanidine NET norepinephrine transporter VMAT vesicular monoamine transporter RT-qPCR real-time quantitative PCR mRNA Messenger-ribonucleic acid GD2 Disialoganglioside2 EMT epithelial–mesenchymal transition MET mesenchymal-epithelial transition ALK Anaplastic lymphoma kinase MRD minimal residual disease ATCC American Type Culture Collection DMEM Dulbecco's Modified Eagle Medium STR short tandem repeat GUS β-glucuronidase B2M Beta-2-microglobulin C T Threshold cycle HBSS Hank’s Balanced Salt Solution IQR interquartile range dox doxycycline Declarations Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate were not sought for this study, as it did not involve human participants or animal subjects. Consent for publication Not applicable. Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Competing Interests The authors declare that they have no competing interests. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Authors’ Contributions Study conception and design: TB JvW RM RL JvN EvdS AvK GT Material preparation and data collection: TB JvW RM RL JvN Data analysis: TB EvdS AvK GT First draft of the manuscript was written by TB AvK GT All authors (TB JvW RM RL JvN EvdS AvK GT) commented on previous versions of the manuscript. All authors (TB JvW RM RL JvN EvdS AvK GT) read and approved the final manuscript. Acknowledgements Robin van Amersfoort, Michelle Muller, and Renate Bezemer worked on this study as part of their scientific internship. 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Beillard, E., et al., Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia, 2003. 17 (12): p. 2474-86. Biedler, J.L., L. Helson, and B.A. Spengler, Morphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture. Cancer Res, 1973. 33 (11): p. 2643-52. Ross, R.A., B.A. Spengler, and J.L. Biedler, Coordinate morphological and biochemical interconversion of human neuroblastoma cells. J Natl Cancer Inst, 1983. 71 (4): p. 741-7. Ciccarone, V., et al., Phenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages. Cancer Res, 1989. 49 (1): p. 219-25. Dong, R., et al., Single-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma. Cancer Cell, 2020. 38 (5): p. 716-733 e6. Kildisiute, G., et al., Tumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell. Sci Adv, 2021. 7 (6). Giammarile, F., et al., EANM procedure guidelines for 131I-meta-iodobenzylguanidine (131I-mIBG) therapy. Eur J Nucl Med Mol Imaging, 2008. 35 (5): p. 1039-47. Smets, L.A., et al., Extragranular storage of the neuron blocking agent meta-iodobenzylguanidine (MIBG) in human neuroblastoma cells. Biochem Pharmacol, 1990. 39 (12): p. 1959-64. Gaze, M.N., et al., Intracellular localization of metaiodobenzyl guanidine in human neuroblastoma cells by electron spectroscopic imaging. Int J Cancer, 1991. 47 (6): p. 875-80. Lashford, L.S., J.P. Hancock, and J.T. Kemshead, Meta-iodobenzylguanidine (mIBG) uptake and storage in the human neuroblastoma cell line SK-N-BE(2C). Int J Cancer, 1991. 47 (1): p. 105-9. Mairs, R.J., M.N. Gaze, and A. Barrett, The uptake and retention of metaiodobenzyl guanidine by the neuroblastoma cell line NB1-G. Br J Cancer, 1991. 64 (2): p. 293-5. Montaldo, P.G., et al., Accumulation of m-iodobenzylguanidine by neuroblastoma cells results from independent uptake and storage mechanisms. Cancer Res, 1991. 51 (16): p. 4342-6. Kolby, L., et al., Uptake of meta-iodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters. Br J Cancer, 2003. 89 (7): p. 1383-8. Supplementary Files SupplementaldataEMT.pdf Supplementary Table 1 Adrenergic and Mesenchymal Signature Scores, and NET, VMAT1, and VMAT2 mRNA Expression in 33 Neuroblastoma Cell Lines This table presents Affymetrix microarray mRNA expression data for 33 human neuroblastoma cell lines. Expression levels for NET (columns 2–3), VMAT1 (columns 4–5), and VMAT2 (columns 6–7) are shown alongside Adrenergic (ADRN) and Mesenchymal (MES) signature scores (columns 8–9). Cell lines highlighted in grey were selected for further experiments, including RT-qPCR and [ 125 I]mIBG uptake and retention studies. Supplemental Fig 1 mRNA expression of NET, VMAT1, VMAT2 using RT-qPCR after doxycycline-induced NOTCH3 expression in SH-SY5Y-NOTCH3 Results are expressed as mean ± SD (n=2, experiments in triplicate).Open black symbols are used for the doxycycline-induced (+dox) timepoints, control (-dox) timepoints are depicted by closed grey symbols. The cell line SH-SY5Y-NOTCH3 is depicted in circles, the wild-type (WT) SH-SY5Y cell line is depicted in rectangles. mRNA expression was measured at baseline (T=0) and after 72 hours. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7480401","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":517600069,"identity":"927bd3db-12da-4830-8a58-c1cf7238b055","order_by":0,"name":"Thomas Blom","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4368-1331","institution":"Princess Maxima Center for Pediatric Oncology: Prinses Maxima Centrum voor Kinderoncologie","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Blom","suffix":""},{"id":517600070,"identity":"5271ab03-685a-4834-9e68-be0778107b1a","order_by":1,"name":"Jalenka van Wijk","email":"","orcid":"","institution":"Sanquin Research","correspondingAuthor":false,"prefix":"","firstName":"Jalenka","middleName":"van","lastName":"Wijk","suffix":""},{"id":517600071,"identity":"117d9889-676c-44c8-a824-f27006df1b63","order_by":2,"name":"Rutger Meinsma","email":"","orcid":"","institution":"Amsterdam UMC - Locatie VUMC: Amsterdam UMC Locatie VUmc","correspondingAuthor":false,"prefix":"","firstName":"Rutger","middleName":"","lastName":"Meinsma","suffix":""},{"id":517600072,"identity":"dbd2354a-f14c-47ae-ba9a-f2124b7473bc","order_by":3,"name":"René Leen","email":"","orcid":"","institution":"Amsterdam UMC - Locatie VUMC: Amsterdam UMC Locatie VUmc","correspondingAuthor":false,"prefix":"","firstName":"René","middleName":"","lastName":"Leen","suffix":""},{"id":517600073,"identity":"b72cf486-20e8-4164-b7ba-6fa2cb2a3932","order_by":4,"name":"Johan van Nes","email":"","orcid":"","institution":"Amsterdam UMC - Locatie AMC: Amsterdam UMC Locatie AMC","correspondingAuthor":false,"prefix":"","firstName":"Johan","middleName":"van","lastName":"Nes","suffix":""},{"id":517600074,"identity":"89ab74fe-c1e9-441e-a50f-6a502a4e56e1","order_by":5,"name":"C. 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1","display":"","copyAsset":false,"role":"figure","size":415566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis figure illustrates the hypothesis that mesenchymal (MES) neuroblastoma cells (in orange), which lack norepinephrine transporters (NET) and vesicular monoamine transporters (VMAT), are unable to effectively concentrate mIBG. As a result, these cells may go undetected on mIBG imaging and evade targeted radionuclide mIBG therapy. In contrast, mIBG uptake in adrenergic (ADRN) neuroblastoma cells (in blue) via NET and VMAT transporters leads to mIBG avidity on imaging. Note: Physiological uptake in the heart, liver, and salivary glands is observed in the non-avid mIBG scan shown. This scan serves as an illustrative example, not as evidence of mesenchymal neuroblastoma cells in a patient.