DOI 10.1007/s11060-016-2289-4
LABORATORY INVESTIGATION
Characterization of the distribution, retention, and efficacy of internal radiation of
188Re-lipid nanocapsules
in an immunocompromised human glioblastoma model
Annabelle Cikankowitz1,2,7 · Anne Clavreul1,3 · Clément Tétaud1,7 · Laurent Lemaire1 · Audrey Rousseau4 · Nicolas Lepareur5 · Djamel Dabli6 · Francis Bouchet6 · Emmanuel Garcion1 · Philippe Menei1,3 ·
Olivier Couturier1,6 · François Hindré1,7
Received: 18 June 2016 / Accepted: 9 October 2016 / Published online: 25 October 2016
© Springer Science+Business Media New York 2016
LNCs by CED led to their complete distribution through- out the tumor and peritumoral space without leakage into the contralateral hemisphere except when large volumes were used. Seventy percent of the 188Re-SSS activity was present in the tumor region 24 h after LNC188Re-SSS injec- tion and no toxicity was observed in the healthy brain.
Double fractionated internal radiotherapy with LNC188Re- SSS triggered survival responses in the immunocompro- mised human GB model with a cure rate of 50 %, which was not observed with external radiotherapy. In conclusion, LNC188Re-SSS can induce long-term survival in an immu- nosuppressive environment, highlighting its potential for GB therapy.
Keywords Internal radiotherapy · Convection-enhanced delivery · Human glioblastoma · Rhenium-188 · Lipid nanocapsules
Abbreviations
BSA Bovine serum albumin CED Convection enhanced-delivery DMEM Dulbecco’s modified Eagle’s medium Ext RT External radiotherapy
FCS Fetal calf serum
GB Glioblastoma
HBSS Hepes buffered saline solution IST Increase in median survival time
LB B lymphocyte
LNCs Lipid nanocapsules
LNC188Re-SSS Lipid nanocapsules loaded with Rhenium-188
mAb Monoclonal antibody MRI Magnetic resonance imaging NK Natural killer
PBS Phosphate buffered saline Abstract Internal radiation strategies hold great promise
for glioblastoma (GB) therapy. We previously developed a nanovectorized radiotherapy that consists of lipid nano- capsules loaded with a lipophilic complex of Rhenium-188 (LNC188Re-SSS). This approach resulted in an 83 % cure rate in the 9L rat glioma model, showing great promise.
The efficacy of LNC188Re-SSS treatment was optimized through the induction of a T-cell immune response in this model, as it is highly immunogenic. However, this is not representative of the human situation where T-cell suppres- sion is usually encountered in GB patients. Thus, in this study, we investigated the efficacy of LNC188Re-SSS in a human GB model implanted in T-cell deficient nude mice.
We also analyzed the distribution and tissue retention of LNC188Re-SSS. We observed that intratumoral infusion of
Electronic supplementary material The online version of this article (doi:10.1007/s11060-016-2289-4) contains supplementary material, which is available to authorized users.
* Anne Clavreul
anne.clavreul@univ-angers.fr
1 INSERM U1066 MINT (Micro et Nanomédecines Biomimétiques), Université d’Angers, Angers, France
2 AMaROC, ONIRIS, Ecole Nationale Véterinaire de Nantes, Nantes, France
3 Service de Neurochirurgie, CHU d’Angers, Angers, France
4 Laboratoire de Pathologie Cellulaire et Tissulaire, CHU d’Angers, Angers, France
5 Centre Régional de Lutte Contre le Cancer (CRLCC) Eugène Marquis, Rennes, France
6 Médecine Nucléaire et Biophysique, CHU d’Angers, Angers, France
7 PRIMEX (Plateforme de Radiobiologie et d’Imagerie Expérimentale), Université d’Angers, Angers, France
Vi Volume of injection Vd Volume of distribution
Introduction
Glioblastoma (GB) is the most frequent and aggressive tumor of the central nervous system. The standard strategy consists of maximal surgical removal of the tumor mass followed by fractionated external radiotherapy of 60 Gy, and adjuvant chemotherapy with temozolomide [1, 2]. The prognosis is poor, with a mean progression-free survival of 7 months and average survival of 12–15 months [2].
Various internal radiation therapy modalities have recently emerged to more precisely target the GB site while avoid- ing adverse effects on healthy tissue [3–6]. We previously developed a nanovectorized radiotherapy consisting of lipid nanocapsules (LNCs) loaded with a lipophilic complex of Rhenium-188 (LNC188Re-SSS; half-life: 16.9 h; β− emitter:
2.12 MeV and γ emitter: 155 keV) [7–9]. It is a low-cost, simple technology, making it highly available and accessi- ble to patients. LNCs are obtained by low-energy emulsifi- cation and can be formulated without organic solvent using excipients that are safe [10]. The 188Re radionuclide can be easily eluted from the 188W/188Re generator [11]. The emis- sion of this radionuclide makes it possible to confirm tar- geting accuracy, follow the behavior, and predict treatment efficacy by determining the 188Re dose distribution within or around the tumor. The dual purpose (imaging/therapy) of this 188Re theranostic represents the future of oncology and nuclear medicine [12, 13].
