Purpose:

Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)—a tumor with an extremely poor prognosis.

Experimental Design:

A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow–derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach.

Results:

A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls.

Conclusions:

A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

Translational Relevance

Glioblastoma (GBM) is the most common malignant type of primary brain tumor with poor prognosis and very limited therapy options. Main challenges in the treatment of GBM include limitations of drug delivery due to the blood–brain barrier, intratumoral and intertumoral heterogeneity, and infiltration into the normal brain parenchyma. Mesenchymal stem cells (MSC) have gained attention as a new treatment platform for the delivery of anticancer cargo, such as therapeutic genes, based on their intrinsic tumor-homing capacity targeting the tumor microenvironment of many solid tumors including GBM. Gene therapy based on the biology of the theranostic sodium iodide symporter (NIS) allows radioiodide application for effective tumor imaging and treatment. A MSC-mediated NIS-based radioiodide therapy approach was performed in a preclinical GBM mouse model demonstrating effective MSC recruitment along with functional NIS expression. A reduced tumor growth and significantly prolonged survival was shown after systemic administration of engineered MSCs and radioiodide application.

Glioblastoma (GBM, grade IV) is the most common and malignant type of primary brain tumor in adults with an extremely poor prognosis (1). The current standard of care includes surgical resection followed by radiochemotherapy with temozolomide and by further adjuvant temozolomide (2). Despite recent advances in characterizing new targets for novel therapies, long-term survival is rare due to a very high recurrence rate (3). The development of more efficacious therapeutic strategies is urgently needed to improve the outcome of patients with GBM.

The major problems that are encountered during the treatment of GBM include the strong therapy resistance of these highly heterogeneous tumors that are notorious for their infiltrative growth pattern (4). An additional important issue is the limitation of most systemically delivered drugs in overcoming the blood–brain barrier (BBB), a structural and functional barrier that separates the peripheral blood from the brain (5). This barrier is partly maintained in GBM (6). Finally, the brain tumor microenvironment is characterized by extensive intratumoral and intertumoral heterogeneity at the cellular and molecular level rendering the process of designing effective targeted, individualized therapies even more complex (7, 8).

A promising treatment approach for GBM is the use of cell-based therapies that can in theory target multiple independent parameters in tumor microenvironments (9). Different types of adult stem cells including neural stem cells (NSC) and mesenchymal stem cells (MSC) have been previously shown to have an inherent ability to specifically migrate into malignant gliomas by overcoming the BBB and have shown the ability to even target single infiltrating tumor cells (10–15).

In their pioneering work, Aboody and colleagues demonstrated that murine NSCs were able to migrate to brain tumors using different routes of implantation: NSC implantation directly into the tumor bed, implantation at an intracranial site distant from the tumor bed in the same or contralateral hemisphere, or intravenous implantation, and led to selective targeting of the brain tumor mass (14). Because the use of NSCs is challenging for clinical application due to the need for high numbers of NSCs needed to meet sufficient dose requirements for human trials, MSCs may represent a more promising alternative source (16). However, to date relatively few early Phase I/II clinical trials (e.g., NCT03896568; NCT04758533) have been conducted using MSCs as delivery vehicles for the treatment of GBM.

MSCs are multipotent progenitor cells with self-renewing capabilities and a high differentiation potential (17). They are well suited for clinical purposes, because they can be easily obtained from different tissue sources (e.g., adipose tissue, umbilical cord, bone marrow), rapidly propagated, and relatively easily genetically modified in vitro. In addition, their low immunogenicity allows allogenic cells to be used (15).

The mechanisms underlying the recruitment of MSCs to tumor sites is still not well understood, but is thought to be driven by the inflammatory micromilieu of the tumor. In this regard, malignant tumors are often described as a chronic injury or “never healing wound” (18). In the course of injury or during chronic inflammation, MSCs are actively recruited to these sites to contribute to tissue remodeling (19).

Current treatment limitations for GBM are often associated with the BBB. MSCs possess leukocyte-like abilities allowing them to transmigrate across the BBB. In addition, the decreased vessel tightness observed in the tumor neovasculature may support diffusion into the tumor parenchyma and lead to passive MSC entrapment in the brain (20).

The therapeutic use of MSCs in cancer is based on their intrinsic tumor-homing capacity that can be exploited for delivery of an antitumor cargo. A number of basic and preclinical studies have used MSCs as cell-based vectors to deliver antitumor proteins, antitumor miRNAs, suicide genes, immunostimulants, and oncolytic viruses (21–26). Recently, research on MSCs has focused more on their use in tumor-targeted gene therapy (27).

Over the past two decades, the sodium iodide symporter (NIS) has emerged as a powerful theranostic gene for the management and treatment of cancer. NIS is a naturally occurring transmembrane glycoprotein usually localized at the basolateral membrane of thyrocytes. It is responsible for the active transport of iodide from the blood into the thyroid as an important prerequisite for thyroid hormone synthesis (28). The dual function of NIS, as reporter and therapy gene, has been widely used in the treatment of differentiated thyroid cancer for 80 years allowing the application of radioiodide as a diagnostic and therapeutic agent (29). Various isotopes can be efficiently transported by NIS that are used for diagnostic purposes (e.g., 123I, 124I, 125I, 99mTc, 18F-tetrafluoroborate) using scintigraphy, single-photon emission computed tomography (SPECT), or PET imaging. In addition, the system is ideal for the therapeutic application of radionuclides (131I, 188Re, 211At). Therapeutic efficacy is enhanced through a bystander effect based on the crossfire effects of the β-emitters 131I and 188Re or the α-emitter 211At that act on NIS-transduced cells as well as neighboring cells (30). Several studies including work by our group have shown the great potential of NIS gene-based therapy using MSCs as delivery vectors for the treatment of distinct nonthyroidal tumors (12, 31–35). In addition to successful MSC delivery to GBM, there are also reports from several preclinical and clinical studies describing the delivery of iodide or alternative isotopes transported by NIS to the brain (12, 36–40).

In the current study, we sought to expand the NIS gene therapy strategy to GBM using a genetically modified murine MSC line, syngeneic to both tumor and host tissue, constitutively expressing NIS driven by a cytomegalovirus (CMV) promoter, using both a subcutaneous and an orthotopic GBM mouse model. The orthotopic GL261 model used in this study has been extensively applied in a series of other experimental studies and shown to recapitulate important histopathologic features of human GBM such as invasive growth and proangiogenic characteristics, and mimics closely the tumor microenvironment as the host immune system is intact (41). In addition, proof of concept was further evaluated in a second subcutaneous U87 xenograft model. We investigated the potential use of NIS as reporter gene to track adoptively applied MSCs in GBM in vivo and ex vivo after systemic delivery followed by therapeutic application using 131I. To this end, in vivo biodistribution of NIS-expressing MSCs (NIS-MSC) was monitored by highly sensitive 124I-PET imaging or 123I-scintigraphy. Finally, 131I application in NIS-MSC–treated GBM mice lead to a significant increase in survival and reduced tumor growth monitored by MRI.