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/3813b6c15848e5e1d00cfac5.png"},{"id":92679544,"identity":"893a3a47-fdee-4ad1-822b-9212e89e68e2","added_by":"auto","created_at":"2025-10-03 01:00:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdrenergic and mesenchymal signature scores with NET, VMAT1, and VMAT2 mRNA expression in neuroblastoma cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA expression levels for NET (panel A), VMAT1 (panel B), and VMAT2 (panel C) are plotted for 33 human neuroblastoma cell lines, in relation to their Adrenergic (ADRN) and Mesenchymal (MES) signature scores (x- and y-axes, respectively). Yellow represents high expression, while purple indicates low expression. Cell lines used in mIBG uptake and retention studies are labeled in panel A.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/55f3c021394241a11b1ff086.png"},{"id":92679542,"identity":"40b0c608-1373-47d2-bbb4-b709ae98ca10","added_by":"auto","created_at":"2025-10-03 01:00:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation between RT-qPCR and Affymetrix microarray mRNA expression data for NET, VMAT1, and VMAT2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePanel a: NET mRNA expression. Panel b: VMAT1 mRNA expression. Panel c: VMAT2 mRNA expression. 2-log-transformed Affymetrix microarray mRNA expression values are plotted on the x-axis, while RT-qPCR mean ΔCT values are plotted on the y-axis. The dotted line represents the best fit, determined by linear regression for the entire group of 12 neuroblastoma cell lines, all of which are labeled in the plots.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/a0c32a9dd506a1a877aac40a.png"},{"id":92681002,"identity":"7553e42a-4929-44b2-bacb-e6307eda2c2e","added_by":"auto","created_at":"2025-10-03 01:08:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime course of RT-qPCR mRNA expression of NET, VMAT1, and VMAT2 following doxycycline-induced PRRX1 expression in SK-N-BE(2)C-PRRX1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are presented as mean ± SD, based on triplicates from a representative experiment, with confirmation from a repeat experiment with fewer time points. Control time points without doxycycline (-dox) are shown in orange, and doxycycline-induced (+dox) time points are shown in blue. Samples were considered ‘negative’ if no amplification was detected within 40 cycles.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/e2f8cf0d67f1493e06606382.png"},{"id":92682461,"identity":"a7477a10-f263-42a5-85dc-941ad72d71ec","added_by":"auto","created_at":"2025-10-03 01:16:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emIBG uptake in ADRN and MES neuroblastoma cell lines under various inhibitory conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emIBG uptake was measured in four ADRN neuroblastoma cell lines (691-ADRN, SK-N-SH, SH-SY5Y, SK-N-BE) and three MES cell lines (691-MES, SH-EP2, GI-ME-N) following 120 minutes of incubation with [125I]mIBG. Uptake was assessed under four conditions: no inhibitor (blue bars), desipramine (NET inhibitor; green bars), reserpine (VMAT inhibitor; orange bars), and combined desipramine plus reserpine treatment (yellow bars). HEK-NET and PC12 cells served as positive controls, while HEK-EV and fibroblasts were negative controls. Data represent mean ± SD from 2–4 independent experiments, each performed in triplicate. mIBG uptake is expressed as nanomoles per milligram of protein per hour. Statistical significance is indicated as ***p ≤ 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/d6cd18fedcd17707689d4435.png"},{"id":92679562,"identity":"a2a8396b-9bdd-4721-aa74-61bff874bc0d","added_by":"auto","created_at":"2025-10-03 01:00:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":253131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emIBG retention in ADRN and MES neuroblastoma cell lines under inhibitor treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emIBG retention was measured in three ADRN neuroblastoma cell lines (691-ADRN, SK-N-SH, SH-SY5Y) and three MES cell lines (691-MES, SH-EP2, GI-ME-N) following 120 minutes of incubation with [125I]mIBG (t = 0). After washing, cells were incubated in mIBG-free medium, and residual cell-associated radioactivity was measured at 4 and 24 hours. Retention was assessed under four conditions: no inhibitor (blue bars), desipramine (NET inhibitor; green bars), reserpine (VMAT inhibitor; orange bars), and combined inhibitor treatment (yellow bars). Data represent mean ± SD from triplicates of a representative experiment, confirmed by a repeat experiment with fewer time points. Note the varying Y-axis scales across panels. Statistical significance is indicated as **p ≤ 0.01; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/1d0ceaa8d53f69f39c93cf22.png"},{"id":98428820,"identity":"e149683b-911a-40f1-acd9-9be0d402beb6","added_by":"auto","created_at":"2025-12-17 16:42:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2005095,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/d8e21df0-091e-4687-9ac9-2aee8cff6be0.pdf"},{"id":92679548,"identity":"d89cd1d6-59a5-4a54-a5fd-05f590e1dcf3","added_by":"auto","created_at":"2025-10-03 01:00:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":223034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1 Adrenergic and Mesenchymal Signature Scores, and NET, VMAT1, and VMAT2 mRNA Expression in 33 Neuroblastoma Cell Lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis table presents Affymetrix microarray mRNA expression data for 33 human neuroblastoma cell lines. Expression levels for NET (columns 2–3), VMAT1 (columns 4–5), and VMAT2 (columns 6–7) are shown alongside Adrenergic (ADRN) and Mesenchymal (MES) signature scores (columns 8–9).\u003c/p\u003e\n\u003cp\u003eCell lines highlighted in grey were selected for further experiments, including RT-qPCR and [\u003csup\u003e125\u003c/sup\u003eI]mIBG uptake and retention studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Fig 1 mRNA expression of NET, VMAT1, VMAT2 using RT-qPCR after doxycycline-induced NOTCH3 expression in SH-SY5Y-NOTCH3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are expressed as mean ± SD (n=2, experiments in triplicate).Open black symbols are used for the doxycycline-induced (+dox) timepoints, control (-dox) timepoints are depicted by closed grey symbols. The cell line SH-SY5Y-NOTCH3 is depicted in circles, the wild-type (WT) SH-SY5Y cell line is depicted in rectangles. mRNA expression was measured at baseline (T=0) and after 72 hours.\u0026nbsp;\u003c/p\u003e","description":"","filename":"SupplementaldataEMT.