Background
Neuroblastoma, the most common extracranial solid tumor of childhood, originates from the early developing embryonic sympathetic nervous system [
1]. Approximately 90% of neuroblastomas express the norepinephrine transporter (NET) [
2], enabling the application of meta-iodobenzylguanidine (MIBG), a structural analogue of norepinephrine, for imaging and treatment purposes. MIBG radiolabeled with iodine-123 is used as a highly NET-selective imaging radiopharmaceutical [
3], whereas MIBG radiolabeled with iodine-131 is being used since 1984 as a therapeutic radiopharmaceutical in patients with neuroblastoma [
4].
Over the years, different strategies have been studied to optimize the application of [
131I]MIBG therapy in neuroblastoma patients [
5]. In the majority of these studies, [
131I]MIBG was used as salvage therapy for refractory or relapsed disease [
6]. However, in the Netherlands [
131I]MIBG has also been extensively used upfront for induction therapy [
7,
8]. Pain relief in the palliative setting is another application of [
131I]MIBG [
9]. Despite the fact that [
131I]MIBG therapy has been used for 37 years in the treatment of neuroblastoma patients, the definitive answer on the optimal role and timing of [
131I]MIBG therapy is not yet given and awaits further prospective trials. Currently, the Children's Oncology Group (COG) is recruiting newly diagnosed high-risk neuroblastoma patients for a randomized Phase 3 study, in which patients receive either standard induction or induction with the addition of [
131I]MIBG therapy (NCT03126916) [
10]. At the same time in Europe, SIOPEN (International Society of Pediatric Oncology, European Neuroblastoma) is investigating whether [
131I]MIBG therapy has a role in intensification treatment strategies for high-risk patients with a poor response to induction chemotherapy (VERITAS study; NCT03165292). A complete novel application is the combined use of [
131I]MIBG therapy with targeted, antibody-based immunotherapy in relapsed and refractory patients. Examples are safety and efficacy studies of [
131I]MIBG therapy with dinutuximab (anti-GD2 antibody; NCT03332667) and with the combination of dinutuximab and Nivolumab (anti-Programmed Cell Death Protein 1; MiNivAN study, NCT02914405).
An unwanted adverse effect of the therapeutical application of [
131I]MIBG has been hematological toxicity, mainly consisting of severe and persistent thrombocytopenia [
11‐
14]. Thrombocytopenia in the pediatric oncology population may lead to interruption of therapy, platelet transfusions, and bleeding complications [
15]. It is conceivable that radiation exposure after selective uptake of MIBG by megakaryocytes is the major cause of MIBG therapy-associated thrombocytopenia. Both human megakaryocytes [
16,
17] and their offspring cells, platelets [
18], express the serotonin transporter (SERT) on their cell membrane. SERT-mediated MIBG uptake by human platelets has previously been described [
19]. More recently, we have demonstrated that selective serotonin reuptake inhibitors (SSRIs) are able to prevent radiotoxic MIBG uptake in platelets without affecting uptake in neuroblastoma tumor [
20]. However, two studies conducted in 1995 and 2002, respectively, have failed to demonstrate MIBG uptake in vitro in megakaryocytic cell lines [
21] and in cultured human megakaryocytes [
22]. Over the years, vast progression has been made in the in vitro culture and differentiation of human megakaryocytes [
23,
24]. The aim of our current study was to investigate whether cultured human megakaryocytes, using an optimized method of differentiation, are capable of selective MIBG uptake.
Discussion
The most common side effect encountered after [
131I]MIBG therapy is hematological toxicity, in particular thrombocytopenia [
33]. Since platelets are anucleate cells with a finite lifespan of 9–11 days [
34], and the observed thrombocytopenia is a delayed toxicity which can persevere for months [
11], we investigated whether platelet precursor cells, i.e., megakaryocytes located in the bone marrow, are the primary targets of radiotoxic MIBG. To date, only circumstantial evidence exists for the capacity of megakaryocytic cells to concentrate MIBG in the form of a case report demonstrating osteomedullary MIBG uptake in a 13-month-old patient with acute megakaryocytic leukemia [
35]. Since both the norepinephrine transporter (NET) and serotonin transporter (SERT) have the capacity for MIBG uptake [
19,
20], we investigated whether SERT, located on the cell membrane of human megakaryocytes [
16,
17], is capable of MIBG uptake in megakaryocytes and whether citalopram, a specific inhibitor of the serotonin transporter (SSRI), could inhibit this uptake.