Cell culture

The murine glioma cell line GL261 was purchased from the NCI (Frederick, MD) and the human glioma cell line U87 (CLS 300367) was purchased from Cell Line GmbH. Both cell lines were cultured in DMEM low glucose (Sigma-Aldrich) supplemented with 10% [volume for volume (v/v)] FBS Superior (Sigma-Aldrich), 1% (v/v) MEM non-essential amino acid solution (Thermo Fisher Scientific), and 1% (v/v) penicillin/streptomycin (Sigma-Aldrich).

Murine and human MSCs were isolated from the bone marrow [in the following referred to as wildtype MSCs (Wt-MSCs)] and are characterized as described previously (31, 32, 42). NIS-MSCs were produced by stable transfection of Wt-MSCs with the expression vector CMV-NIS-pcDNA3, containing the constitutively active CMV promoter coupled to full-length NIS cDNA. NIS functionality, assessed by iodide uptake ability, and tumor-tropic migratory capacity of these NIS-MSCs has been shown by our group previously (31, 32). MSCs were cultured in RPMI (Sigma-Aldrich) containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin and selection of murine NIS-MSCs was maintained with 100 μg/mL G418 (Sigma-Aldrich) and 500 μg/mL G418 for human MSCs. Preparation of MSCs for injection into mice was performed as described previously (43).

All cells were maintained in an incubator at 37°C in a humidified atmosphere of 95% and 5% CO2. The cell lines were examined for Mycoplasma and viruses according to the FELASA (Federation of European Laboratory Animal Science Associations) guidelines by Charles River Research Animal Diagnostic Services (Mouse essential panel) prior to in vivo transplantation.

Animals

Female C57Bl/6 mice were purchased from Charles River and used for subcutaneous (6-week-old) or orthotopic (8-week-old) GL261 cell implantation. For subcutaneous U87 cell injection (6-week-old), female CD-1 nu/nu mice were purchased from Charles River. Mice were kept under specific pathogen-free conditions with access to chow and water ad libitum. Tumor-harboring mice were treated with drinking water supplemented with 5 mg/mL levothyroxine (L-T4, Sigma-Aldrich) in 0.01% (v/v) BSA (Sigma-Aldrich) to suppress intrinsic thyroidal tracer uptake, in addition to iodide-deficient diet (ssniff Spezialdiäten GmbH) 10 days prior to the imaging experiments and during 131I therapy experiments.

All animal experiments were approved by the local governmental commission for animals (Government of Upper Bavaria/Regierung von Oberbayern) and were conducted in accordance with institutional guidelines of animal welfare of the Klinikum rechts der Isar, Technical University of Munich. Mice were sacrificed at defined presymptomatic timepoints or at defined humane endpoints (significant weight loss; neurologic symptoms; changes in drinking, eating, or cleaning behavior; signs of pain).

Establishment of subcutaneous and orthotopic syngeneic GBM mouse models

Subcutaneous syngeneic GL261 and xenograft U87 tumors were generated by subcutaneous injection of 1 × 106 cells into the right flank. Tumors were regularly measured using a caliper and the tumor volumes were estimated using the equation volume = length × width × height × 0.52.

For orthotopic brain tumor implantation, mice were anesthetized with ketamine and xylazine and immobilized in a stereotactic frame (David Kopf Instruments) in a flat-skull position. A middle line skin incision was made on the top of the skull and a hole was carefully drilled into the skull with a 21G needle 1 mm anterior and 1.5 mm to the right of the bregma. A blunt Hamilton syringe (22G, Hamilton) was stereotactically inserted 4 mm deep and retracted 1 mm. GL261 cells were implanted (1 × 105 cells/μL PBS) into the brain in a total volume of 1 μL within 2 minutes. The syringe was removed slowly in 1 mm/minute steps and the skin was sutured. Mice were preoperatively and postoperatively treated with meloxicam (0.5 mg/kg; Boehringer Ingelheim Vetmedica GmbH).

Radioiodide biodistribution studies in vivo in subcutaneous GBM tumors using 123I-scintigraphy

Once subcutaneous GL261 and U87 tumors had reached a volume of approximately 500 mm³, mice received three applications of MSCs in 2-day intervals, followed by intraperitoneal injection of 18.5 MBq (0.5 mCi) 123I (GE Healthcare) 72 hours later. Serial scanning 1 to 8 hours after tracer application was performed on a gamma camera using a low-energy high-resolution collimator (ecam, Siemens). As a control for NIS-specific radioiodide accumulation in the tumor, perchlorate (NaClO4; 2 mg per mouse), a competitive inhibitor for NIS-mediated iodide transport, was injected intraperitoneally 30 minutes before 123I administration. Regions of interest were drawn with HERMES Gold software (HERMES Medical Solutions) and tumoral iodide uptake was determined and calculated as percentage of the injected radionuclide dose per gram tumor (% ID/g). Tumor weight was assessed after removal of the tumors at the end of the imaging study. Dosimetry for 131I was calculated with a RADAR dose factor (www.doseinfo-radar.com) using the Medical Internal Radiation DOSE (MIRD) concept.

Radioiodide biodistribution studies in vivo in orthotopic GBM tumors using 124I-PET/CT imaging

Three-dimensional serial PET imaging was performed in syngeneic orthotopic GL261 tumor-bearing mice. NIS- or Wt-MSCs were systemically applied via the tail vein: three MSC applications were given in 2-day intervals 1.5–2 weeks after intracranial tumor cell inoculation, followed by 124I-PET imaging 72 hours after the last MSC administration. In addition, shortened application regimes using a single MSC application 2–2.5 weeks after intracranial tumor cell implantation, followed by radioiodide PET 48 or 72 hours later, were applied. Mice received 10 MBq of 124I (Perkin Elmer or DSD Pharma GmbH) intravenously and serial acquisition was performed 1, 3, and 5 hours after 124I application using a preclinical small-animal Inveon P120 PET/CT scanner (Siemens). PET images were reconstructed with Inveon Acquisition Workplace (Siemens) and volumes of interest of the whole tumor were drawn using Inveon Research Workplace software (Siemens) and stated as fraction of the whole injected 124I dose per tumor volume (% ID/mL). Tumor volumes determined ex vivo (see below) were used. Dosimetry for 131I was calculated with a RADAR dose factor (www.doseinfo-radar.com) using the MIRD concept.