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7480401/v1/b8e6f8fb85193798b310cc1e.pdf"}],"financialInterests":"","formattedTitle":"Mesenchymal Neuroblastoma Cells Lack Critical Transporters for mIBG Uptake and May Evade Detection and Therapy","fulltext":[{"header":"Background","content":"\u003cp\u003eNeuroblastoma is the most common extracranial solid tumor in children and arises from neural crest cells. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Intertumor heterogeneity is a hallmark of neuroblastoma and underlies its highly variable clinical course, which ranges from spontaneous regression to aggressive metastatic disease and death. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] Despite recent advances in survival due to anti-GD2 immunotherapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], only about 50% of high-risk neuroblastoma patients survive long-term. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] Relapsed disease remains a major therapeutic challenge in neuroblastoma, with post-relapse survival being exceedingly rare. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eApproximately 90% of neuroblastomas express the norepinephrine transporter (NET) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which allows for the application of meta-iodobenzylguanidine (mIBG), a norepinephrine analog, in both diagnostic imaging and therapy. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] mIBG labeled with iodine-123 is a highly NET-selective radiopharmaceutical used for imaging [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while iodine-131-labeled mIBG has been employed therapeutically since 1984 to treat neuroblastoma. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Previous studies have linked NET mRNA expression to mIBG avidity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and subsequent data indicate a correlation between NET protein expression and mIBG uptake in tumors. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] However, inconsistencies have been observed: certain mIBG-non-avid tumors demonstrate high NET expression, while some mIBG-avid tumors show unexpectedly low levels of NET expression. The vesicular monoamine transporters (VMAT1 and VMAT2) may also contribute to mIBG uptake by enabling vesicular storage of mIBG [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], yet the mechanisms driving the heterogeneous expression of these transporters remain poorly understood. Interestingly, a recent clinical study by Batra et al. found that lower NET and VMAT2 protein expression were unexpectedly associated with a higher likelihood of response to [\u003csup\u003e131\u003c/sup\u003eI]mIBG therapy in patients with relapsed or refractory neuroblastoma, challenging traditional assumptions about the predictive value of transporter expression for therapeutic efficacy. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe epithelial\u0026ndash;mesenchymal transition (EMT) is a cellular program that confers motility and invasiveness [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], playing a key role in tumor progression, metastasis, and therapy resistance. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] While EMT and its reverse process, mesenchymal-epithelial transition (MET), are widely studied in various cancers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], these processes are also implicated in the heterogeneity of neuroblastoma. [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Mesenchymal (MES) neuroblastoma cells exhibit increased motility [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and resistance to chemotherapy compared to lineage-committed adrenergic (ADRN) cells. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] Moreover, MES cells lack the oncogene \u003cem\u003eALK\u003c/em\u003e, rendering them resistant to ALK inhibitors [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and show reduced expression of GD2, leading to resistance against anti-GD2 antibody therapy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], a cornerstone of high-risk neuroblastoma treatment.\u003c/p\u003e\u003cp\u003eOur previous work demonstrated that MES neuroblastoma cells can escape detection in minimal residual disease (MRD) assays that rely solely on ADRN-specific markers. To address this, we developed a mesenchymal-specific MRD panel. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] In this study, we investigated the expression of NET, VMAT1, and VMAT2, as well as mIBG uptake in MES and ADRN neuroblastoma cells \u003cem\u003ein vitro\u003c/em\u003e. We hypothesized that MES cells lack the transporters necessary for mIBG uptake, rendering them invisible to mIBG imaging and resistant to mIBG-based therapies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eCell lines and culture conditions\u003c/p\u003e\u003cp\u003eHuman neuroblastoma cell lines (ATCC\u0026reg;, Manassas, USA) were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; GIBCO\u0026reg;, Thermo Fisher Scientific, Waltham, USA), supplemented with fetal calf serum, L-glutamine, penicillin-streptavidin, and non-essential amino acids as described previously. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. The patient-derived 691-MES (AMC691T) and 691-ADRN (AMC691B) neuroblastoma cell lines were cultured under previously reported conditions. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] SK-N-BE(2)-C and SH-SY5Y neuroblastoma cells with inducible \u003cem\u003ePRRX1A\u003c/em\u003e and \u003cem\u003eNOTCH3\u003c/em\u003e expression, respectively, were used as described in earlier studies. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Doxycycline (100 ng/ml) was added to induce \u003cem\u003ePRRX1A\u003c/em\u003e or \u003cem\u003eNOTCH3\u003c/em\u003e transgene expression. HEK293 cells (ATCC\u0026reg; CRL-3216\u0026trade;, Manassas, USA) were transiently transfected with a human NET plasmid (addgene plasmid #15475; HEK-NET) or an empty vector (HEK-EV) as controls. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] Cell identity was verified by short tandem repeat (STR) analysis, and mycoplasma contamination was routinely checked.\u003c/p\u003e\u003cp\u003eMesenchymal and adrenergic signature scores and monoamine transporter gene expression\u003c/p\u003e\u003cp\u003eThe MES and ADRN gene signature scores were developed as described by Van Groningen and colleagues. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] These scores were based on mRNA expression data from four isogenic MES and ADRN neuroblastoma cell line pairs, identifying 485 MES-specific and 369 ADRN-specific genes. Signature scores for each cell line were calculated by averaging expression levels across the genes in each signature. Signature scores and gene expression data for norepinephrine transporter (NET, \u003cem\u003eSLC6A2\u003c/em\u003e), vesicular monoamine transporter 1 (VMAT1, \u003cem\u003eSLC18A1\u003c/em\u003e), and vesicular monoamine transporter 2 (VMAT2, \u003cem\u003eSLC18A2\u003c/em\u003e) were extracted from a neuroblastoma cell line panel available on the R2 genomics platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://r2.amc.nl/\u003c/span\u003e\u003cspan address=\"http://r2.amc.nl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; GEO accession number GSE28019). [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] The data cover 33 neuroblastoma cell lines, which are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. Expression data were log2-transformed for statistical analysis and figure presentation.