Neuroblastoma xenograft models demonstrate the therapeutic potential of ¹⁷⁷Lu-octreotate

Neuroblastoma (NB) represents 7–9% of all tumors detected in children [1, 2]. NB are neuroendocrine tumors (NETs), deriving from primitive nerve cells in the sympathetic nervous system. Approximately two thirds of the patients develop tumor in the abdominal region with the adrenal glands being the primary organ [1], although it can also originate from the thorax, pelvis, spine or neck. Metastatic disease is present in more than 50% of patients, with metastases most commonly located in regional lymph nodes, bone marrow, bone, liver, and skin [3]. NB is a heterogeneous cancer type with various characteristics and features, and with diverse outcomes. Patients with NB are divided into different risk-assessment groups, and The International Neuroblastoma Risk Group (INRG) applies a classification system based on key NB criteria, such as age, stage, tumor histology, MYCN amplification (MNA), 11q deletion and ploidy to define very low-, low-, intermediate-, and high-risk (HR) groups according to 5-year event-free survival (EFS) [4]. Patients with HR-NB have a 5-year survival rate of only 40–50%, as compared to 90–100% for those with low-risk NB [5, 6]. HR-NB harbors genetic aberrations such as deletion of chromosome arm 1p, 17q-gain, amplification of MYCN and deletion of parts of chromosome 11q. Two NB groups are considered as HR, the MYCN amplified (29–25%) and the 11q-deletions (35–45%) groups [7]. Surgery alone leads to a good prognosis for patients with resectable low-risk NB. For patients with HR-NB, multimodal therapy including targeted therapy, surgery, chemotherapy, radiation therapy and autologous peripheral blood stem cell transplantation (ASCT) are included [8]. In cases of disseminated disease systemic treatments are needed.

Diagnosis and therapy using the norepinephrine analogue metaiodobenzylguanidine (MIBG) is an already established radiopharmaceutical option for patients with NB. ¹³¹I-MIBG therapy have response rates of 20–37% in patients with refractory and relapsed NB [9, 10]. Treatments with doxorubicin in combination with surgery or inhibitors such as ixazomib have proven anti-tumor effects in preclinical studies [11, 12]. Further chemotherapy regimens that have been suggested for HR-NB, due to a randomized phase-3 trial, is busulfan in combination with melphalan [13]. The combination of topotecan, cyclophosphamide, and etoposide have also demonstrated promising results for relapsed and untreated NB in a phase-II trial [14]. Dinutuximab is an anti-GD2 monoclonal antibody which is both EMA- and FDA-approved for first line treatment of HR-NB. Immunotherapy with dinutuximab in combination with granulocyte-macrophage colony-stimulating factor, interleukin-2 and isotretinoin was compared to treatment with isotretinoin for HR-NB patients and resulted to a 2-year EFS of 66 and 46%, respectively [15]. By observing survival rates over the past decades, it is clear that the treatment methods for NB patients have improved. However, survival rates for HR-NB remain low [5], which strengthens the need for more effective treatment methods.

Systemic radionuclide therapy with ¹⁷⁷Lu-octreotate could be a potential treatment option for HR-NB. NETs often overexpress somatostatin (SS) receptors (SSTRs), which enables the use of radiolabeled SS analogues for diagnostics and therapy of SSTR-expressing NETs. There are five different subtypes of SSTRs, SSTR1–5. All but SSTR1 are internalized into the cell after binding with an appropriate agonist [16]. Previous studies demonstrate the expression of SSTRs in NBs, most prominently SSTR1 and SSTR2 [17‐21]. Prominent proliferation and MIBG-avidity in NBs are factors that seems to correlate with high SSTR2 expression [19, 22]. Octreotate is an SS analogue, with highest affinity towards SSTR2 and somewhat lower to SSTR4 and SSTR5 [23]. Today, radionuclide therapy using ¹⁷⁷Lu-octreotate is an EMA and FDA approved treatment option for patients with gastroenteropancreatic NETs. ¹⁷⁷Lu-octreotate has recently been included in a phase II study for patients with relapsed or refractory metastatic HR-NB with modest results [24]. Reviewing the treatment schedule and optimizing the amount of activity administered were factors that could have impacted the results. The benefits of a personalized approach are seen in a study where a refractory metastatic HR-NB-patient received an adapted ¹⁷⁷Lu-octreotate treatment which prevented the tumor progression [25].