Mouse brain tissue preparation

After sacrifice, mice were transcardially perfused using PBS (Sigma-Aldrich) followed by 4% formaldehyde solution (Pharmacy, University Hospital LMU Munich, Munich, Germany). Brains were dissected, incubated in 4% formaldehyde for 24 hours at room temperature, and then transferred into 30% sucrose solution at 4°C until the brain sank to the bottom of the tube. After brains were embedded in Tissue-Tek OCT compound (Sakura Finetek), whole brains were sliced in 10-μm-thick horizontal sections using a cryotome. Sections were stored at −20°C before further processing.

Ex vivo tumor size determination

For tumor size analysis, hematoxylin and eosin (H&E) staining was performed on horizontal sections of the brain with defined stereotactic coordinates (at 0.72 to 5.52 mm from the dural surface) according to the mouse brain atlas (44). Stained H&E slides were scanned and the tumor area within each brain section was determined using Aperio ImageScope software (Leica Biosystems). The tumor volume quantification was done as previously described by Zhao and colleagues (45).

Immunofluorescence analysis of NIS and CD31

Frozen sections of brain tumors and sections of control organs (liver, lung, kidney, spleen) were subjected to immunofluorescence staining using rabbit anti-NIS (EUD4101, Origene; 1:1000) and rat anti-CD31 (blood vessel density; BD Pharmingen; 1:100) primary antibodies. A secondary anti-rabbit Alexa488-conjugated antibody (Jackson ImmunoResearch) for NIS staining, Cy3-conjugated anti-rat (Jackson ImmunoResearch) for CD31 staining, and Hoechst bisbenzimide (5 μg/mL) to counterstain nuclei were used. Finally, sections were mounted with fluorescence mounting medium (Dako).

All slides were digitalized by whole-slide scanning in a Panoramic MIDI II slide scanner and images were taken with the aid of the software CaseViewer (Version 2.4, 3DHISTECH Ltd.). Quantification of NIS-positive cells (percentage of NIS-positive cells in the tumor) was obtained by evaluation of six visual fields (20× magnification) per tumor using ImageJ software (NIH, Bethesda, MD).

Ex vivo mRNA analysis by qRT-PCR

Total RNA of frozen brain tumor sections and non-target organs (liver, lung, kidney, and spleen) derived from 124I-PET imaging experiments was extracted using TRIzol Reagent (Invitrogen Inc.) according to the manufacturer's instructions. Single-stranded cDNA was generated using LunaScript RT SuperMix Kit (New England Biolabs). qRT-PCR was conducted on a Lightcycler 96 System (Roche) using SybrGreen PCR master mix (Qiagen). The following primers were used: human NIS forward 5′-TGCGGGACTTTGCAGTACATT-3′ and reverse 5′-TGCAGATAATTCCGGTGGACA-3′, neomycin (selection cassette detecting NIS-MSCs) forward 5′-ATGCCCGACGGCGAGGATCT-3′ and reverse 5′-ATACCGTAAAGCACGAGGAAGCG-3′, and as internal controls human/mouse r18S forward 5′-CAGCCACCCGAGATTGAGCA-3′ and reverse 5′-TAGTAGCGACGGGCGGTGTG-3′ and mouse ACTB forward 5′-AAGAGCTATGAGCTGCCTGA-3′ and reverse 5′-TACGGATGTCAACGTCACAC-3′. The mRNA expression level of the target genes were normalized to the internal controls and relative expression was calculated using the ΔΔCt method.

Radioiodide therapy studies in vivo

A therapy trial of orthotopic GL261 tumors was started 5–6 days after intracranial tumor implantation. Tumor growth was assessed using a preclinical small animal 7T-MRI scanner (Agilent & GE Healthcare MR Discovery 901 with Bruker AVANCE III HD electronics) using a volume resonator together with a dedicated two-channel brain coil (RAPID Biomedical). Mice were included in the therapy and randomly distributed to all groups as soon as the inclusion criterion was met (tumor volume of 0.6–2.1 mm³; day 0). Tumor growth was then monitored twice a week by MRI and in vivo tumor volume determined as described previously (38).

On the basis of imaging results, an application regime with one MSC application followed by intraperitoneal injection of 55.5 MBq 131I (GE Healthcare or Rotop Pharmaka GmbH) 48 hours later was employed. This therapy cycle was repeated three times, with 2 days between each cycle. Four treatment cohorts were used: the therapy group received NIS-MSCs + 131I (n = 5) and controls received either Wt-MSCs + 131I (n = 5), NIS-MSCs + saline (NaCl; n = 6), or NaCl only (n = 5).

Indirect immunofluorescence analysis of CD31/Ki67

After 131I therapy, mice were transcardially perfused and brain tissue was processed as described above. Frozen brain tumor sections were subjected to immunofluorescence staining using rabbit anti-Ki67 (proliferation fraction; ab16667, Abcam; 1:200) and rat anti-CD31 (blood vessel density; BD Pharmingen; 1:100) antibodies. Secondary anti-rabbit Alexa488-conjugated antibody (Jackson ImmunoResearch) for Ki67 staining, Cy3-conjugated anti-rat (Jackson ImmunoResearch) for CD31 staining, and Hoechst bisbenzimide (5 μg/mL) to counterstain nuclei were used. Finally, sections were mounted with fluorescence mounting medium (Dako).

All slides were scanned as described above (see immunofluorescence analysis of NIS and CD31). Quantification of Ki67-positive cells (percentage of proliferating cells in the tumor) and blood vessel density (CD31-positive area in the tumor) was examined by evaluation of four visual fields (20× magnification) per tumor using ImageJ software (NIH).

Statistical analysis

Results are expressed as mean ± SEM, mean-fold change ± SEM, or percent. Statistical significance was generally determined by two-tailed Student t test.

For the therapy study, one-way ANOVA was performed for tumor volumes followed by post hoc Tukey honestly significant difference test. Kaplan–Meier plots were used for survival curves and statistical significance was analyzed by log-rank test. P-values <0.05 were considered as significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).

Data availability

The data generated in this study are available upon request from the corresponding author.

In vivo radioiodide biodistribution after MSC-mediated NIS gene transfer

To evaluate the general efficacy of MSC-mediated NIS gene delivery to GBM, functional NIS expression was determined in a syngeneic subcutaneous GL261 mouse model by 123I-scintigraphy. Murine MSCs were administered intravenously three times in 2-day intervals followed by radioiodide injection 72 hours after the last MSC application.