\u003c/p\u003e\u003cp\u003emRNA expression using real-time quantitative polymerase chain reaction (RT-qPCR)\u003c/p\u003e\u003cp\u003eNET, VMAT1, and VMAT2 mRNA expression was analyzed across 12 human neuroblastoma cell lines (691-ADRN, 691-MES, SH-SY5Y, SH-EP2, GI-ME-N, IMR-32, N206, SJNB-1, SJNB-8, NGP, SK-N-SH, and SK-N-BE) using RT-qPCR. RNA was extracted using TRIzol (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s protocol. Methods for cDNA synthesis and RT-qPCR were performed as described previously. (van Wezel, Zwijnenburg et al. 2015) Normalization of NET/VMAT expression was done using β-glucuronidase (GUS) as the reference gene, with the formula: normalized cycle threshold (ΔC\u003csub\u003eT\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;C\u003csub\u003eT\u003c/sub\u003e GUS\u0026thinsp;\u0026minus;\u0026thinsp;C\u003csub\u003eT\u003c/sub\u003e NET/VMAT. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] In \u003cem\u003ePRRX1\u003c/em\u003e-inducible SK-N-BE(2)-C cells, Beta-2-microglobulin (B2M) served as the internal control due to its stable expression. Samples without amplification after 40 cycles were scored as negative. All qPCR reactions were performed in triplicate, and results are presented as ΔC\u003csub\u003eT\u003c/sub\u003e values (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e\u003cp\u003ePrimers and probes for VMAT1 and VMAT2 were designed based on previously published gene sequences (ENSEMBL: VMAT1 transcript ENST00000276373.10; VMAT2 transcript ENST00000644641.2). [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] Oligonucleotides were synthesized by Eurogentec (Li\u0026egrave;ge, Belgium), with detailed sequences provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Primer/probe sets for β-glucuronidase (GUS), Beta-2-microglobulin (B2M), and the norepinephrine transporter (NET) were adopted from earlier studies. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimer and probe combinations for VMAT1 and VMAT2\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVMAT1 (\u003cem\u003eSLC18A1\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVesicular monoamine transporter 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eExon\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eForward primer\u003c/p\u003e\u003cp\u003eReverse primer\u003c/p\u003e\u003cp\u003eProbe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;- GCT TTT TCT GGG ACC TAT ACT CTA CTC T \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\u003cp\u003e5\u0026rsquo;- CAA GAC CTG CAA CAG ATG AAA ATG \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e\u003cp\u003e5\u0026rsquo;FAM- TGT GGC CCG AAC CCT TCA AGG C \u0026minus;\u0026thinsp;3\u0026rsquo;TAMRA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003cp\u003e6\u003c/p\u003e\u003cp\u003e5\u0026ndash;6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVMAT2 (\u003cem\u003eSLC18A2\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVesicular monoamine transporter 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eForward primer\u003c/p\u003e\u003cp\u003eReverse primer\u003c/p\u003e\u003cp\u003eProbe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026rsquo;- GCT GCT GAA GGA CCC GTA CA -3\u0026rsquo;\u003c/p\u003e\u003cp\u003e5\u0026rsquo;- GCG ATG CCC ATG TTT GC -3\u0026rsquo;\u003c/p\u003e\u003cp\u003e5\u0026rsquo;FAM \u0026ndash; CTC ATT GCT GCA GGC TCC ATC TGC TT \u0026minus;\u0026thinsp;3\u0026rsquo;TAMRA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003cp\u003e10\u003c/p\u003e\u003cp\u003e9\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e[\u003csup\u003e125\u003c/sup\u003eI]mIBG uptake and retention experiments\u003c/p\u003e\u003cp\u003eHuman neuroblastoma cells were grown in 12-well plates at 80\u0026ndash;90% confluence for uptake experiments. Culture medium was replaced with fresh medium containing 1.0 \u0026micro;M of mIBG and a fixed concentration of [\u003csup\u003e125\u003c/sup\u003eI]mIBG (3.7 kBq/ml; specific activity\u0026thinsp;\u0026asymp;\u0026thinsp;0.65 TBq/mmol; Chelatec, Saint-Herblain, France). For some conditions, NET inhibitor (desipramine) or VMAT inhibitor (reserpine) was added at 1.0 \u0026micro;M (all inhibitors Sigma-Aldrich, Burlington, USA). Cells were incubated for 120 minutes at 37\u0026deg;C with 5% CO2. Following incubation, cells were washed with ice-cold Hank\u0026rsquo;s Balanced Salt Solution (HBSS) to halt further uptake and preserve intracellular content, then lysed with 0.2 M sodium hydroxide. Cell-associated radioactivity was measured in Ultima Gold\u0026reg; scintillation fluid (PerkinElmer, Waltham, USA) using a PerkinElmer Tri-Carb liquid scintillation counter (4910TR) and normalized to total protein content. Uptake was expressed as nanomoles of mIBG per milligram of protein per hour. All assays were performed in triplicate.\u003c/p\u003e\u003cp\u003eFor retention studies, neuroblastoma cells were preloaded with radioactive mIBG for 2 hours under the same conditions. After washing, fresh non-radioactive medium was added, with or without inhibitors. Radioactivity retained by cells was measured after 4 and 24 hours of incubation. Control experiments included PC-12 rat pheochromocytoma cells, fibroblasts, and HEK293 cells transfected with NET (HEK-NET) or empty vector (HEK-EV).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Group means were compared using Welch\u0026rsquo;s t-test. Pearson\u0026rsquo;s correlation test was used to evaluate the relationship between microarray and RT-qPCR expression data. Statistical analyses were performed using GraphPad Prism 9.3.0 (GraphPad Software, San Diego, USA). Statistical significance was indicated by p-values: *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026le;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eMES neuroblastoma cells lack NET, VMAT1, and VMAT2 mRNA expression\u003c/p\u003e\u003cp\u003eTo investigate the relationship between adrenergic (ADRN) and mesenchymal (MES) gene signature scores and the expression of NET, VMAT1, and VMAT2 mRNA, we visualized the data using the R2 Genomics Analysis and Visualization platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://r2.amc.nl/\u003c/span\u003e\u003cspan address=\"http://r2.amc.nl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Data from 33 neuroblastoma cell lines were analyzed; raw expression values and signature scores are listed in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. In the