Today, there is a lack of knowledge in how ¹⁷⁷Lu-octreotate is distributed in NB patients. In our previous in vitro studies, the binding and internalization of ¹⁷⁷Lu-octreotate to the two NB cell lines IMR-32 and CLB-BAR were investigated (Binding and internalization of ¹⁷⁷Lu-octreotate in cell lines of neuroblastoma, breast cancer, and non-small cell lung cancer, submitted). The results indicated a high specific binding and internalization of ¹⁷⁷Lu-octreotate for both cell lines. These promising results encouraged us to study this further in human NB xenograft models.

This paper explores the biodistribution of ¹⁷⁷Lu-octreotate in NB xenograft bearing nude mice and evaluates the potential of ¹⁷⁷Lu-octreotate as a treatment option for patients with NB. The study was performed on three human NB xenograft animal models. To our knowledge this is the first biodistribution study of ¹⁷⁷Lu-octreotate in mice bearing human NB xenografts.

In general, high concentration levels of ¹⁷⁷Lu were observed in all human NB tumor xenograft tissues investigated (Tables 1, 2 and 3). The adrenal glands, kidneys and lungs reached higher ¹⁷⁷Lu activity concentration levels than the other normal organs. Dose- and time-dependent differences in the ¹⁷⁷Lu concentration levels were observed. A decrease of the ¹⁷⁷Lu concentration over time was observed for all tissues, but retention was higher in the tumor tissue compared with that of other organs.

There have been great advances in the treatment of patients with NB over the last decades. The overall survival rate for all NB patients has increased to approximately 80%, but for HR-NB patients overall survival rate is still only about 40% [5]. This patient group has not responded as well as low-risk NB patients to the newly introduced treatment methods. There is thus a need for novel treatment modalities for patients with HR-NB, where ¹⁷⁷Lu-octreotate could be a potential option if necessary dosimetric and radiosensitivity requirements are fulfilled. Knowledge of biodistribution and biokinetics of the radiopharmaceutical is needed in order to be able to determine if a compound is suitable for diagnostic and therapeutic purposes for each new type of cancer. When it comes to radionuclide therapy, it is most often the absorbed dose to the critical organs that will limit the activity level possible to administer. The present study was performed in order to examine the biokinetics and biodistribution of ¹⁷⁷Lu-octreotate in three aggressive human NB xenograft models: CLB-BAR, CLB-GE and IMR-32. The results demonstrated high ¹⁷⁷Lu activity concentration levels in the xenograft tumor tissue arising from all three NB cell lines. The differences in ¹⁷⁷Lu activity concentration between the NB cell lines may be due to differences in overall SSTR expression, SSTR subtype expression and receptor saturation for higher amounts of octreotate administered. CLB-GE xenograft tumors had the highest uptake, while CLB-BAR xenografts demonstrated saturation effects at the higher activity levels and hence had the lowest uptake. IMR-32 xenograft tumors had lower uptake of ¹⁷⁷Lu-octreotate in comparison with CLB-GE, but still displayed a relatively long retention rate of ¹⁷⁷Lu-octreotate.

In all but one experiment, the ¹⁷⁷Lu concentration in the tumor tissues decreased with time during the 168 h interval studied, regardless of the amount injected. However, when the CLB-GE xenograft bearing mice were injected with 15 MBq ¹⁷⁷Lu-octreotate, the ¹⁷⁷Lu concentration in tumor did not decrease from 1 h to 168 h p.i, a finding not detected after 0.15 MBq. One explanation can be a higher accumulation in tumor tissue due to a therapeutic effect resulting in tumor volume reduction, a phenomenon we have observed in our previous studies on the human small intestinal NET GOT1 model [35, 38‐41]. For patients with advanced midgut NETs, ¹⁷⁷Lu-octreotate has proven to be a treatment that increases the progression-free survival and the overall survival [42]. Therefore, ¹⁷⁷Lu-octreotate (Lutathera®) is now FDA and EMA approved for treatment of SSTR-positive midgut, foregut and hindgut NETs. Hence it is of interest to compare data from our NB models with similar from the GOT1-model. Solely observing the concentration levels of ¹⁷⁷Lu in the tumor tissues in the different tumor models it becomes clear that all xenografts derived from NB cell lines have similar or higher tumor uptake relative to other NET models (Table 5). The differences between the cell lines are evident also when the T/B and the T/K values are compared (Fig. 4).