In vivo whole-body 123I-scintigraphy showed high levels of NIS-mediated radionuclide accumulation in subcutaneous GL261 tumors after systemic injection of NIS-MSCs (Fig. 1A). Tumors accumulated a maximum of 8.0% ± 1.1% ID/g with an average biological half-life of 3.7 ± 0.4 hours (Fig. 1B). Dosimetric calculations showed a tumor-absorbed dose of 46.5 ± 5.8 mGy/MBq for 131I. In addition, 123I uptake was also observed in organs that physiologically express NIS like the thyroid, salivary glands, and stomach as well as the urinary bladder due to mainly renal tracer excretion. To confirm NIS specificity of tumoral iodide uptake, the competitive NIS inhibitor perchlorate was injected to a subset of mice treated with NIS-MSCs (Fig. 1C). Perchlorate treatment resulted in a reduction of NIS-mediated tumoral radioiodide accumulation in addition to a reduction in physiologic uptake by thyroid, salivary glands, and stomach. Administration of Wt-MSCs as an additional control resulted in no tumoral radioiodide accumulation above background level (Fig. 1D). A second model, the subcutaneous U87 model, was used to demonstrate proof of concept in an immunocompromised background. 123I-scintigraphy showed a tumoral iodide accumulation of U87 tumors after three NIS-MSC applications of 5.1% ± 0.6% ID/g (Fig. 1E and F). Injection of perchlorate (Fig. 1G) and Wt-MSCs (Fig. 1H) served as controls to demonstrate NIS specificity and showed similar results as compared with the GL261 model.

Figure 1.

123I-scintigraphy revealed high tumoral radioiodide uptake after systemic NIS-MSC application in subcutaneous GL261 and U87 tumors. Three systemic injections of NIS-MSCs in mice harboring subcutaneous GL261 tumors (A) resulted in a maximum of 8.0% ± 1.1% of the injected dose per gram tumor (ID/g; n = 8, B). Iodide uptake of tumors and endogenous NIS-expressing organs was blocked upon treatment with the competitive NIS inhibitor perchlorate (n = 3, C). Treatment with Wt-MSCs showed no tumor-specific radioiodide uptake (n = 4, D). Tumoral iodide uptake of subcutaneous U87 (E) revealed a maximum of 5.1% ± 0.6% ID/g (n = 8, F) after three systemic NIS-MSC injections, which was reduced to background level using perchlorate (n = 2, G). Injection of Wt-MSCs resulted in no tumoral 123I accumulation (n = 3, H). Physiologic 123I accumulation was seen in thyroid, salivary glands (sg), stomach, and in the bladder due to renal excretion (A, C, D, E, G, H). For each group, one representative picture at 2 hours after tracer application is shown. The tumor is encircled in red. Data are represented as mean values ± SEM.

Figure 1.

123I-scintigraphy revealed high tumoral radioiodide uptake after systemic NIS-MSC application in subcutaneous GL261 and U87 tumors. Three systemic injections of NIS-MSCs in mice harboring subcutaneous GL261 tumors (A) resulted in a maximum of 8.0% ± 1.1% of the injected dose per gram tumor (ID/g; n = 8, B). Iodide uptake of tumors and endogenous NIS-expressing organs was blocked upon treatment with the competitive NIS inhibitor perchlorate (n = 3, C). Treatment with Wt-MSCs showed no tumor-specific radioiodide uptake (n = 4, D). Tumoral iodide uptake of subcutaneous U87 (E) revealed a maximum of 5.1% ± 0.6% ID/g (n = 8, F) after three systemic NIS-MSC injections, which was reduced to background level using perchlorate (n = 2, G). Injection of Wt-MSCs resulted in no tumoral 123I accumulation (n = 3, H). Physiologic 123I accumulation was seen in thyroid, salivary glands (sg), stomach, and in the bladder due to renal excretion (A, C, D, E, G, H). For each group, one representative picture at 2 hours after tracer application is shown. The tumor is encircled in red. Data are represented as mean values ± SEM.

Close modal

This proof-of-concept study was then expanded to a clinically more relevant orthotopic GL261 model. Once mice had developed brain tumors, the same application regimen as used in the subcutaneous model was applied for NIS imaging—with three cycles of MSC administration at 2-day intervals, followed by a single radionuclide injection (Fig. 2A). Functional NIS expression and, thus, MSC homing to brain tumors was monitored using three-dimensional, high-resolution small-animal 124I-PET imaging (10 MBq 124I, i.v.) allowing a better discrimination of exogenous and endogenous NIS-mediated signals in the head region. As determined by serial scanning, tumoral iodide uptake amounted to a maximum of 2.6% ± 0.2% ID/mL after NIS-MSC injection with an average biological half-life of 7.6 ± 2.5 hours (Fig. 2G). A tumor-absorbed dose of 31.1 ± 12.2 mGy/MBq for 131I was calculated. Injection of Wt-MSCs in a subset of mice resulted in a 124I uptake comparable with background level showing that the tumoral iodide uptake was NIS-MSC-mediated (Fig. 2D). Analogous to 123I-scintigraphy, endogenous NIS expression by the thyroid, salivary glands, and stomach as well as iodide elimination via the urinary bladder were also observed using 124I-PET imaging. The thyroid gland accumulated approximately 6.3%–10.9% ID/mL of 124I (Supplementary Fig. S1).

Figure 2.

Enhanced tumoral radioiodide accumulation after systemic NIS-MSC injection in syngeneic orthotopic GBM tumors. AF, Exemplary whole-body 124I-PET/CT scans (sagittal planes) and 2× magnification of the brain (sagittal and horizontal planes; tumor is located on the right) 3 hours after 124I injection are displayed. The brain areas are highlighted by red dotted lines and tumors are indicated by white arrows.124I-PET imaging revealed high tumoral radioiodide uptake after three (A, n = 6) or one (B, n = 4; C, n = 3) NIS-MSC application(s). Treatment with Wt-MSCs did not result in tumoral radioiodide accumulation above background level (D, n = 3; E, n = 2; F, n = 3). G, Quantification of serial 124I-PET imaging of tumoral radioiodide accumulation over 5 hours used for determination of radionuclide retention time. Results are expressed as mean values ± SEM. Two-tailed Student t test was performed for statistical analysis of NIS-MSCs versus Wt-MSCs of the same application schedule and was analyzed at each given timepoint after radionuclide injection (*, P < 0.05; **, P < 0.01); sg, salivary glands.

Figure 2.

Enhanced tumoral radioiodide accumulation after systemic NIS-MSC injection in syngeneic orthotopic GBM tumors. AF, Exemplary whole-body 124I-PET/CT scans (sagittal planes) and 2× magnification of the brain (sagittal and horizontal planes; tumor is located on the right) 3 hours after 124I injection are displayed. The brain areas are highlighted by red dotted lines and tumors are indicated by white arrows.124I-PET imaging revealed high tumoral radioiodide uptake after three (A, n = 6) or one (B, n = 4; C, n = 3) NIS-MSC application(s). Treatment with Wt-MSCs did not result in tumoral radioiodide accumulation above background level (D, n = 3; E, n = 2; F, n = 3). G, Quantification of serial 124I-PET imaging of tumoral radioiodide accumulation over 5 hours used for determination of radionuclide retention time. Results are expressed as mean values ± SEM. Two-tailed Student t test was performed for statistical analysis of NIS-MSCs versus Wt-MSCs of the same application schedule and was analyzed at each given timepoint after radionuclide injection (*, P < 0.05; **, P < 0.01); sg, salivary glands.