bottom-right quadrant of the plots\u0026mdash;characterized by high ADRN and low MES scores\u0026mdash;NET expression was markedly elevated, with a median of 8.2 (interquartile range [IQR] 7.4\u0026ndash;9.0; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, the top-left quadrant, representing high MES and low ADRN scores, showed significantly lower NET expression (median 1.9, IQR 1.3\u0026ndash;2.8; p\u0026thinsp;=\u0026thinsp;0.0006). A similar pattern was observed for VMAT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with expression levels of 5.4 (IQR 3.6\u0026ndash;7.4) in ADRN-high/MES-low cell lines, compared to 2.7 (IQR 1.3\u0026ndash;3.3) in MES-high/ADRN-low cell lines (p\u0026thinsp;=\u0026thinsp;0.0004). VMAT2 expression followed the same trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), with 5.1 (IQR 4.6\u0026ndash;8.4) in ADRN-high/MES-low cells and 3.0 (IQR 2.2\u0026ndash;3.5) in MES-high/ADRN-low cells (p\u0026thinsp;=\u0026thinsp;0.0006). These data indicate that NET, VMAT1, and VMAT2 mRNA expression is significantly lower in MES-type neuroblastoma cell lines, with NET showing the strongest association with the ADRN phenotype.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eValidation of NET, VMAT1, and VMAT2 mRNA expression\u003c/p\u003e\u003cp\u003eTo validate the differential expression of NET, VMAT1, and VMAT2 mRNA, we performed RT-qPCR on 12 human neuroblastoma cell lines, including two isogenic pairs (SH-SY5Y with SH-EP2, and 691-ADRN with 691-MES) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). RT-qPCR results showed a strong correlation with microarray data for NET (r\u0026thinsp;=\u0026thinsp;0.9451; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Low NET expression (reflected by low ΔC\u003csub\u003eT\u003c/sub\u003e values) was consistently observed in MES-type cell lines (691-MES, SH-EP2, and GI-ME-N). Among the ADRN-type lines, SJNB-8 stood out as an outlier (ΔC\u003csub\u003eT\u003c/sub\u003e = \u0026minus;\u0026thinsp;11.4), lacking a clear ADRN or MES identity (ADRN score \u0026minus;\u0026thinsp;0.43; MES score 0.20). Similarly, strong correlations were found for VMAT1 (r\u0026thinsp;=\u0026thinsp;0.8884; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) and VMAT2 (r\u0026thinsp;=\u0026thinsp;0.8074; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) between RT-qPCR and microarray expression data. These findings confirm that MES neuroblastoma cell lines exhibit significantly lower mRNA expression of NET, VMAT1, and VMAT2, and strongly support the reliability of the microarray data.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDownregulation of NET, VMAT1, and VMAT2 expression during ADRN-to-MES reprogramming\u003c/p\u003e\u003cp\u003eTo further investigate whether acquisition of a MES phenotype is associated with downregulation of NET, VMAT1, and VMAT2, we analyzed their mRNA expression during ADRN-to-MES reprogramming in two inducible cell models. In SK-N-BE(2)C cells, reprogramming was triggered by doxycycline-induced expression of PRRX1, with successful MES transition confirmed by upregulation of the mesenchymal marker POSTN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Over a 28-day time course, NET (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), VMAT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), and VMAT2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) mRNA levels were significantly reduced in PRRX1-induced cells (+\u0026thinsp;dox) compared to non-induced controls (\u0026ndash;dox), with significant differences already apparent by day 3 (p\u0026thinsp;=\u0026thinsp;0.0082, p\u0026thinsp;=\u0026thinsp;0.0013, and p\u0026thinsp;=\u0026thinsp;0.0362, respectively), and further decreases observed through day 28.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsistent results were obtained in a second model, SH-SY5Y-NOTCH3 cells, in which NOTCH3 induction led to marked downregulation of NET, VMAT1, and VMAT2 mRNA as early as day 3. No such changes were observed in wild-type SH-SY5Y cells treated with doxycycline (\u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e). A mesenchymal marker was not assessed in this model.\u003c/p\u003e\u003cp\u003eTogether, these findings show that NET, VMAT1, and VMAT2 are consistently downregulated during the ADRN-to-MES transition, supporting their strong association with the ADRN cell state.\u003c/p\u003e\u003cp\u003eMES neuroblastoma cells lack functional mIBG uptake\u003c/p\u003e\u003cp\u003eTo assess whether MES neuroblastoma cells possess functional transporters required for mIBG uptake, we performed uptake assays using [\u003csup\u003e125\u003c/sup\u003eI]mIBG (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). ADRN cell lines (691-ADRN, SK-N-SH, SH-SY5Y, and SK-N-BE) showed robust mIBG uptake, with SK-N-SH exhibiting the highest uptake (0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 nmol/mg/h) and SH-SY5Y the lowest (0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 nmol/mg/h). Uptake in these lines was largely NET-dependent, as the NET inhibitor desipramine significantly reduced mIBG uptake (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, the VMAT inhibitor reserpine significantly inhibited mIBG uptake in the pheochromocytoma cell line PC12 (p\u0026thinsp;=\u0026thinsp;0.00073) but had no significant effect on uptake in ADRN neuroblastoma cells, suggesting minimal VMAT involvement in these lines. MES cell lines (691-MES, SH-EP2, GI-ME-N) demonstrated minimal mIBG uptake, comparable to negative controls lacking monoamine transporters (HEK-EV and fibroblasts), and this uptake was not affected by NET or VMAT inhibition. Extending incubation times did not increase mIBG uptake in MES cells. These findings indicate that MES neuroblastoma cells lack functional NET-mediated mIBG uptake, highlighting a key functional difference between MES and ADRN phenotypes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMES neuroblastoma cells show no mIBG retention\u003c/p\u003e\u003cp\u003eTo assess mIBG retention, cells were pre-loaded with [\u003csup\u003e125\u003c/sup\u003eI]mIBG, washed, and then incubated with fresh medium for either 4 or 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). ADRN cell lines retained significant amounts of mIBG (\u0026gt;\u0026thinsp;59% after 4 hours and \u0026gt;\u0026thinsp;51% after 24 hours), as long as NET function was preserved (no desipramine). Inhibition of VMAT with reserpine did not affect retention. In contrast, MES cell lines (691-MES, SH-EP2, GI-ME-N) showed no mIBG retention, mirroring the behavior of control cells lacking monoamine transporters (HEK-EV, fibroblasts). No retention was observed under any inhibitory conditions. These findings confirm that MES neuroblastoma cells lack the ability to retain mIBG, and that VMAT does not significantly contribute to mIBG retention in ADRN neuroblastoma cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the impact of mesenchymal differentiation in neuroblastoma on the uptake of meta-iodobenzylguanidine (mIBG) through the norepinephrine transporter (NET) and vesicular monoamine transporter (VMAT). Our findings indicate that MES neuroblastoma cell lines exhibit either undetectable or low mRNA expression levels of NET, VMAT1, and VMAT2 compared to cell lines that exhibit a lineage-committed adrenergic (ADRN) phenotype. Additionally, the reprogramming of ADRN cell lines into a mesenchymal phenotype via \u003cem\u003ePRRX1\u003c/em\u003e and \u003cem\u003eNOTCH3\u003c/em\u003e induction resulted in a dramatic decrease in mRNA expression of NET, VMAT1, and VMAT2 over time. Crucially, MES neuroblastoma cells are unable to concentrate or retain mIBG \u003cem\u003ein vitro\u003c/em\u003e due to the absence of these essential monoamine neurotransmitter transporters.