Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma

Purpose Meningioma recurrence rates can be reduced by optimizing surgical resection with the use of intraoperative molecular fluorescence guided surgery (MFGS). We evaluated the potential of the fluorescent tracer 800CW-TATE for MFGS using in vitro and in vivo models. It targets somatostatin receptor subtype 2 (SSTR2), which is overexpressed in all meningiomas. Methods Binding affinity of 800CW-TATE was evaluated using [177Lu] Lu-DOTA-Tyr3-octreotate displacement assays. Tumor uptake was determined by injecting 800CW-TATE in (SSTR2-positive) NCI-H69 or (SSTR2-negative) CH-157MN xenograft bearing mice and FMT2500 imaging. SSTR2-specific binding was measured by comparing tumor uptake in NCI-H69 and CH-157MN xenografts, blocking experiments and non-targeted IRDye800CW-carboxylate binding. Tracer distribution was analyzed ex vivo, and the tumor-to-background ratio (TBR) was calculated. SSTR2 expression was determined by immunohistochemistry (IHC). Lastly, 800CW-TATE was incubated on frozen and fresh meningioma specimens and analyzed by microscopy. Results 800CW-TATE binding affinity assays showed an IC50 value of 72 nM. NCI-H69 xenografted mice showed a TBR of 21.1. 800CW-TATE detection was reduced after co-administration of non-fluorescent DOTA-Tyr3-octreotate or administration of IRDye800CW. CH-157MN had no tumor specific tracer staining due to absence of SSTR2 expression, thereby serving as a negative control. The tracer bound specifically to SSTR2-positive meningioma tissues representing all WHO grades. Conclusion 800CW-TATE demonstrated sufficient binding affinity, specific SSTR2-mediated tumor uptake, a favorable biodistribution, and high TBR. These features make this tracer very promising for use in MFGS and could potentially aid in safer and a more complete meningioma resection, especially in high-grade meningiomas or those at complex anatomical localizations. Supplementary Information The online version contains supplementary material available at 10.1007/s11060-021-03739-1.


Immunohistochemistry on formalin fixed paraffin embedded (FFPE) sections
SSTR 2 expression in H69 and CH-157MN cells in vitro was determined and confirmed by immunohistochemistry (IHC), by analyzing sections of Cellient blocks stained with the primary antibody mouse-anti-SSTR 2 (MAB4224, R&D systems, Abingdon, United Kingdom; 1:100). Sections of 4 µm were deparaffinized in xylene and rehydrated in ethanol. Tris/EDTA buffer (pH 9.0) was used for antigen retrieval for seven minutes in a microwave. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxidase for 30 minutes. The primary antibody SSTR 2 (MAB4224, R&D systems; 1:100) was incubated for one hour at room temperature.

H&E staining
Sections were deparaffinized in xylene and rehydrated in ethanol. Slices were incubated in hematoxylin for ten minutes, washed with tap water and incubated with eosin for two minutes. Finally, they were washed and dehydrated in ethanol, mounted with a cover slip and scanned using NanoZoomer 2.0 HT multi-slider scanner (Hamamatsu, Hamamatsu City, Japan).

Xenograft mice models
H69 cells (5 million cells) were inoculated in six to eight-week-old BALB/c-nu mice (Janvier Laboratories, Le Genest-Saint-Isle, France) in a 300 µl 1:1 mix of Matrigel (Corning, #354248) and medium. [1-3] CH-157MN cells were inoculated by injection of 1.5 million cells in medium. In both models, the cells were subcutaneously injected between the scapulae. Tumor growth was monitored biweekly using a caliper. The Institutional Review Board of the University of Groningen approved the study and animal care complied with the Guide for the Care and Use of Laboratory Animals.

Tracer uptake, biodistribution and imaging
Mice bearing xenografts with a volume of approximately 500 mm 3 were divided into treatment groups for imaging. The animals were anesthetized with 5% isoflurane and using 2.5% isoflurane for maintenance. Indicated doses of tracer or DOTA-TATE were retro-orbitally injected. Tumor uptake of 800CW-TATE was determined by longitudinal imaging for 1, 2, and 4 hours post tracer injection using the FMT2500 (PerkinElmer, Waltham, nmol, 50 µl). Mice were terminated by heart puncture and cervical dislocation for further postmortem evaluation.

Massachusetts, United States
Xenografts, organs and fluids were harvested and macroscopically imaged using the PEARL scanner (LI-COR Biosciences) to determine ex vivo tumor uptake and biodistribution. Tumor, brain, skull, muscle, skin, kidney, and liver tissue were sectioned in sequential slides, and tracer fluorescence was imaged using an Odyssey CLx (LI-COR Biosciences). Consecutive slides were processed for fluorescence microscopy and anti-SSTR 2 and H&E staining as described above.
Data was analyzed using ImageJ and GraphPad Prism 8.0. Statistical significance was tested using a one-way ANOVA with Bonferroni post-hoc analysis; a P-value of ≤ 0.05 was considered statistically significant. Fluorescence specificity values are reported as mean±SEM. The TBR was defined as the ratio of the mean fluorescence intensity (MFI) of the tumor, and the MFI of brain tissue. TBRs are shown as mean with 95% confidence intervals in brackets.

Post-mortem intra-operative molecular fluorescence guided surgery
One H69 xenograft bearing mouse (500 mm 3 ) was intravenously injected with 800CW-TATE (3 µg, 1.36 nmol, 50 µl) for post-mortem fluorescence guided surgery. Draping was used to block fluorescence of the kidneys. The xenograft was first resected using white light guidance, followed by fluorescent guided resection of residual tissue with the SurgVision (SurgVision BV 't Harde, The Netherlands). Images were processed using ImageJ, and the TBR was calculated by dividing the MFI of the tumor by the MFI of surrounding tissue. The background MFI was measured within a spherical volume of interest positioned in an area of physiological muscle tissue.

Analyses of human meningioma specimens
Meningioma specimens resected at the University Medical Center Groningen between 2006 and 2012 were available, and most of these have been described previously: two WHO grade III meningiomas have been added to the current dataset, leading to ten analyzed samples.
[5] The Institutional Review Board approved the experimental protocol and informed consent. If informed consent could not be obtained, the Code of Conduct for responsible use of human tissue was followed.
Frozen sections (4 µm) were incubated with 10 -6 M 800CW-TATE for one hour, mounted with Prolong Antifade, and imaged using the inverted microscope as described above. Sequential sections were stained with either H&E or anti-SSTR 2 . For the latter, the primary antibody mouse-anti-SSTR 2 (MAB4224, R&D systems; 1:100) incubated for one hour at room temperature, followed by incubation with the secondary antibody rabbit anti-mouse Alexa Fluor 647(A21239, Invitrogen, Carlsbad, California, United States; 1:75). Slides were mounted using ProLong Antifade and imaged using a Leica SP8 Confocal Laser Scan Microscope (Leica Biosystems, Wetzlar, Germany).
For whole specimen analysis, two freshly resected meningioma specimen were cut into 3 mm sections and transferred to a 6-well plate containing 2 ml DMEM/F12, supplemented with 1% penicillin/streptomycin and 5 × 10 -