We previously showed the utility of LNC188Re-SSS for the treatment of GB and hepatocellular carcinomas [8, 9].
Fractionated internal radiation with LNC188Re-SSS trig- gered a remarkable survival response (cure rate of 83 %) in the 9L rat glioma model [8]. This survival response was not observed using free-188Re treatment, probably due to its rapid elimination in urine and feces after intracranial delivery [8]. The efficacy of LNC188Re-SSS treatment in the 9L glioma model was optimized through the induction of T-cell mediated immunity. This immune response can be encountered in the 9L glioma model because of its immu- nogenic properties [14, 15] but is unlikely to be encoun- tered in GB patients. T-cell suppression is often observed in GB patients partially due to the recruitment of myeloid derived suppressor cells and regulatory T cells to the tumor site [16, 17]. We investigated the efficacy of LNC188Re- SSS in the human Lab1 GB model implanted in nude mice, in which T cell effectors were absent to better represent the clinical situation. Lab1 GB cells came from a recurrent GB that relapsed 6 months after treatment of the initial tumor by fractionated radiotherapy (60 Gy). We also analyzed the
tissue distribution and retention of LNC188Re-SSS in this model.
Materials and methods Glioblastoma cell culture
The human Lab1 cell line was derived from a primary cul- ture of a recurrent GB (Service de Neurochirurgie, CHU, Angers, France) [18]. Cells were grown to 80 % confluence in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Verviers, Belgium) containing 10 % fetal calf serum (FCS) and 1 % antibiotics (Sigma-Aldrich, Saint-Quentin Fal- lavier, France) at 37 °C under a 5 % CO2 atmosphere. All experiments were performed with cells between passages 29 and 31.
The orthotopic Lab1 GB model
Female nude-NMRI mice, aged 9 weeks, were obtained from Janvier-Labs (Le Genest Saint Isle, France). Experi- ments were approved by the Ethics of Animal Experiments of the ‘Pays de la Loire’ committee (Permit No. CEEA.
2012.56). Animals were anesthetized by intraperitoneal injection of xylazine (13 mg/kg body weight) and ketamine (100 mg/kg body weight) and were placed in a Stoelting stereotactic instrument.
On day zero (D0), mice received an intracranial injec- tion into the right striatum (coordinates: +0.5 mm anterior from the bregma, −2.1 mm lateral and −3 mm deep from the outer border of the cranium) of 5000 Lab1 cells in 5 µL HBSS containing Ca2+ and Mg2+.
Production and characterization of the LNC188Re-SSS complex
Blank-LNCs and LNC188Re-SSS were obtained using a phase inversion process as previously described [8, 19].
Before intracranial injection, LNCs were filtered (0.2 µm) and 1 mL of the formulation measured using the dose calibrator to precisely determine the volumic activity. The mean size, polydispersity index, and zeta potential were determined using a Malvern Zetasizer® Nano Serie DTS 1060 (Malvern Instruments S.A., Worcestershire, UK) 1 week after the formulation.
LNC distribution analysis by fluorescence and autoradiography
The distribution of LNCs in the orthotopic GB Lab1 model was followed by fluorescence (LNC-DID) and
autoradiography (LNC188Re-SSS) as described in supple- mentary data (Figure S1 and supplementary methods 1).
Evaluation of LNC188Re-SSS therapy on the orthotopic Lab1 GB model and comparison with external
radiotherapy
On D0, 5000 Lab1 cells, in 5 µL HBSS with Ca2+ and Mg2+, were implanted into the striatum of mice as described above (n = 20). On D12, either 3 µL Blank- LNCs (n = 7) or 3 µL LNC188Re-SSS (3 MBq, n = 8) were injected into the center of the tumor via CED with a flow rate of 0.5 µL/min. This injection protocol was repeated on D19 with a volume of 5 µL Blank-LNCs or LNC188Re- SSS. For the external beam radiation protocol, a fraction- ated regimen of 6 Gy (whole brain targeting, dose rate of 2.9 Gy/min with an XRAD 225 Cx X-ray irradiation sys- tem, 225 keV, 13 mA, collimator 15 mm, Nantes) was used on D12 and D19 following Lab1 cell implantation (n = 5).
Tumor volume was measured by MRI every week and the mice were killed when they lost more than 10 % of their body weight.
Long-term survivors obtained from fractionated internal radiation therapy (D12 and D19) were re-challenged with 5000 Lab1 cells in the left striatum (coordinates: +0.5 mm anterior from the bregma, +2.1 mm lateral and −3 mm deep from the outer border of the cranium) on D100.
MRI
Tumor progression and volume were verified and measured by MRI as described in supplementary methods 2.
Evaluation of the effect of LNC188Re-SSS CED on tumor growth and its microenvironment
Mice were injected on D0 with Lab1 cells and received Blank-LNCs (n = 4) or LNC188Re-SSS (3 MBq, n = 8) via CED on D12. They were killed on D19 and brains were snap-frozen in isopentane cooled by liquid nitro- gen to assess the tumor histology, apoptosis, proliferative intratumoral Ki67+ cells, CD31+ vessels, and immune reaction. Histological analyses were performed by a neuropathologist.