\u003c/p\u003e\u003cp\u003eNeuroblastoma cell lines can adopt either a lineage-committed ADRN phenotype or an immature MES phenotype based on distinct lineage-specific core regulatory networks. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] These findings align with previous studies highlighting the morphological and biochemical heterogeneity of neuroblastoma cells \u003cem\u003ein vitro\u003c/em\u003e. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] Biedler and colleagues originally classified neuroblastoma cells into two phenotypes: N-type (neuroblast-like cells exhibiting catecholamine activity) and S-type (large, flattened, adhesive cells). Subsequent research revealed spontaneous bidirectional interconversion between these cell types [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] and described an intermediate type (I-type) representing a transitional state in the trans-differentiation process. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] More recently, studies have shown that MES neuroblastoma cells are not detectable by current minimal residual disease mRNA marker panels. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Notably, MES cells exhibit relative resistance to chemotherapy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and those harboring ALK mutations show resistance to ALK inhibitor treatment. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] Therefore, MES cells are prime candidates for therapeutic escape, underscoring the need to evaluate and optimize detection methods for their sensitivity to MES cells.\u003c/p\u003e\u003cp\u003eWhile our findings demonstrate that MES neuroblastoma cells are unable to concentrate or retain mIBG in vitro, the clinical implications of this observation remain uncertain. The timing and localization of mesenchymal phenotype acquisition by neuroblastoma cells \u003cem\u003ein vivo\u003c/em\u003e have yet to be clearly established. Notably, single-cell RNA sequencing studies have not identified MES-like cells in primary neuroblastoma tumors. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] Nonetheless, key questions persist regarding the role of MES neuroblastoma cells in treatment-induced selection, metastatic dissemination, and drug-resistant disease recurrence following an initial complete response. It remains speculative whether MES neuroblastoma cells can evade detection by mIBG imaging, potentially contributing to mIBG non-avid disease and reduced sensitivity to targeted radionuclide therapy. If MES cells exist as a minor subpopulation within primary tumors, their resistance to radiation may be mitigated by cross-fire effects from high-energy β-emissions of [\u003csup\u003e131\u003c/sup\u003eI]mIBG absorbed by neighboring ADRN cells. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] However, if MES cells occur as solitary, migratory entities with metastatic potential\u0026mdash;possibly driving relapse after apparent remission, as some data suggest [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] \u0026ndash; their lack of mIBG uptake becomes a more pressing concern. Understanding the \u003cem\u003ein vivo\u003c/em\u003e behavior and therapeutic susceptibility of MES neuroblastoma cells is therefore critical for improving the efficacy of mIBG-based treatment strategies.\u003c/p\u003e\u003cp\u003eOur results indicate that mIBG retention in ADRN cell lines relies solely on the efficient re-uptake of accumulated mIBG by NET, rather than vesicular sequestration by VMAT. This finding aligns with earlier studies demonstrating that mIBG loading in neuroblastoma cells is predominantly governed by cytoplasmic retention and efficient recycling by NET located on the plasma membrane, rather than VMAT-controlled vesicular sequestration. [\u003cspan additionalcitationids=\"CR40 CR41 CR42\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] While NET has long been regarded as the primary transporter responsible for mIBG uptake in neuroblastoma, recent research has identified a positive correlation between VMAT2 protein expression and mIBG tumor avidity, suggesting a potential role for VMAT2-mediated vesicular mIBG sequestration. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] However, this study primarily provides statistical correlation between VMAT2 expression and mIBG avidity without elucidating the underlying mechanisms governing mIBG transport in neuroblastoma cells or the specific role of VMAT in this process. Therefore, while VMAT's role in mIBG uptake and retention is well established in pheochromocytomas and carcinoid tumors [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], our study suggests a minor role for VMAT in neuroblastoma tumors, indicating that low VMAT expression in MES neuroblastoma cells may be of limited significance.\u003c/p\u003e\u003cp\u003eWe recognize several limitations to our study. Primarily, our findings are based on \u003cem\u003ein vitro\u003c/em\u003e experiments using neuroblastoma cell lines, which may not fully capture the complexity of tumor behavior \u003cem\u003ein vivo\u003c/em\u003e. Further research is needed to determine whether, when, and where neuroblastoma cells adopt a mesenchymal phenotype in patients. Functional studies using mesenchymal tumor organoids or in vivo xenograft models may offer valuable insight into the biological role of MES cells and their relevance for mIBG avidity. In addition, mIBG uptake and retention experiments were not performed in the two transgenic adrenergic cell lines containing inducible regulators of mesenchymal transformation. Including these models in future studies could provide important mechanistic insights into the regulation of NET function and mIBG uptake during the ADRN-to-MES transition.