In general, lower ¹⁷⁷Lu concentration levels in tumors were observed after administration of 15 MBq ¹⁷⁷Lu-octreotate compared with lower amounts administered. One explanation for this finding may be saturation of the SSTRs. Although internalization of SSTRs occurs after a few minutes, 2.5 min in rats [47], saturation effects are still prominent [48]. Saturation of SSTRs are prevented by fractionated administration, which may also result in upregulation of SSTR expression that may also further increase the uptake and hence therapeutic effect [16].

The high uptake of ¹⁷⁷Lu-octreotate in CLB-GE tumors compared with that of the CLB-BAR and IMR-32 xenografts may be explained by the high SSTR2 expression of the CLB-GE xenograft tumors demonstrated by IHC, since octreotate has a higher affinity for SSTR2 than for the other SSTR subtypes [23]. However, assuming solely that high receptor expression leads to high tumor uptake and absorbed dose is a somewhat simplified picture of reality. This is demonstrated when the SSTR2 expression for CLB-BAR and IMR-32 xenografts is compared. Our IHC data indicate that CLB-BAR xenografts has a higher expression of SSTR2 than IMR-32 xenografts, while the mean absorbed dose for IMR-32 tumors was higher than for CLB-BAR. There are many reasons that may explain these discrepancies, e.g. that the affinity to the other SSTR subtypes are of importance, primarily SSTR4 and SSTR5, together with other functional differences between SSTR subtypes, such as receptor internalization, receptor cycling, receptor saturation and radionuclide retention. However, the main reason is most probably that binding of ¹⁷⁷Lu-octreotate can only occur to SSTRs expressed on the outside of cell membrane, while the IHC images visualize all SSTRs expressed in tissue, also the intracellularly distributed receptors. Thus no quantitative analyses were performed on the IHC images.

A prerequisite for the therapeutic use of radiolabeled SS analogs is high expression of SSTRs. Our results demonstrate high expression of SSTR in these NB xenograft models. Previous study demonstrated frequent expression of SSTR subtypes in NB xenograft models: SH-SY5Y, SK-N-DZ, SK-N-AS, IMR-32 and KELLY, which is in line with our results [17]. High expression of SSTR2 has also been reported in other NB xenograft models [22, 49]. A case-study of 54 NB patients reported SSTR2 expression in 44 patients, with 19 of 27 HR-NB patients expressing SSTR2 [18], confirming similar studies and conveys SSTR2 expression in HR-NB [17, 19, 20]. A recent study could, through personalized treatment with ⁹⁰Y-, ¹¹¹In- and/or ¹⁷⁷Lu-labeled octreotate in combination with chemotherapy, demonstrate partial responses in all four HR-NB patients who previously did not responded to multimodal treatments [20]. Our results and prior research highlights the diagnostic and therapeutic potentials of targeting SSTR2 in NB patients with radiolabeled SS analogues. Analyzing the R2 database demonstrates that SSTR2 is the most abundantly expressed SSTR subtype in NB tumors (http://​r2.​amc.​nl). Furthermore, previous data on the transcriptional expression of the various SSTR subtypes in CLB-BAR, CLB-GE and IMR-32 support the high SSTR2 expression in our IHC data (Fig. 7) [50, 51]. Our IHC findings may also indicate, in contrast to the mRNA expression, that SSTR3 could be a useful target for treatment. In that regard the SS analogue, octreotide, is preferable due to its higher affinity to SSTR3 in comparison with octreotate [23]. Mapping the SSTR subtypes of the tumor before treatment can promote the choice of tumor seeking agents.