Immunofluorescence
Cryosections were fixed with ethanol (95 %)/acetic acid (5 %) or acetone. Sections were incubated in PBS contain- ing 4 % BSA and 10 % normal goat serum to block non- specific binding. Sections were incubated overnight at 4 °C with isotype controls and primary antibodies against endothelial cells (mouse CD31, Biosciences, Le Pont de
Claix, France), macrophages (mouse F4/80, eBioscience, Paris, France), B lymphocytes (mouse CD45R, eBiosci- ence), granulocytes (mouse Ly6-G, eBioscience), NK cells (mouse CD335, Ozyme, Saint Quentin en Yvelines, France) and proliferative cells (human Ki67, Dako, Trap- pes, France). Primary antibodies were detected using bioti- nylated secondary antibodies and the signal was amplified with streptavidin-FITC (Dako). Nuclei were counterstained with DAPI (Sigma). Apoptotic cells were detected based on the Trevigen® kit protocol: TACS 2 TdT-Fluor In Situ Apoptosis detection kit according to the manufacturer’s instructions (R&D systems, Lille, France).
Cryosections from three mice of each group (Blank- LNCs, LNC188Re-SSS) were analyzed under a fluorescence microscope (Axioscope® 2 optical). Immune and apop- totic cells were counted using the MetaView computer- ized image-analysis system. Results are expressed as the mean number of positively stained cells per mm2 for each group ± SEM.
Statistical analysis
Results are expressed as the mean ± SEM. The Kruskal- Wallis test was used for statistical analyses. The survival advantage was analyzed by log-rank tests, based on the Kaplan-Meier method. Differences were considered to be significant for p < 0.05.
Results
Analysis of LNC distribution in the orthotopic Lab1 GB model
Two approaches were used to follow the distribution of LNCs in the orthotopic Lab1 GB model: fluorescence (LNC-DID) and autoradiography (LNC188Re-SSS). On D12, the Lab1 tumor volume was approximately 2–3 µL by MRI and fluorescence (DAPI). Injection of 3 µL LNC-DID into the tumor with a flow rate of 0.5 µL/min resulted in a volume of distribution (Vd) of 10.2 ± 1.9 µL 1 h after injection (Fig. 1a). The Vd increased to 15.7 ± 1.6 µL when the flow rate was 0.25 µL/min. The LNC-DID were distributed throughout the tumor and accumulated in the peritumoral space (Fig. 1b). Injection of 10 or 15 µL LNC-DID yielded a Vd of approximately 36 µL resulting in a wide distribution of LNCs through- out the hemisphere and leakage into the left hemisphere via the corpus-callosum (Fig. 1b). The Lab1 tumor vol- ume was 23–24 µL on D18–19. Injection of 5 µL of LNC- DID into the tumor by CED with a flow rate at 0.5 µL/
min yielded a Vd of 32.1 ± 7.1 µL. Autoradiography anal- ysis of LNC distribution showed higher Vd values than
those measured by immunofluorescence (Fig. 1b). These values are not representative and may be explained by the difficulty to eliminate the influence of the background on the radiation emission print in this small tumor model.
Based on these data, we chose a volume of injection (Vi) of 3 µL with a flow rate of 0.5 µL/min at D12 and a Vi of 5 µL with a flow rate of 0.5 µL/min on D19 to optimally target the tumor.
Analysis of the retention of 188Re-SSS activity within mouse brains
We analyzed the activity of LNC188Re-SSS in each brain hemisphere and slices on D12 (3 µL, 3 MBq, 0.5 µL/min) and D19 (5 µL, 3 MBq, 0.5 µL/min) one and 24 h after injection into the Lab1 tumor localized in the right brain hemisphere (Fig. 2a). One hour after CED infusion, 68 % of the expected activity was detected in the brain of which 69 % was confined to the right brain hemisphere, where the tumor was present. After 24 h, 73 % of the expected activ- ity was still detected in the brain of which 76 % was in the right brain hemisphere (Fig. 2c). The activity distribution of LNC188Re-SSS in brain slices is presented in Fig. 2b.
As previously described [5], approximatively 15 % of the LNCs were distributed throughout the body 24 h after CED administration (Fig.S2).
Analysis of the efficacy of internal radiotherapy on the Lab1 GB model and comparison with external radiotherapy
Lab1 tumor-bearing mice were treated by a CED injection of LNC188Re-SSS or Blank-LNCs on D12 (3 µL, 3 MBq, 0.5 µL/min) and D19 (5 µL, 3 MBq, 0.5 µL/min) (Fig. 3a).
Mice receiving no treatment or Blank-LNCs died within 27 days, with a median survival of 21 and 20 days, respec- tively (Fig. 3). In contrast, mice treated with LNC188Re-SSS had a median survival time of 180 % relative to mice treated with Blank-LNCs. In addition, four mice (50 %) treated with LNC188Re-SSS were long-term survivors (Fig. 3d).