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our findings indicate that MES neuroblastoma cells lack mIBG uptake \u003cem\u003ein vitro\u003c/em\u003e due to the absence of NET expression. This has significant clinical implications, as MES neuroblastoma cells may evade detection through mIBG imaging and potentially escape targeted radionuclide mIBG therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eADRN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eadrenergic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eMES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003emesenchymal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003emIBG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003emeta-iodobenzylguanidine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eNET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003enorepinephrine transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eVMAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003evesicular monoamine transporter\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eRT-qPCR\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003ereal-time quantitative PCR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003emRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eMessenger-ribonucleic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eGD2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eDisialoganglioside2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eEMT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eepithelial\u0026ndash;mesenchymal transition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eMET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003emesenchymal-epithelial transition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eALK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAnaplastic lymphoma kinase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eMRD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eminimal residual disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eATCC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eAmerican Type Culture Collection\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eDMEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eDulbecco\u0026apos;s Modified Eagle Medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eSTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eshort tandem repeat\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eGUS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003e\u0026beta;-glucuronidase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eB2M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eBeta-2-microglobulin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eC\u003csub\u003eT\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eThreshold cycle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eHBSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eHank\u0026rsquo;s Balanced Salt Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eIQR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003einterquartile range\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003edox\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003edoxycycline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable. Ethics approval and consent to participate were not sought for this study, as it did not involve human participants or animal subjects.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eAvailability of data and material\u003c/h3\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026rsquo; Contributions\u003c/h3\u003e\n\u003cp\u003eStudy conception and design:\u0026nbsp;TB JvW RM RL JvN EvdS AvK GT\u003c/p\u003e\n\u003cp\u003eMaterial preparation and data collection: TB JvW RM RL JvN\u003c/p\u003e\n\u003cp\u003eData analysis: TB\u0026nbsp;EvdS AvK GT\u003c/p\u003e\n\u003cp\u003eFirst draft of the manuscript was written by TB\u0026nbsp;AvK GT\u003c/p\u003e\n\u003cp\u003eAll authors (TB JvW RM RL JvN EvdS AvK GT) commented on previous versions of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors (TB JvW RM RL JvN EvdS AvK GT) read and approved the final manuscript.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eRobin van Amersfoort, Michelle Muller, and Renate Bezemer worked on this study as part of their scientific internship. We would like to express our gratitude for their efforts and commitment to this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaris, J.M., et al., \u003cem\u003eNeuroblastoma.\u003c/em\u003e Lancet, 2007. \u003cstrong\u003e369\u003c/strong\u003e(9579): p. 2106-20.\u003c/li\u003e\n\u003cli\u003eMatthay, K.K., et al., \u003cem\u003eNeuroblastoma.\u003c/em\u003e Nat Rev Dis Primers, 2016. \u003cstrong\u003e2\u003c/strong\u003e: p. 16078.\u003c/li\u003e\n\u003cli\u003eYu, A.L., et al., \u003cem\u003eAnti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma.\u003c/em\u003e N Engl J Med, 2010. \u003cstrong\u003e363\u003c/strong\u003e(14): p. 1324-34.\u003c/li\u003e\n\u003cli\u003eYu, A.L., et al., \u003cem\u003eLong-Term Follow-up of a Phase III Study of ch14.18 (Dinutuximab) + Cytokine Immunotherapy in Children with High-Risk Neuroblastoma: COG Study ANBL0032.\u003c/em\u003e Clin Cancer Res, 2021. \u003cstrong\u003e27\u003c/strong\u003e(8): p. 2179-2189.\u003c/li\u003e\n\u003cli\u003eMatthay, K.K., et al., \u003cem\u003eLong-term results for children with high-risk neuroblastoma treated on a randomized trial of myeloablative therapy followed by 13-cis-retinoic acid: a children\u0026apos;s oncology group study.\u003c/em\u003e J Clin Oncol, 2009. \u003cstrong\u003e27\u003c/strong\u003e(7): p. 1007-13.\u003c/li\u003e\n\u003cli\u003eLondon, W.B., et al., \u003cem\u003eClinical and biologic features predictive of survival after relapse of neuroblastoma: a report from the International Neuroblastoma Risk Group project.\u003c/em\u003e J Clin Oncol, 2011. \u003cstrong\u003e29\u003c/strong\u003e(24): p. 3286-92.\u003c/li\u003e\n\u003cli\u003eStreby, K.A., et al., \u003cem\u003eNothing but NET: a review of norepinephrine transporter expression and efficacy of 131I-mIBG therapy.\u003c/em\u003e Pediatr Blood Cancer, 2015. \u003cstrong\u003e62\u003c/strong\u003e(1): p. 5-11.\u003c/li\u003e\n\u003cli\u003eSharp, S.E., et al., \u003cem\u003eMIBG in Neuroblastoma Diagnostic Imaging and Therapy.\u003c/em\u003e Radiographics, 2016. \u003cstrong\u003e36\u003c/strong\u003e(1): p. 258-78.\u003c/li\u003e\n\u003cli\u003eVik, T.A., et al., \u003cem\u003e(123)I-mIBG scintigraphy in patients with known or suspected neuroblastoma: Results from a prospective multicenter trial.\u003c/em\u003e Pediatr Blood Cancer, 2009. \u003cstrong\u003e52\u003c/strong\u003e(7): p. 784-90.\u003c/li\u003e\n\u003cli\u003eTreuner, J., et al., \u003cem\u003eTreatment of neuroblastoma with metaiodobenzylguanidine: results and side effects.\u003c/em\u003e Med Pediatr Oncol, 1987. \u003cstrong\u003e15\u003c/strong\u003e(4): p. 199-202.\u003c/li\u003e\n\u003cli\u003eCarlin, S., et al., \u003cem\u003eDevelopment of a real-time polymerase chain reaction assay for prediction of the uptake of meta-[(131)I]iodobenzylguanidine by neuroblastoma tumors.\u003c/em\u003e Clin Cancer Res, 2003. \u003cstrong\u003e9\u003c/strong\u003e(9): p. 3338-44.