Close modal

In addition, a time-sparing treatment schedule that was more applicable in this rapidly growing tumor model was assessed for tumoral radioiodide uptake using PET imaging after application of a single MSC injection. MSCs were applied either 48 or 72 hours before 124I injection for imaging. In vivo124I-PET imaging revealed high levels of NIS-mediated radionuclide accumulation in brain tumors both at 48 or 72 hours after a single NIS-MSC application (Fig. 2B and C), whereas no radionuclide accumulation above background level was detected in the tumors of mice that had received Wt-MSCs (Fig. 2E and F). When PET imaging was performed at 48 hours after NIS-MSC injection, tumors of mice accumulated 3.5% ± 1.0% ID/mL of 124I with an average biological half-life of 13.3 ± 2.7 hours (Fig. 2G). On the basis of the imaging data, a tumor-absorbed dose of 60.3 ± 18.8 mGy/MBq for 131I was determined. When PET imaging was conducted 72 hours after a single MSC application, a tumoral 124I uptake of 2.5% ± 0.2% ID/mL with an average biological half-life of 8.2 ± 2.9 hours was reached. A tumor-absorbed dose of 32.7 ± 11.4 mGy/MBq for 131I was calculated.

The tumoral radioiodide quantification showed no significant differences between the different application schedules of NIS-MSC–treated mice. This finding implies that a single MSC application led to sufficient radioiodide uptake of brain tumors.

Ex vivo analysis of NIS expression

H&E staining of horizontal brain sections was used to visualize the mass of GL261 tumors at the area of implantation in the right caudate putamen (Fig. 3A). Ex vivo analysis of NIS protein expression showed high NIS-specific immunoreactivity throughout the tumor stroma of GL261 tumors (Fig. 3BD) after one or three systemic NIS-MSC injections. This demonstrated efficient MSC homing to orthotopic GBM tumors and functional NIS transgene expression by the engineered MSCs. NIS-specific immunoreactivity was observed at the plasma membrane and in the cytoplasm of engineered MSCs, which were most abundant in perivascular regions. Normal brain tissue, nontreated tumors, and brain tumors of mice that received Wt-MSCs showed no NIS protein expression above background level (Fig. 3B, E, and N). Non-target organs (liver, kidney, spleen served as controls) showed no detectable NIS protein expression (Fig. 3FK). Only a few spots were found to be affected with a small number of MSCs in the lungs after systemic application as shown by NIS-specific immunostaining (Fig. 3LN). The presence of MSCs in the lung may result from the pulmonary first-pass effect as adoptively employed MSCs move through the circulation before they reach the tumor site (31, 42). Quantification of NIS immunostaining (Fig. 3N) showed results consistent with NIS mRNA analysis data (Fig. 3O).

Figure 3.

Ex vivo analysis of GL261 brain tumors and control organs after systemic MSC application. A, Representative H&E images of horizontal sections of the brain for visualization of the tumor mass. The area of implantation in the right caudate putamen is shown and the tumor is circled in yellow. BE, NIS-specific immunofluorescence staining (green) and CD31 (red; labeling blood vessels) was performed on cryosections of brains. Nuclei are counterstained with Hoechst (blue). C and D, NIS protein expression is demonstrated in tumors of NIS-MSC–treated mice by high NIS immunoreactivity throughout the tumor stroma, which was most prominent near blood vessels and found on the cellular membrane and in the cytoplasm (white arrows). No NIS protein expression was detected in tumors after Wt-MSC injection (E) and in non-target organs after NIS-MSC application (FK; liver, spleen, kidney). A small number of NIS-expressing MSCs was detected in the lung of mice that received three (L) or one (M) NIS-MSC application(s). One representative image is shown each; scale bar = 40 μm. N, Quantification of NIS-positive cells was determined of tumors and the lungs after NIS-MSC injection and tumors of mice after Wt-MSCs injection as well as of normal brain tissue as compared with untreated brain tumors showing only low background level (which was arbitrarily set to one). NIS (O) and neomycin (P; selection marker of NIS-MSCs) mRNA expression was detected by qPCR in GL261 tumors after NIS-MSC application, while only a low background level of NIS and neomycin mRNA expression was found in tumors of mice that had received Wt-MSCs or compared with untreated tumors (which was arbitrarily set to one; NIS mRNA levels of untreated ΔΔCt = 0.0003 and neomycin mRNA levels of untreated ΔΔCt = 0.002). In addition, no NIS or neomycin mRNA expression was detectable in non-target organs such as the liver, spleen, kidney, and lung (see Supplementary Fig. S2). Data are represented as mean-fold change ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

Ex vivo analysis of GL261 brain tumors and control organs after systemic MSC application. A, Representative H&E images of horizontal sections of the brain for visualization of the tumor mass. The area of implantation in the right caudate putamen is shown and the tumor is circled in yellow. BE, NIS-specific immunofluorescence staining (green) and CD31 (red; labeling blood vessels) was performed on cryosections of brains. Nuclei are counterstained with Hoechst (blue). C and D, NIS protein expression is demonstrated in tumors of NIS-MSC–treated mice by high NIS immunoreactivity throughout the tumor stroma, which was most prominent near blood vessels and found on the cellular membrane and in the cytoplasm (white arrows). No NIS protein expression was detected in tumors after Wt-MSC injection (E) and in non-target organs after NIS-MSC application (FK; liver, spleen, kidney). A small number of NIS-expressing MSCs was detected in the lung of mice that received three (L) or one (M) NIS-MSC application(s). One representative image is shown each; scale bar = 40 μm. N, Quantification of NIS-positive cells was determined of tumors and the lungs after NIS-MSC injection and tumors of mice after Wt-MSCs injection as well as of normal brain tissue as compared with untreated brain tumors showing only low background level (which was arbitrarily set to one). NIS (O) and neomycin (P; selection marker of NIS-MSCs) mRNA expression was detected by qPCR in GL261 tumors after NIS-MSC application, while only a low background level of NIS and neomycin mRNA expression was found in tumors of mice that had received Wt-MSCs or compared with untreated tumors (which was arbitrarily set to one; NIS mRNA levels of untreated ΔΔCt = 0.0003 and neomycin mRNA levels of untreated ΔΔCt = 0.002). In addition, no NIS or neomycin mRNA expression was detectable in non-target organs such as the liver, spleen, kidney, and lung (see Supplementary Fig. S2). Data are represented as mean-fold change ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

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To assess relative NIS mRNA expression after systemic MSC application, mRNA was extracted from brain tumors and controls. Significant levels of NIS mRNA were detected in tumors of mice after one NIS-MSC application as compared with tumors from mice treated with Wt-MSCs or untreated tumors, and interestingly levels were higher as compared with mice receiving three rounds of MSCs (Fig. 3O). In addition, neomycin resistance control mRNA expression (expressed for vector selection) showed the same effect in brain tumors after MSC treatment (Fig. 3P). Control organs did not show any detectable levels of NIS or neomycin resistance mRNA expression (Supplementary Fig. S2) coherent with the NIS immunofluorescence staining.