Monitoring of Lab1 tumor growth by MRI showed that the tumor volume was approximately 2 µL before treatment (D11). The Lab1 tumor grew in control mice (no treat- ment and Blank-LNCs) until D25 with a diffuse morphol- ogy (Fig. 3c). Lab1 tumor growth was significantly reduced (approximatively half) in mice treated with LNC188Re-SSS 6 days after the first injection (12.72 ± 5.28 µL) relative to Blank-LNCs (30.00 ± 4.60 µL) (Fig. 3). MRI or histological analysis did not show a detectable brain tumor at D100 in long-term surviving animals. The long-term survivors were re-challenged by implantation of 5000 Lab1 cells in the left striatum. There was no increase in their median survival time. They died within 27 days as did the animals of the control group.
Fig. 1 Analysis of LNC distribution by fluorescence (LNC-DID) and autoradiography (LNC188Re-SSS). a Different volumes of LNC-DID or LNC188Re-SSS (Vi) were injected via CED at two flow rates on D12 and D18 after Lab1 cell implantation. Mice (three per condition) were sacrificed 1 h later and the LNC volume of distribution (Vd) was determined by fluorescence analysis or autoradiography. The val-
ues are presented in the table. b Fluorescence images of LNC-DID (red) 1 h after their injection into the Lab1 tumor by CED (Vi from 3 to 15 µL on D12 and a Vi of 5 µL on D18). Nuclei were stained with DAPI. The border of the tumor is indicated in white (scale bar = 500 µm)
We irradiated mice bearing an intracranial Lab1 tumor with 6 Gy on D12 and D19 to investigate the relative effi- cacy of fractionated D12-D19 internal radiotherapy ver- sus external beam radiation. This treatment slowed tumor progression, but unlike internal radiotherapy, none of the tumor-bearing mice were cured by external radiation (Fig. 3).
Effect of LNC188Re-SSS treatment on the tumor and its microenvironment
On D19 (7 days after the first intratumoral injection of LNC188Re-SSS), we observed a difference in the histol- ogy of the tumors treated with LNC188Re-SSS relative to those treated by injection of Blank-LNCs. The Lab1 tumor treated with Blank-LNCs showed high cellular- ity, whereas the tumor treated with LNC188Re-SSS was smaller and composed of atypical cells with bigger nuclei.
Tumors treated with LNC188Re-SSS had few Ki67+
cells, disorganized CD31+ vessels, and a higher number of apoptotic cells (Fig. 4). Injection of LNC188Re-SSS induced intratumoral infiltration of CD45R+ B lympho- cytes and increased infiltration of F4/80+ macrophages and Ly6-G+ granulocytes (Fig. 4). We did not detect any NK cells in untreated or treated tumors. On D26 (7 days after the second intratumoral injection of LNC188Re- SSS), the tumoral tissue was further weakened, disinte- grating, and composed of more atypical cells (data not shown). We observed no tumors on D100 in long-term survivors, as indicated above, and the tissue was normal by histology.
We analyzed the periphery of the tumor after LNC188Re-SSS treatment. The structure of this area was similar to that observed in the contralateral hemisphere, and the vessels were well organized after the first and second injection of LNC188Re-SSS. We observed no apoptotic cell figures (data not shown).
Fig. 2 Determination of the 188Re-SSS activity in Lab1 tumor- bearing mice. A Vi of 3 or 5 µL LNC188Re-SSS corresponding to an activity of 3 MBq was injected on D12 and D19 after Lab1 cell implantation, respectively. After 1 and 24 h, brains were cut into eight, 1 mm slices and the 188Re-SSS activity measured. a Mouse brain-top view showing slices from the front (number 1) to the back
(number 8). bGraph representing the distribution of 188Re-SSS activ- ity (MBq) throughout the brain (n = number of mice analyzed). c Table showing the 188Re-SSS activity obtained within the entire brain and in each hemisphere [L (left) and R (right)]. Activity results are corrected for decay of the 188Re at the time of brain harvest (1 or 24 h) and are expressed in MBq
Discussion
Internal radiation therapy modalities resulting in local- ized radiation for the treatment of GB are in full develop- ment [3–6]. One strategy consists of using radiolabeled mAbs, such as those against tenascin C or the EGFR. Sev- eral phase I/II trials have been performed to date but their efficacy was limited due in part to heterogeneous expres- sion of the antigens, catabolism of the immunoglobulins, and an unfavorable biodistribution of the mAb leading to
a high radiation burden in normal tissue [6, 20–23]. In this study, we have developed another internal radiation therapy strategy that consists of LNC188Re-SSS. We first analyzed the distribution and retention of LNC188Re-SSS in the Lab1 GB model. The intratumoral injection of LNC188Re- SSS was performed using CED technology, a bulk flow- driven process which permits the infusion of clinically relevant amounts of therapeutic agents directly into the tumor, bypassing the blood-brain barrier [24–28]. There is little information about the distribution of nanocarriers
Fig. 3 Efficacy of fractionated D12–D19 internal radiotherapy using LNC188Re-SSS after administration by CED in the Lab1 GB model.