Our study demonstrated that in vitro suspension-cultured human megakaryocytes using a standardized differentiation protocol are capable of selective plasma membrane transport of MIBG. After 10 days of differentiation, megakaryocytic cell cultures from three different healthy adult HSPC donors showed on average 9.2 ± 2.4 nmol of MIBG uptake per milligram protein per hour after incubation with 10
–7 M MIBG (range: 6.6 ± 1.0 nmol/mg/h to 11.2 ± 1.0 nmol/mg/h). In comparison, serotonin uptake was on average 61.99 ± 23.8 nmol per milligram protein per hour after incubation with 10
–7 M serotonin (range: 35.7 ± 3.0 nmol/mg/h to 82.2 ± 7.9 nmol/mg/h). Co-incubation with the SSRI citalopram led to a significant reduction (30.1–41.5%) in MIBG uptake, implying SERT-specific uptake of MIBG. A strong correlation between the number of mature megakaryocytes, expressing glycoprotein Ib (CD42b) [
36] assessed by flow cytometry phenotyping, and specific substrate uptake (both MIBG and serotonin) was discovered. This suggests that the most mature megakaryocytes have the highest uptake capacity for MIBG. Unfortunately, it was not possible to perform uptake and inhibition experiments with purified mature megakaryocytes, as the fragility of these cells prevented sorting via, e.g., FACS or MACS (magnetic-activated cell sorting).
Our results demonstrated for the first time the selective uptake of MIBG via the serotonin transporter by cultured human megakaryocytes and thereby offer an alternative explanation to the hematologic toxicity encountered after
131I-MIBG therapy. Early studies reported that the whole-body radiation dose could serve as an excellent predictor of thrombocytopenia after therapeutic
131I-MIBG [
37,
38], and this association was confirmed by more recent studies [
11,
39,
40]. Although a large study performed by Trieu et al. could not find significant association between both entities, this was attributed to the high-level and narrow range of MIBG activity applied (16.3 mCI/kg ± 5.9 [mean ± SD]). However, the whole-body radiation dose cannot explain the thrombocytopenia encountered in neuroblastoma patients treated with
125I-MIBG, a radiopharmaceutical emitting Auger electrons with an insufficient range to interact with surrounding untargeted cells [
41], which was more severe than in
131I-MIBG-treated patients in the same study who received higher radiation doses [
38]. Our finding that megakaryocytes are capable of selective MIBG uptake may explain these findings. Our group described earlier that peripheral blood stem cell (PBSC) apheresis is feasible after upfront
131I-MIBG therapy in neuroblastoma patients, although delayed platelet reconstitution occurred after reinfusion in MIBG-treated patients in comparison with chemotherapy-only-treated patients [
42]. Interestingly, no association was found between bone marrow tumor infiltration at diagnosis and platelet reconstitution. We hypothesize that
131I-MIBG taken up by megakaryocytes in the bone marrow might damage neighboring hematopoietic stem cells via cross-fire irradiation and radiation-induced biological bystander effects [
41,
43], leading to delayed platelet reconstitution after reinfusion. These two radiobiological effects may also partly account for the widespread and enduring hematological toxicity seen after
131I-MIBG therapy.