NIS-mediated 131I therapy study of GBM

The therapeutic efficacy of MSC-based NIS-mediated 131I therapy was then evaluated in the syngeneic orthotopic GL261 model. On the basis of the NIS imaging data (Fig. 2A–G), the therapy study was performed with three cycles of a single MSC application followed by 131I administration 48 hours later with a 2-day break after each cycle. This relatively short treatment regime was chosen due to the aggressive nature of tumor growth in this model. For standardized inclusion (initial tumor volume of 0.6–2.1 mm³) of the mice and tumor growth monitoring, screenings on a small animal 7T-MRI scanner were performed twice a week. A significant delay in tumor growth and reduction of the tumor burden was observed in the mice of the therapy group (NIS-MSCs + 131I; Fig. 4A and E) as compared with control groups (Wt-MSCs + 131I; Fig. 4B and E; NIS-MSCs + NaCl; Fig. 4C and E; NaCl + NaCl; Fig. 4D and E) determined by MRI 10 days after therapy start. Most mice from all three controls reached humane endpoint (significant weight loss; neurologic symptoms; changes in drinking, eating or cleaning behavior; signs of pain) before all treatment cycles were completed (day 11 after therapy start; Fig. 4F). Therapy mice demonstrated significantly prolonged survival. After day 13 (therapy start), 60% of mice in the therapeutic schedule were still alive, while all control mice had already reached the predefined endpoint. Median survival (MS) of the therapy group was 15 days as compared with 10.5 days of NIS-MSCs + NaCl–treated and 10 days of Wt-MSCs + 131I–treated mice, while the NaCl + NaCl group showed the shortest survival (MS = 9 days).

Figure 4.

131I therapy study after MSC-mediated NIS gene transfer in vivo. Mice harboring orthotopic GL261 tumors were treated with three cycles of a single MSC intravenous injection followed by a single 131I intraperitoneal injection 48 hours later (days 1/3, 5/7, 9/11, respectively). Tumor growth was monitored twice per week by MRI. Representative MR images of tumors 10 days after therapy start from a NIS-MSCs + 131I–treated (A), a Wt-MSCs + 131I–treated (B), a NIS-MSC + NaCl–treated (C), and a NaCl + NaCl–treated (D) mouse are shown. Tumors are circled by yellow dotted lines. E, Mice treated with NIS-MSCs + 131I (n = 5) showed a delay in tumor growth as compared with control groups Wt-MSCs + 131I (n = 5; *, P < 0.05), NIS-MSC + NaCl (n = 6; **, P < 0.01) and NaCl + NaCl (n = 5; *, P < 0.05). F, Treatment with NIS-MSCs + 131I led to a significantly prolonged survival (**, P < 0.01) as compared with all control groups. Data are represented as mean ± SEM (*, P < 0.05; **, P < 0.01).

Figure 4.

131I therapy study after MSC-mediated NIS gene transfer in vivo. Mice harboring orthotopic GL261 tumors were treated with three cycles of a single MSC intravenous injection followed by a single 131I intraperitoneal injection 48 hours later (days 1/3, 5/7, 9/11, respectively). Tumor growth was monitored twice per week by MRI. Representative MR images of tumors 10 days after therapy start from a NIS-MSCs + 131I–treated (A), a Wt-MSCs + 131I–treated (B), a NIS-MSC + NaCl–treated (C), and a NaCl + NaCl–treated (D) mouse are shown. Tumors are circled by yellow dotted lines. E, Mice treated with NIS-MSCs + 131I (n = 5) showed a delay in tumor growth as compared with control groups Wt-MSCs + 131I (n = 5; *, P < 0.05), NIS-MSC + NaCl (n = 6; **, P < 0.01) and NaCl + NaCl (n = 5; *, P < 0.05). F, Treatment with NIS-MSCs + 131I led to a significantly prolonged survival (**, P < 0.01) as compared with all control groups. Data are represented as mean ± SEM (*, P < 0.05; **, P < 0.01).

Close modal

At the end of the therapy, brains were dissected and ex vivo immunofluorescence analysis was performed on cryopreserved tissue of tumors with similar size (Fig. 5). The intratumoral cell proliferation index (Ki67; Fig. 5A and E) of the therapeutically treated cohort was significantly lower as compared with controls (Fig. 5BD and E). Interestingly, mice treated with saline only showed a significantly higher proliferation potential in comparison with mice treated with MSCs plus saline. Blood vessel density (CD31; Fig. 5AD and F) analysis demonstrated a trend of reduced tumor vascularization, even though not statistically significant, in the therapeutically treated animals as compared with all controls.

Figure 5.

Ex vivo analysis of GBM brain tumors after MSC-mediated NIS gene therapy. Ki67 (proliferation index; green) and CD31 (blood vessels; red) immunofluorescence staining was performed on frozen brain tissue sections derived from mice that had received NIS-MSCs +131I (A), Wt-MSCs + 131I (B), NIS-MSCs + NaCl (C), and saline only (D) at the end of the therapy study. Nuclei were counterstained with Hoechst (blue). An exemplary image is shown each at 40× magnification (scale bar = 40 μm). Quantification of the proliferation index (E) shows a significantly reduced intratumoral cell proliferation as a result of NIS-MSC + 131I treatment and a nonsignificant decrease in blood vessel density in comparison with all control groups (F). Data are expressed as mean ± SEM (*, P < 0.05; **, P < 0.01).

Figure 5.

Ex vivo analysis of GBM brain tumors after MSC-mediated NIS gene therapy. Ki67 (proliferation index; green) and CD31 (blood vessels; red) immunofluorescence staining was performed on frozen brain tissue sections derived from mice that had received NIS-MSCs +131I (A), Wt-MSCs + 131I (B), NIS-MSCs + NaCl (C), and saline only (D) at the end of the therapy study. Nuclei were counterstained with Hoechst (blue). An exemplary image is shown each at 40× magnification (scale bar = 40 μm). Quantification of the proliferation index (E) shows a significantly reduced intratumoral cell proliferation as a result of NIS-MSC + 131I treatment and a nonsignificant decrease in blood vessel density in comparison with all control groups (F). Data are expressed as mean ± SEM (*, P < 0.05; **, P < 0.01).