a Experimental design of the treatment of the Lab1 GB model with LNC188Re-SSS. The presence and localization of the Lab1 tumor were verified by MRI on D11 after Lab1 cell injection. Animals in which the tumor was incorrectly localized or insufficiently devel- oped (<1.5 µL) were excluded from the study. On D12, mice were infused with 3 µL of Blank-LNCs or LNC188Re-SSS (3 MBq,) at a flow rate of 0.5 µL/min. This protocol was repeated on D19 with an infusion of 5 µL of Blank-LNCs or LNC188Re-SSS with the same flow rate. Tumor progression was followed weekly by MRI.
b Kaplan-Meier survival curves for Lab1 tumor-bearing mice with indicated treatments. [No treatment (n = 5, black line), Blank-LNCs
(n = 7, blue line), LNC188Re-SSS (n = 8, orange line), external radio- therapy (6 Gy) (n = 5, purple line)]. Fifty percent of mice treated with LNC188Re-SSS survived long-term. c Representative T2-weighted MRI images of mice treated with Blank-LNCs or LNC188Re-SSS.
d Table representing the efficacy of LNC188Re-SSS therapy on the xenograft Lab1 GB model and comparison with external radiother- apy. The external radiotherapy (2 × 6 Gy) was performed on D12 and D19 after Lab1 cell injection. The increase in median survival time (IST median) is calculated relative to the control Blank-LNC group (100 %). The tumor volume (µL) on D11 and D18 after the intracer- ebral injection of Lab1 cells was measured by analysis of the ROI by MRI (mean ± SD; *p = 0.03 < 0.05) (n = number of animals)
using CED in brain tumors. The injection volume gener- ally used for CED in mouse brain is between 5 and 18 µL, but is mostly set at 10 µL with a flow rate of 0.5 µL/min [29–33]. In our study, fluorescence analysis showed that an
injection of 10 µL LNC-DID, at a flow rate of 0.5 µL/min, into a small Lab1 tumor (volume about 2–3 µL) resulted in wide distribution of LNCs throughout the hemisphere and leakage into the left hemisphere via the corpus-callosum.
Fig. 4 Evaluation of LNC188Re-SSS treatment on tumor growth and its microenvironment. a Histology and immunofluorescence analyses of the Lab1 tumor 17 days (D19) after the treatment of Lab1 bear- ing mice with Blank-LNCs or LNC188Re-SSS. Three brains for each group were used for these analyses. Ki67, CD31, CD45R, F4/80, and
Ly6-G markers are in green and nuclei are in blue (scale bar = 100 µm). b Quantitative results for immune cells and apoptosis. Results are expressed as the mean ± SEM of number of immune or apoptotic cells in the tumor/mm2. *Significantly different from the Blank-LNC group (p < 0.05)
Injections of 3 and 5 µL LNCs, at a flow rate of 0.5 µL/min, led to a Vd of approximately 10 and 13 µL, respectively, largely sufficient to cover the entire tumor volume and its peripheral environment. The Vd increased with a lower flow rate fixed at 0.25 µL/min as previously described [34]. We also observed that the Vd positively correlated with tumor size for a given Vi and flow rate. This obser- vation is different from that of Saucier-Sawyer et al who investigated intratumoral CED infusions of “brain-pene- trating” nanoparticles (BPNPs) in animals bearing either U87 or RG2 intracranial tumors [35]. They observed that the Vd of BNPPs did not change significantly with tumor size. This suggests that tumor distribution may depend on the nanocarrier system used, highlighting the need to address the tumor distribution of each nanocarrier system before initiating a therapeutic efficacy study. The distribu- tion data obtained by fluorescence could not be validated by autoradiography due to the small tumor model and the difficulty to eliminate the influence of the background on the radiation emission print. However, it provided a pre- liminary qualitative approach to study the LNC188Re-SSS volume and distance of action. Analysis of the retention of LNC188Re-SSS activity in mouse brain showed that approx- imately 70 % was present in the region of interest 24 h after LNC injection, in agreement with our previous results in the 9L glioma model [8]. The half-life of 188Re is 17 h. A retention time of 24 h is sufficient for this radionuclide to exert a continuous radiobiological effect at a maximum dis- tance of 10 mm (mean penetration range of 3.1 mm) while minimizing toxicity risks.
Fractionated internal radiotherapy by LNC188Re-SSS resulted in survival in the Lab1 model with cure rates of 50 %. The tumors treated with LNC188Re-SSS had a lower proportion of proliferative Ki67+ cells and more highly dis- organized vessels than tumors treated with Blank-LNCs.
We observed more apoptotic cells and greater infiltra- tion of innate immune cells, such as macrophages and granulocytes, and B lymphocytes in the tumors treated with LNC188Re-SSS than those treated with Blank-LNCs.
Apoptosis and immune tumor infiltration have already been observed after irradiation [3, 36–38]. We also observed multinucleated giant cells with a flattened morphology in the tumors treated with LNC188Re-SSS. These giant non- apoptotic cells may be endopolyploid cells described after severe genotoxic insult with irradiation or chemotherapy [39, 40]. Most of these cells stop endoreduplication and undergo senescence, whereas some release para-diploid mitotic sub-cells, which commence clonogenic growth and reenter the mitotic cell cycle prior to disease recurrence [41–45]. The induction of these cells by LNC188Re-SSS treatment may explain why 50 % of animals bearing Lab1 tumors showed only a prolonged median survival. Further studies are needed to determine the involvement of these
giant cells in LNC188Re-SSS ionizing radiation resistance.