The kidneys, lungs and adrenal glands were consistently the three organs with the highest concentrations of ¹⁷⁷Lu. The adrenals and kidneys are known to express SSTRs. High kidney uptake of ¹⁷⁷Lu-octreotate has been observed in biodistribution studies before [35, 46, 52], demonstrating the well-known fact that the kidneys are one of the main organs at risk. This is due to the fact that mouse kidneys express SSTR1–5 as well as the tubular reabsorption process [53, 54]. Also the lungs in mice have demonstrated expression of SSTR1–5 [55], which may explain the relative high uptake of ¹⁷⁷Lu-octreotate in the present study. Furthermore, for these three tissue types the ¹⁷⁷Lu concentration decreased significantly with higher amounts of ¹⁷⁷Lu-octreotate injected, demonstrating a potential saturation of SSTRs, a phenomenon previously demonstrated in non-tumor bearing C57 mice, but also for ¹¹¹In-octreotide in tumor-bearing nude mice [48, 52]. In general, these tissues had lower retention rate of ¹⁷⁷Lu compared with tumor tissue, resulting in increasing tumor-to-normal-tissue values with time. The clearly different biokinetic pattern of ¹⁷⁷Lu in lungs and kidneys, respectively, for the CLB-BAR and CLB-GE xenograft bearing mice remains to be clarified. The majority of the normal tissues demonstrated a decrease in ¹⁷⁷Lu concentration with time. Exceptions were found in the CLB-BAR mice for the kidneys (1.5 MBq) and adrenals (0.15 MBq) with maxima at 24 h p.i.

Two important parameters related to the potential success in treatment with ¹⁷⁷Lu-octreotate are tumor-to-blood (T/B) and tumor-to-kidney (T/K) values, since the bone marrow and the kidneys are the main risk organs in this treatment modality. T/B increased with time in most experiments, and T/K in all experiments, which is highly beneficial for future therapy. Furthermore, T/B and T/K for the CLB-GE and IMR-32 xenografts were higher than corresponding values reported for the human small-intestinal NET GOT1 animal model, which we have long-term experience of as a realistic model for the patient situation, and where curative effects are obtained with ¹⁷⁷Lu-octreotate [38, 40]. The estimated absorbed dose confirmed the high tumor uptake for all NB xenografts, with the tumor receiving higher dose in comparison with the kidneys in all experiments except for one. Altogether, the biokinetic data for the examined NBs and potential therapeutic effects for CLB-GE already at these low amounts, clearly demonstrate that ¹⁷⁷Lu-octreotate therapy can be very effective in these types of NB.

The present study was performed in immunocompromised Balb/c nude mice xenografted with human tumor cells, which is a well-established model type frequently used in translational research and enables in vivo studies on human tumor tissue. In recent years, the importance of the immune system on the radiation induced tumor response in radiation therapy has been investigated [56], and the choice of animal strain for preclinical models could then be of importance. However, the present study is only related to biodistribution of the radiopharmaceutical, which is highly related to the SSTR-expression at the tumor cell surface, which most probably is less dependent on the immune system. Furthermore, our previous similar biodistribution studies on other neuroendocrine tumor types in this nude mouse model have demonstrated similar data in the animal models and in the patients who donated the tumor tissue [57], which supports the interpretation and translation of the findings in the present study.

In future studies, we will examine the potential of combining two different radiopharmaceuticals. In theory it would be beneficial to combine ¹³¹I-MIBG and ¹⁷⁷Lu-octreotate, since the biokinetics in risk organs differs between the two radiopharmaceuticals. Then, the absorbed dose to the tumor tissue might be higher, while keeping the absorbed dose to the risk organs acceptable.

In conclusion, the biodistribution studies of ¹⁷⁷Lu-octreotate in all three investigated human NB xenograft mouse models demonstrated very high uptake in tumors compared with that in normal tissues. Therefore, ¹⁷⁷Lu-octreotate should be considered a potential systemic treatment option, especially in high risk NB patients with high expression of SSTRs and with low response to present treatment options.