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Glioblastoma is the most common type of primary brain tumor and shows an extremely poor prognosis with limited current treatment options. Because of its highly complex and aggressive nature, GBM is characterized by several mechanisms that help the tumor evade effective treatment underscoring the urgent need for new therapy options (1–4). MSCs have emerged as promising cellular vectors for the delivery of therapeutic genes into the tumor microenvironment due to their robust and innate tumor-homing capacity. Several studies have examined the migratory and homing capacity of MSCs to GBM including their ability to cross the BBB (12, 13, 15). Different routes of MSC administration have been reported to precisely and selectively target malignant brain tumors, including intracranial (10), intra-arterial (15), and intravenous delivery (46, 47). Intravenous injection would be optimal for clinical translation providing broad biodistribution and easy access, while local administration routes harbor increased risk and side effects including tissue injury (48). We and others have shown the feasibility of MSCs as cellular vectors for the delivery of the theranostic NIS gene after systemic application in various preclinical tumor models (31, 32, 34, 35, 49). On the basis of this experience, we sought to expand the MSC-mediated NIS gene therapy concept to GBM in the current study.

Monitoring the biological behavior of MSCs including migration, distribution, and fate are important and highly desirable for the design of an effective MSC-mediated treatment strategy. An essential advantage of the NIS gene therapy concept is the application of NIS as a reporter gene allowing noninvasive in vivo imaging of NIS-MSCs, for example, using scintigraphy, SPECT, or PET imaging. In a proof-of-concept study, we were able to demonstrate tumor-selective recruitment of NIS-MSCs to subcutaneous GL261 and subcutaneous U87 tumors using 123I-scintigraphy. Maximal NIS-specific tumoral iodide uptakes were comparable with data obtained in previous studies using a hepatocellular cancer model which resulted in successful therapeutic application (31). Because therapeutic options for GBM are limited by the BBB and the brain tumor microenvironment, the orthotopic GL261 model, which additionally might mimic more closely the growth and immune response of human GBM, was instrumental to address these challenges. In vivo124I-PET imaging demonstrated (throughout all MSC application schemes) a remarkable level of MSC recruitment to brain tumors, which was quantified by 124I-uptake in NIS transgene expressing MSCs. Conversely, Bexell and colleagues found no MSCs in gliomas 2 and 7 days following systemic intravenous MSC injection in a rat glioma model suggesting intratumoral MSC administration as the route of choice. In this context, the authors were able to show more efficient distribution of rat bone marrow–derived MSCs highly specific to tumor tissue after a single intratumoral MSC injection and substantial migration of MSCs to distant tumor microsatellites (10). However, Nakamizo and colleagues reported brain tumor–specific MSC delivery after systemic MSC injection into the carotid artery in a U87 xenograft GBM mouse model (15). In line with our findings, Shi and colleagues performed successful microSPECT/CT imaging using 125I as radiotracer to monitor NIS functional activity to follow MSC fate after three rounds of intravenously injected bone marrow–derived GFP-NIS-MSCs in a xenograft U87 glioma model (12). Differences in the extent of MSCs engrafted into the tumor may stem from the MSC populations used, the sources of MSCs, and isolation protocols that may help explain the divergent results reported (50).

In the study reported here, a single intravenous MSC application followed by radioiodide administration 48 hours later yielded the most promising results in the diagnostic imaging series. Compared with the administration of radioiodide 72 hours after a single, or multiple MSC injections as determined by 124I-PET imaging, the maximum radioiodide uptake was found to be higher, the efflux from the tumor setting more moderate, and the average biological half-life within the tumor environment was longer resulting in an increased calculated tumor-absorbed dose for 131I. Ex vivo analysis of NIS expression correlated with the in vivo data demonstrating a higher amount of NIS-positive cells 48 hours after a single MSC application as compared with 72 hours after receiving a total of three MSC injections. Consistent with these findings, we had reported a single MSC application to yield high tumoral radioiodide levels in an endogenous pancreatic ductal adenocarcinoma (PDAC) model as reported earlier (32). Evaluation of NIS-engineered MSCs in the PDAC tumors showed a higher number of NIS-positive cells in the group receiving only one MSC injection as compared with three rounds of MSCs using the same time points and same murine NIS-MSCs as the current study. These findings were reflected by a significant delay in tumor growth reported following 131I administration (32).

In addition, the tumoral radioiodide retention time in intracranial tumors was longer as compared with the subcutaneous model. The situation in glioblastoma is unique in comparison with peripheral or subcutaneous tumors—underlying mechanisms of accurate tracer influx and clearance of the brain remain partially unknown. An interplay of different parameters such as loss of BBB integrity, aberrant perfusion, diffusion, and permeability accompanied with a dysfunctional brain lympathic system, reported in rodent models as well as in patients, may contribute to an increased average biological half-life of radioiodide seen in the orthotopic brain tumors (51–54). In this circumstance, a slow brain “washout” is therapeutically advantageous based on potentially longer retention of radioiodide in the brain (55).

On the basis of our previous experience in the PDAC model, we hypothesized that the tumoral iodide uptake and calculated tumor absorbed dose for 131I should be sufficient to obtain a therapeutic effect in the comparably aggressive GBM tumor model. Indeed, a significantly prolonged survival was observed in the therapy group that received NIS-MSCs + 131I as compared with the three control groups conducting the therapy study with the most optimal application regime based on the imaging data. Tumor growth was significantly delayed in the therapy group most prominently after full completion of two therapy cycles (10 days after therapy start).

In accordance with former studies by our group using experimental hepatocellular carcinoma or colon carcinoma metastasis models, a significant decrease in proliferating cells was observed in tumors of the therapy group as compared with all control groups (31, 33, 34, 56). While safety concerns for the use of MSCs have been assessed in a series of clinical trials, several central issues remain to be addressed (50). Major concerns regarding the safety or caveats for MSCs use in clinical trials include the contradictory results researchers found regarding their protumorigenic or antitumorigenic effects. Pavon and colleagues reported tumor dissemination, glial invasiveness, and vascular proliferation following injection of MSCs in the caudal vein—none of which were seen in mice that did not receive MSCs (57). In contrast, unmodified MSCs prolonged the survival of 9L glioma-bearing rats after intracranial administration compared with untreated rats in a further study, indicating an antitumor effect of the MSCs used (13). In the current study, no protumorigenic effect was apparent, as no difference in tumor growth or survival of GBM mice was observable in the group that received MSCs + NaCl compared with the NaCl only group. However, the Ki67 proliferation index in brain tumors from MSC-treated mice (+NaCl) was significantly reduced as compared with mice treated with NaCl only.

In addition to the lack of tumor-promoting effects by MSCs in our study, adoptively applied NIS-expressing MSCs are effectively eliminated after application of therapeutically active radioisotopes. The use of NIS transgene also helps address the concern of using MSCs in the context of cancer therapy, that is, their poor persistence and retention time after transplantation (50). In our therapeutic regime, long-term survival of adoptively applied NIS-MSCs is negligible as they are eliminated by accumulation of 131I and therapeutic efficacy is enhanced by a new cycle of MSC load and treatment with iodide.