\u003c/li\u003e\n\u003cli\u003eDubois, S.G., et al., \u003cem\u003eEvaluation of Norepinephrine Transporter Expression and Metaiodobenzylguanidine Avidity in Neuroblastoma: A Report from the Children\u0026apos;s Oncology Group.\u003c/em\u003e Int J Mol Imaging, 2012. \u003cstrong\u003e2012\u003c/strong\u003e: p. 250834.\u003c/li\u003e\n\u003cli\u003eTemple, W., et al., \u003cem\u003eVesicular monoamine transporter protein expression correlates with clinical features, tumor biology, and MIBG avidity in neuroblastoma: a report from the Children\u0026apos;s Oncology Group.\u003c/em\u003e Eur J Nucl Med Mol Imaging, 2016. \u003cstrong\u003e43\u003c/strong\u003e(3): p. 474-481.\u003c/li\u003e\n\u003cli\u003eBatra, V., et al., \u003cem\u003eNorepinephrine transporter and vesicular monoamine transporter 2 tumor expression as a predictor of response to 131I‐MIBG in patients with relapsed/refractory neuroblastoma.\u003c/em\u003e Pediatric Blood \u0026amp; Cancer, 2024. \u003cstrong\u003e71\u003c/strong\u003e(1): p. e30743.\u003c/li\u003e\n\u003cli\u003eKalluri, R. and R.A. 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Helson, and B.A. Spengler, \u003cem\u003eMorphology and growth, tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture.\u003c/em\u003e Cancer Res, 1973. \u003cstrong\u003e33\u003c/strong\u003e(11): p. 2643-52.\u003c/li\u003e\n\u003cli\u003eRoss, R.A., B.A. Spengler, and J.L. Biedler, \u003cem\u003eCoordinate morphological and biochemical interconversion of human neuroblastoma cells.\u003c/em\u003e J Natl Cancer Inst, 1983. \u003cstrong\u003e71\u003c/strong\u003e(4): p. 741-7.\u003c/li\u003e\n\u003cli\u003eCiccarone, V., et al., \u003cem\u003ePhenotypic diversification in human neuroblastoma cells: expression of distinct neural crest lineages.\u003c/em\u003e Cancer Res, 1989. \u003cstrong\u003e49\u003c/strong\u003e(1): p. 219-25.\u003c/li\u003e\n\u003cli\u003eDong, R., et al., \u003cem\u003eSingle-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma.\u003c/em\u003e Cancer Cell, 2020. \u003cstrong\u003e38\u003c/strong\u003e(5): p. 716-733 e6.\u003c/li\u003e\n\u003cli\u003eKildisiute, G., et al., \u003cem\u003eTumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell.\u003c/em\u003e Sci Adv, 2021. \u003cstrong\u003e7\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eGiammarile, F., et al., \u003cem\u003eEANM procedure guidelines for 131I-meta-iodobenzylguanidine (131I-mIBG) therapy.\u003c/em\u003e Eur J Nucl Med Mol Imaging, 2008. \u003cstrong\u003e35\u003c/strong\u003e(5): p. 1039-47.\u003c/li\u003e\n\u003cli\u003eSmets, L.A., et al., \u003cem\u003eExtragranular storage of the neuron blocking agent meta-iodobenzylguanidine (MIBG) in human neuroblastoma cells.\u003c/em\u003e Biochem Pharmacol, 1990. \u003cstrong\u003e39\u003c/strong\u003e(12): p. 1959-64.\u003c/li\u003e\n\u003cli\u003eGaze, M.N., et al., \u003cem\u003eIntracellular localization of metaiodobenzyl guanidine in human neuroblastoma cells by electron spectroscopic imaging.\u003c/em\u003e Int J Cancer, 1991. \u003cstrong\u003e47\u003c/strong\u003e(6): p. 875-80.\u003c/li\u003e\n\u003cli\u003eLashford, L.S., J.P. Hancock, and J.T. Kemshead, \u003cem\u003eMeta-iodobenzylguanidine (mIBG) uptake and storage in the human neuroblastoma cell line SK-N-BE(2C).\u003c/em\u003e Int J Cancer, 1991. \u003cstrong\u003e47\u003c/strong\u003e(1): p. 105-9.\u003c/li\u003e\n\u003cli\u003eMairs, R.J., M.N. Gaze, and A. Barrett, \u003cem\u003eThe uptake and retention of metaiodobenzyl guanidine by the neuroblastoma cell line NB1-G.\u003c/em\u003e Br J Cancer, 1991. \u003cstrong\u003e64\u003c/strong\u003e(2): p. 293-5.\u003c/li\u003e\n\u003cli\u003eMontaldo, P.G., et al., \u003cem\u003eAccumulation of m-iodobenzylguanidine by neuroblastoma cells results from independent uptake and storage mechanisms.\u003c/em\u003e Cancer Res, 1991. \u003cstrong\u003e51\u003c/strong\u003e(16): p. 4342-6.\u003c/li\u003e\n\u003cli\u003eKolby, L., et al., \u003cem\u003eUptake of meta-iodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters.\u003c/em\u003e Br J Cancer, 2003. \u003cstrong\u003e89\u003c/strong\u003e(7): p. 1383-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuroblastoma, meta-iodobenzylguanidine (mIBG), norepinephrine transporter (NET), vesicular monoamine transporter (VMAT1, VMAT2) adrenergic, mesenchymal","lastPublishedDoi":"10.21203/rs.3.rs-7480401/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7480401/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNeuroblastoma exhibits phenotypic heterogeneity with two main transcriptional states: adrenergic (ADRN) and mesenchymal (MES). MES neuroblastoma cells are linked to increased motility and chemoresistance, but their capacity to accumulate meta-iodobenzylguanidine (mIBG)\u0026mdash;a cornerstone in neuroblastoma imaging and therapy\u0026mdash;remains unclear. We hypothesized that MES cells fail to take up mIBG due to reduced expression of the norepinephrine transporter (NET) and vesicular monoamine transporters (VMAT1/2). To investigate this, we analyzed NET, VMAT1, and VMAT2 mRNA expression in 33 neuroblastoma cell lines and correlated results with ADRN and MES transcriptional signatures. Validation was performed by real-time quantitative PCR (RT-qPCR) in 12 lines (3 MES, 9 ADRN). Additionally, two ADRN cell lines (SK-N-BE(2)C-PRRX1 and SH-SY5Y-NOTCH3) were induced to undergo MES transition and monitored by time-series RT-qPCR. Functional uptake and retention of [\u003csup\u003e125\u003c/sup\u003eI]mIBG were assessed in seven cell lines (3 MES, 4 ADRN), with desipramine and reserpine used to confirm transporter specificity.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eMES cell lines showed markedly lower expression of NET, VMAT1, and VMAT2 compared with ADRN lines. Induction of a MES phenotype in ADRN cells led to a progressive reduction in transporter expression. Functional assays demonstrated that MES cells lacked the ability to accumulate or retain mIBG.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eMES neuroblastoma cells are deficient in critical transporters required for mIBG uptake. These findings suggest that MES-predominant tumors may escape detection by mIBG imaging and resist mIBG-based radionuclide therapy, underscoring the need to account for tumor cell state in clinical imaging and treatment planning.\u003c/p\u003e","manuscriptTitle":"Mesenchymal Neuroblastoma Cells Lack Critical Transporters for mIBG Uptake and May Evade Detection and Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 01:00:03","doi":"10.21203/rs.3.rs-7480401/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"03703b7f-db9b-40e3-ba56-de09c8c5cf97","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T13:54:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 01:00:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7480401","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7480401","identity":"rs-7480401","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}