Long-term survivors rechallenged with Lab1 GB cells did not show longer median survival than naive mice with the tumor, in contrast to the 9L glioma model, indicating the importance of T cells in the induction of a memory immune response against the tumor. The peritumoral zone had a normal histology after LNC188Re-SSS treatment, showing neither immune cell infiltration nor apoptosis, highlighting the limited side effects of this treatment on healthy brain after local delivery.
In conclusion, we show that LNC188Re-SSS can be dis- tributed throughout the tumor by CED and retained in the region of interest for at least 24 h with no toxicity to healthy brain. LNC188Re-SSS are functional in an immunosuppres- sive environment, which may be useful given the heteroge- neity of the immune status of GB patients. The next step is to conduct a phase I study in patients with relapsed/refrac- tory GB to assess the feasibility of delivering LNC188Re- SSS by CED in a volume and dose adapted to the patient, sufficient to provide coverage of the entire tumor and the paths followed by the infiltrated tumoral cells at the appro- priate dose.
Ackowledgements We thank neurosurgeons from CHU Angers for providing the Lab1 tumor sample. We also thank Dr Catherine Ibisch and Dr Jérôme Abadie (AMaROC, ONIRIS, Nantes), Pierre Legras and Jérôme Roux (Service Commun d’Animalerie Hospitalo- Universitaire, Angers), Pr Jean-Pierre Benoit and Aurélien Contini (INSERM U1066-MINT, Angers), Dr Florence Franconi (PRIMEX, Angers) and Dr Franck Lacoeuille (Médecine Nucléaire et Biophy- sique, CHU d’Angers) for allowing us to use their facilities. This work was supported by the French National Research Agency through the RADIOHEAD program (ANR-12-EMMA-0033-01), “La Région Pays-de-la-Loire” through the Nuclear Technology for Health project (NucSan) and IRAD programs, “La Ligue Nationale Contre le Can- cer” through an “Equipe Labellisée 2012” grant, the “Institut National de la Santé et de la Recherche Médicale” (INSERM), the “Axe Vec- torisation et Radiothérapies”, and the “Réseau Gliome Grand Ouest”
(ReGGO) of the “Cancéropôle Grand-Ouest”. The co-authors of this manuscript are also members of the Labex IRON “Innovative Radi- opharmaceuticals in Oncology and Neurology” as part of the French government program “Investissements d’Avenir”. A. Ci. received a fellowship from the NucSan program.
Compliance with ethical standards
Conflict of interest We have no potential conflicts of interest to de- clare.
References
1. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumour and Radiotherapy Groups, National Cancer Insti- tute of Canada Clinical Trials Group (2005) Radiotherapy plus
concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996
2. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K, Hau P, Brandes AA, Gijtenbeek J, Marosi C, Vecht CJ, Mokhtari K, Wesseling P, Villa S, Eisenhauer E, Gorlia T, Weller M, Lacombe D, Cairncross JG, Mirimanoff RO, European Organi- sation for Research and Treatment of Cancer Brain Tumour and Radiation Oncology Groups, National Cancer Institute of Can- ada Clinical Trials Group (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10(5):459–466
3. Vanpouille-Box C, Hindre F (2012) Nanovectorized radiother- apy: a new strategy to induce anti-tumor immunity. Front Oncol 2:136
4. Oh DS, Adamson DC, Kirkpatrick JP (2012) Targeted radio- therapy for malignant gliomas. Curr Drug Discov Technol 9(4):268–279