When using MSCs constitutively expressing the NIS transgene, MSC migration to non-tumor tissue might be disadvantageous due to off-target toxicity (16, 42). In the study presented here, in vivo tracking via 124I-PET imaging of NIS-expressing MSCs could not demonstrate significant off-target recruitment. Relatively small numbers of NIS-expressing MSCs were detected in the lung of the mice assessed by NIS immunofluorescence staining. This general phenomenon has been observed in several studies where the presence of MSCs in the lung was higher at earlier timepoints after injection potentially due to entrapment within the microvascular system, but was reduced by later timepoints (12, 58).

We demonstrate here the great potential of MSCs engineered to express the theranostic NIS gene as an anticancer agent for the treatment of GBM. While MSC-mediated NIS gene therapy led to a significant prolongation of median survival up to 67% after three treatment cycles as compared with control groups, we did not achieve a complete tumor regression. Nevertheless, this survival increase in a preclinical study is a promising result that is comparable with previous preclinical studies made in different glioma models including GL261 (59–61). An increase of the therapeutic efficacy might be obtained through the use of the α-emitter 211At as alternative isotope also transported by NIS that results in a higher dose rate based on a shorter half-life and higher energy as compared with 131I (30). The syngeneic GL261 model is often used in the context of cancer immunotherapy based on the intact host immune system. While ongoing studies reveal promising preclinical results, data have to be interpreted with caution due to potential moderate immunogenicity of the GL261 model (62–65). On the basis of the currently available data, combination of NIS-based radionuclide therapy with immunotherapy seems to be another promising approach for future studies to increase and foster therapeutic efficacy in this still deadly disease.

The combination of MSC-mediated NIS gene therapy as an adjuvant for standard treatment strategies may represent a viable approach for clinical translation to enhance therapeutic efficacy. Major obstacles in the treatment of GBM include its invasive growth pattern, which means that infiltrative tumor extensions reach into the surrounding brain parenchyma leading to growth of distant tumor microsatellites. This precludes complete surgical resection and is often responsible for tumor relapse. Bexell and colleagues found that MSCs injected intratumorally in a preclinical 3000 N32 glioma model during partial resection were able to migrate efficiently within glioma remnants, even though they were not able to show long-distance engraftment of their MSCs (11). Building upon these studies the combination of NIS-MSC–based radionuclide therapy with surgical excision of the tumor may have the potential to reduce the risk of postsurgical relapse.

In addition to surgery, radiotherapy is a standard treatment for many solid tumors including GBM. An enhancement of MSC homing following irradiation pretreatment of tumors by a radiation-induced enhancement of the inflammatory response has been described after intravascular administration (66). Synergistic effects of tumor irradiation and MSC-mediated cancer treatment have been reported in hepatocellular carcinoma, breast cancer, colon cancer, and glioma (12, 58, 66–68). On the basis of our previous studies in hepatocellular cancer and the above mentioned studies in preclinical GBM, the combination of tumor irradiation and MSC-mediated NIS gene therapy might be a promising approach in GBM to reduce the risk of tumor recurrence.

In conclusion, we demonstrate the potential of ex vivo genetically engineered MSCs as a tumor-selective vector system for NIS gene transfer in a syngeneic GBM model after systemic intravenous injection. In vivo biodistribution studies with 123I-scintigraphy or 124I-PET imaging showed selective MSC recruitment to subcutaneous and in particular to orthotopic brain tumors using NIS a potent and well-characterized reporter gene. A critically high number of NIS-MSCs was recruited to the tumor yielding a significantly prolonged survival and reduced tumor growth after 131I treatment. NIS gene cancer therapy employing MSCs as a targeting vector opens the prospect of a very promising new treatment approach for newly diagnosed as well as refractory brain tumors due to the opportunity of combining conventional treatment methods with easily modifiable NIS-expressing MSCs to enhance general therapeutic efficacy.

C. Kitzberger reports grants from Deutsche Forschungsgemeinschaft SFB 824 during the conduct of the study. R. Spellerberg reports grants from Deutsche Forschungsgemeinschaft during the conduct of the study. C. Stauss reports grants from SFB during the conduct of the study. R.E. Kälin reports grants from DFG GL691/2-4 and Anni-Hofmann Stiftung during the conduct of the study. C. Spitzweg reports grants from Deutsche Forschungsgemeinschaft and Wilhelm-Sander-Stiftung during the conduct of the study. No disclosures were reported by the other authors.

C. Kitzberger: Conceptualization, formal analysis, investigation, visualization, methodology, writing–original draft. R. Spellerberg: Investigation. Y. Han: Investigation. K.A. Schmohl: Conceptualization, writing–review and editing. C. Stauss: Investigation. C. Zach: Formal analysis. R.E. Kälin: Funding acquisition, methodology, writing–review and editing. G. Multhoff: Resources, funding acquisition, methodology. M. Eiber: Resources, funding acquisition. F. Schilling: Resources, funding acquisition, methodology, writing–review and editing. R. Glass: Resources, funding acquisition, methodology, writing–review and editing. W.A. Weber: Resources, writing–review and editing. E. Wagner: Supervision. P.J. Nelson: Conceptualization, supervision, funding acquisition, validation, methodology, writing–review and editing. C. Spitzweg: Conceptualization, resources, supervision, funding acquisition, validation, project administration, writing–review and editing.

We owe special thanks to Sybille Reder, Markus Mittelhäuser, Hannes Rolbieski, and Sandra Sühnel (Department of Nuclear Medicine, School of Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany) for their valuable help in performing imaging studies. We appreciate the help from Dr. Stefan Stangl and Dr. Cai Linzhi for establishing the orthotopic glioblastoma mouse model. Furthermore, we thank Dr. Julia Mayerle, Dr. Ivonne Regel, and Dr. Ujjwal Mahajan for allowing us to use their lab equipment.

This work was performed as partial fulfillment of the doctoral thesis of C. Kitzberger at the faculty for Chemistry and Pharmacy of the LMU Munich.

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) within the Collaborative Research Center SFB 824, to C. Spitzweg (project C8), R. Glass (B2), F. Schilling (Z3), G. Multhoff (B4), M. Eiber (B11) and within the Priority Program SPP1629, to C. Spitzweg and P.J. Nelson, as well as a grant from the Wilhelm-Sander-Stiftung to C. Spitzweg (2014.129.1). R.E. Kälin and R. Glass are supported by the DFG (GL691/2; SFB824), the “Wilhelm Sander-Stiftung,” the “Anni-Hofmann Stiftung,” and the “Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München” (WiFoMed). R. Glass acknowledges funding by DFG grant INST 409/223-1 FUGG.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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