Our results are in apparent contrast with a previous study by Tytgat et al. showing the inability of specific uptake of MIBG in cultured megakaryocytes [
22]. It is conceivable that the discrepancy between our results and those reported by Tytgat et al. is due to differences in differentiation protocols which may have led to less mature megakaryocytes and therefore less SERT expression on the cell surface. First, the composition of the differentiation medium used is different. It is known that culture medium composition is critical for proper differentiation and maturation of HPSCs [
27]. Second, while Tytgat et al. used thrombopoietin as sole growth factor, without the addition of cytokines [
22], our optimized and reproducible differentiation protocol [
23,
25] includes, besides Nplate, a thrombopoietin analog, stem cell factor (SCF), and the combination of cytokines interleukin-1β and -6 (IL-1β and IL-6). Third, the differentiation of the megakaryocyte cultures was determined solely based on the expression of CD61, or glycoprotein IIIa, a non-specific surface marker present on platelets, megakaryocytes, monocytes, macrophages, and endothelial cells [
44]. While CD61 expression is associated with commitment to the megakaryocytic lineage, it is not a specific marker for megakaryocytic maturation [
45]. It is, therefore, unclear whether and to what extent the differentiation protocol led to proper maturation of megakaryocytes in this study [
22].
The MIBG uptake in megakaryocytic cultures proved to be 5.4–7.3-fold lower compared with the serotonin uptake. Previously, we showed that the affinity of SERT for MIBG was considerable lower than for the natural ligand serotonin (K
M 9.7 µM and K
M 3.6 µM, respectively) [
20]. Moreover, the catalytic efficiency of SERT, defined as the ratio
\(\frac{{V}_{max}}{{K}_{M}}\), was 4.2 × higher for serotonin than for MIBG, implying that SERT has a substantial higher ability to transport serotonin than MIBG. Interactions between SERT protein and ligand may account for these observed differences. Recently, critical interactions between SERT protein and ligand were elucidated [
46‐
48]. These interactions provide a structural explanation for the observed uptake differences of serotonin and MIBG by SERT. Although, on the one hand, striking similarities in interactions between the transporter protein and ligands are evident among the neurotransmitter:sodium symporter (NSS) family that includes both SERT and NET [
47‐
49]. There are other SERT protein–ligand interactions, on the other hand, that are critical for membrane transport [
47], that can only be established by a bicyclic indole compound as serotonin and not by the catecholamine-related MIBG.
Another mechanism that may lead to the observed differences in serotonin and MIBG uptake in megakaryocytes is that plasma membrane SERT density and, subsequently, serotonin uptake, increases after exposure to increasing extracellular serotonin levels. This regulatory mechanism was first discovered in platelets [
50,
51]. Whether a similar regulatory mechanism exists in megakaryocytes, and whether exposure to MIBG leads to similar up-regulatory effects on SERT, is not known.
Differences in SERT transporter expression might explain why the MIBG uptake in megakaryocytic cultures was 4.5–7.6-fold lower than the MIBG uptake in HEK293-SERT transfected cells. Caution should be exercised, however, when comparing a highly heterogeneous population of cells, i.e., the megakaryocytic cell cultures derived from healthy adult HSPC donors after 10 days of differentiation, with a homogenous population of transfected HEK-cells overexpressing SERT. This difference between heterogeneous and homogenous cell populations might also explain why the heterogeneous, megakaryocyte cultures in our uptake experiments were not “able to concentrate large amounts of administered MIBG (…) equivalent to that observed for serotonin” as homogenous, donor-derived platelets were in earlier studies [
19]. Furthermore, the difference in uptake capacity between platelets and their progenitor megakaryocytes might be explained by the absence of dense granules (DG) in the latter cells. DGs have the capacity to accumulate monoamines, like serotonin [
50], but also catecholamine-related substances as MIBG [
52]. For a long time, DGs were thought to be fully matured within megakaryocytes [
53,
54]. Recent evidence suggested that DGs are not formed in megakaryocytes but instead in proplatelets [
55]. Therefore, comparing the uptake capacity of megakaryocytes after only 10 days of maturation with full-grown platelets might be an attempt to draw similarities between two things that are not similar.
In conclusion, our data demonstrate that human megakaryocytes cultured in vitro is capable of SERT-selective MIBG uptake. Presumably, the most mature megakaryocytes in the bone marrow are primarily responsible for this uptake. The concomitant application of a SSRI in neuroblastoma patients treated with [131I]MIBG therapy seems a promising strategy to prevent radiotoxic MIBG uptake by these cells and the onset of thrombocytopenia. Future studies should examine the effect of citalopram on platelet count, reconstitution, and need for transfusions in [131I]MIBG-treated neuroblastoma patients in a placebo-controlled clinical trial.
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