5. Barani IJ, Larson DA (2015) Radiation therapy of glioblastoma.
Cancer Treat Res 163:49–73
6. von Neubeck C, Seidlitz A, Kitzler HH, Beuthien-Baumann B, Krause M (2015) Glioblastoma multiforme: emerging treat- ments and stratification markers beyond new drugs. Br J Radiol 88(1053):20150354
7. Allard E, Hindre F, Passirani C, Lemaire L, Lepareur N, Noiret N, Menei P, Benoit JP (2008) 188Re-loaded lipid nanocapsules as a promising radiopharmaceutical carrier for internal radio- therapy of malignant gliomas. Eur J Nucl Med Mol Imaging 35(10):1838–1846
8. Vanpouille-Box C, Lacoeuille F, Belloche C, Lepareur N, Lemaire L, LeJeune JJ, Benoit JP, Menei P, Couturier OF, Garcion E, Hindre F (2011) Tumor eradication in rat gli- oma and bypass of immunosuppressive barriers using inter- nal radiation with (188)Re-lipid nanocapsules. Biomaterials 32(28):6781–6790
9. Vanpouille-Box C, Lacoeuille F, Roux J, Aube C, Garcion E, Lepareur N, Oberti F, Bouchet F, Noiret N, Garin E, Benoit JP, Couturier O, Hindre F (2011) Lipid nanocapsules loaded with rhenium-188 reduce tumor progression in a rat hepatocellular carcinoma model. PLoS One 6(3):e16926
10. Hureaux J, Lagarce F, Gagnadoux F, Rousselet MC, Moal V, Urban T, Benoit JP (2010) Toxicological study and efficacy of blank and paclitaxel-loaded lipid nanocapsules after i.v. adminis- tration in mice. Pharm Res 27(3):421–430
11. Lepareur N, Ardisson V, Noiret N, Boucher E, Raoul JL, Clem- ent B, Garin E (2011) Automation of labelling of Lipiodol with high-activity generator-produced 188Re. Appl Radiat Isot 69(2):426–430
12. Guo R, Zhang M, Xi Y, Ma Y, Liang S, Shi S, Miao Y, Li B (2014) Theranostic studies of human sodium iodide symporter imaging and therapy using 188Re: a human glioma study in mice. PLoS One 9(7):e102011
13. Srivastava SC (2012) Paving the way to personalized medi- cine: production of some promising theragnostic radionu- clides at Brookhaven National Laboratory. Semin Nucl Med 42(3):151–163
14. Barth RF, Kaur B (2009) Rat brain tumor models in experimen- tal neuro-oncology: the C6, 9L, T9, RG2, F98, BT4C, RT-2 and CNS-1 gliomas. J Neurooncol 94(3):299–312
15. Stojiljkovic M, Piperski V, Dacevic M, Rakic L, Ruzdijic S, Kanazir S (2003) Characterization of 9L glioma model of the Wistar rat. J Neurooncol 63(1):1–7
16. Dubinski D, Wolfer J, Hasselblatt M, Schneider-Hohendorf T, Bogdahn U, Stummer W, Wiendl H, Grauer OM (2016) CD4 +
T effector memory cell dysfunction is associated with the accu- mulation of granulocytic myeloid-derived suppressor cells in glioblastoma patients. Neuro. Oncol 18(6):807–818
17. See AP, Parker JJ, Waziri A (2015) The role of regulatory T cells and microglia in glioblastoma-associated immunosuppression. J Neurooncol 123(3):405–412
18. Clavreul A, Jean I, Preisser L, Chassevent A, Sapin A, Micha- lak S, Menei P (2009) Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy.
In Vitro Cell Dev Biol Anim 45(9):500–511
19. Heurtault B, Saulnier P, Pech B, Proust JE, Benoit JP (2002) A novel phase inversion-based process for the preparation of lipid nanocarriers. Pharm Res 19(6):875–880
20. Reardon DA, Quinn JA, Akabani G, Coleman RE, Friedman AH, Friedman HS, Herndon JE 2nd, McLendon RE, Pegram CN, Provenzale JM, Dowell JM, Rich JN, Vredenburgh JJ, Desjar- dins A, Sampson JH, Gururangan S, Wong TZ, Badruddoja MA, Zhao XG, Bigner DD, Zalutsky MR (2006) Novel human IgG2b/
murine chimeric antitenascin monoclonal antibody construct radiolabeled with 131I and administered into the surgically cre- ated resection cavity of patients with malignant glioma: phase I trial results. J Nucl Med 47(6):912–918
21. Casacó A, López G, García I, Rodríguez JA, Fernández R, Figueredo J, Torres L, Perera A, Batista J, Leyva R, Peña Y, Amador Z, González A, Estupiñan B, Coca M, Hernández A, Puig M, Iglesias M, Hernández A, Ramos M, Rodríquez L, Suarez N (2008) Phase I single-dose study of intracavitary- administered Nimotuzumab labeled with 188 Re in adult recur- rent high-grade glioma. Cancer Biol Ther 7(3):333–339
22. Li L, Quang TS, Gracely EJ, Kim JH, Emrich JG, Yaeger TE, Jenrette JM, Cohen SC, Black P, Brady LW (2010) A Phase II study of anti-epidermal growth factor receptor radioimmuno- therapy in the treatment of glioblastoma multiforme. J Neurosurg 113(2):192–198
23. Reulen HJ, Poepperl G, Goetz C, Gildehaus FJ, Schmidt M, Tatsch K, Pietsch T, Kraus T, Rachinger W (2015) Long-term outcome of patients with WHO Grade III and IV gliomas treated by fractionated intracavitary radioimmunotherapy. J Neurosurg 123(3):760–770
24. Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH (1994) Convection-enhanced delivery of macromol- ecules in the brain. Proc Natl Acad Sci USA 91(6):2076–2080 25. Allard E, Passirani C, Benoit JP (2009) Convection-enhanced
delivery of nanocarriers for the treatment of brain tumors. Bio- materials 30(12):2302–2318
26. Jahangiri A, Chin AT, Flanigan PM, Chen R, Bankiewicz K, Aghi MK (2016) Convection-enhanced delivery in glioblastoma:
a review of preclinical and clinical studies. J Neurosurg:1–10.
27. Vogelbaum MA, Aghi MK (2015) Convection-enhanced deliv- ery for the treatment of glioblastoma. Neuro. Oncol 17(Suppl 2):ii3–ii8
28. Healy AT, Vogelbaum MA (2015) Convection-enhanced drug delivery for gliomas. Surg Neurol Int 6(Suppl 1):S59–S67 29. Shultz MD, Wilson JD, Fuller CE, Zhang J, Dorn HC, Fatouros
PP (2011) Metallofullerene-based nanoplatform for brain tumor brachytherapy and longitudinal imaging in a murine orthotopic xenograft model. Radiology 261(1):136–143
30. Kaluzova M, Bouras A, Machaidze R, Hadjipanayis CG (2015) Targeted therapy of glioblastoma stem-like cells and tumor non- stem cells using cetuximab-conjugated iron-oxide nanoparticles.
Oncotarget 6(11):8788–8806
31. Bernal GM, LaRiviere MJ, Mansour N, Pytel P, Cahill KE, Voce DJ, Kang S, Spretz R, Welp U, Noriega SE, Nunez L, Larsen G, Weichselbaum RR, Yamini B (2014) Convection-enhanced delivery and in vivo imaging of polymeric nanoparticles for the treatment of malignant glioma. Nanomedicine 10(1):149–157
32. Hadjipanayis CG, Machaidze R, Kaluzova M, Wang L, Schuette AJ, Chen H, Wu X, Mao H (2010) EGFRvIII antibody-conju- gated iron oxide nanoparticles for magnetic resonance imaging- guided convection-enhanced delivery and targeted therapy of glioblastoma. Cancer Res 70(15):6303–6312
33. Chen PY, Ozawa T, Drummond DC, Kalra A, Fitzgerald JB, Kir- potin DB, Wei KC, Butowski N, Prados MD, Berger MS, For- sayeth JR, Bankiewicz K, James CD (2013) Comparing routes of delivery for nanoliposomal irinotecan shows superior anti-tumor activity of local administration in treating intracranial glioblas- toma xenografts. Neurooncology 15(2):189–197
34. Chen MY, Lonser RR, Morrison PF, Governale LS, Oldfield EH (1999) Variables affecting convection-enhanced delivery to the striatum: a systematic examination of rate of infusion, cannula size, infusate concentration, and tissue-cannula sealing time. J Neurosurg 90(2):315–320
35. Saucier-Sawyer JK, Seo YE, Gaudin A, Quijano E, Song E, Saw- yer AJ, Deng Y, Huttner A, Saltzman WM (2016) Distribution of polymer nanoparticles by convection-enhanced delivery to brain tumors. J Control Release 232:103–112
36. Demaria S, Golden EB, Formenti SC (2015) Role of Local Radiation Therapy in Cancer Immunotherapy. JAMA Oncol 1(9):1325–1332
37. Sridharan V, Schoenfeld JD (2015) Immune effects of targeted radiation therapy for cancer. Discov Med 19(104):219–228 38. Maier P, Hartmann L, Wenz F, Herskind C (2016) Cellular path-
ways in response to Ionizing radiation and their targetability for tumor radiosensitization. Int J Mol Sci 14:17 (1)
39. Illidge TM, Cragg MS, Fringes B, Olive P, Erenpreisa JA (2000) Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage. Cell Biol Int 24(9):621–633
40. Kaur E, Rajendra J, Jadhav S, Shridhar E, Goda JS, Moiyadi A, Dutt S (2015) Radiation-induced homotypic cell fusions of innately resistant glioblastoma cells mediate their sustained sur- vival and recurrence. Carcinogenesis 36(6):685–695
41. Ivanov A, Cragg MS, Erenpreisa J, Emzinsh D, Lukman H, Ill- idge TM (2003) Endopolyploid cells produced after severe gen- otoxic damage have the potential to repair DNA double strand breaks. J Cell Sci 116(Pt 20):4095–4106
42. Erenpreisa J, Ivanov A, Wheatley SP, Kosmacek EA, Ianzini F, Anisimov AP, Mackey M, Davis PJ, Plakhins G, Illidge TM (2008) Endopolyploidy in irradiated p53-deficient tumour cell lines: persistence of cell division activity in giant cells express- ing Aurora-B kinase. Cell Biol Int 32(9):1044–1056
43. Erenpreisa J, Salmina K, Huna A, Kosmacek EA, Cragg MS, Ianzini F, Anisimov AP (2011) Polyploid tumour cells elicit paradiploid progeny through depolyploidizing divisions and reg- ulated autophagic degradation. Cell Biol Int 35(7):687–695 44. Mirzayans R, Andrais B, Scott A, Wang YW, Murray D (2013)
Ionizing radiation-induced responses in human cells with differ- ing TP53 status. Int J Mol Sci 14(11):22409–22435
45. Schwarz-Finsterle J, Scherthan H, Huna A, Gonzalez P, Muel- ler P, Schmitt E, Erenpreisa J, Hausmann M (2013) Volume increase and spatial shifts of chromosome territories in nuclei of radiation-induced polyploidizing tumour cells. Mutat Res 756(1–2):56–65
46. Lainé AL, Clavreul A, Rousseau A, Tétaud C, Vessieres A, Gar- cion E, Jaouen G, Aubert L, Guilbert M, Benoit JP, Toillon RA, Passirani C (2014) Inhibition of ectopic glioma tumor growth by a potent ferrocenyl drug loaded into stealth lipid nanocapsules.
Nanomedicine 10(8):1667–1677
