Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies

Abstract

Purpose Recent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs. Procedures The irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography. Results No binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed. Conclusions This study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.

The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses

“…However, in the context of this study, no significant differences were observed compared to 7D12-PS ( Figure S4). Together with the fact that the monovalent nanobody format is expected to distribute more homogenously than a bivalent format [11], this study was focused on one conjugate only, i.e., 7D12-PS.…”

“…The use of a NB for PDT brings a series of advantages over antibody-targeted PDT. Their small size enables rapid tumor accumulation, with a homogeneous distribution, and a rapid clearance from circulation when unbound [9][10][11]. In the context of PDT, this allows the application of the light shortly after conjugate administration, ensures more extensive tumor damage, and reduces the chances of phototoxicity present in both PDT and antibody-targeted PDT protocols.…”

“…Due to its slow internalization, after binding to EGFR, 7D12-PS is mostly present on the cell membrane when light is applied, thereby inducing membrane damage and subsequent cell death [8]. In vivo, this nanobody has been described to present rapid tumor accumulation and homogenous distribution [10,11] and, upon illumination, induces extensive tumor damage, as well as vascular effects [7,15]. In order to take into account possible inter-tumor heterogeneity of target expression, two different tumor cell lines were…”

Development of in vitro-grown spheroids as a 3D tumor model system for solid-state NMR spectroscopy

“…7D12 and R2 were expressed, purified by IMAC and conjugated to indicated dyes as described elsewhere (Oliveira et al 2012 ; Beltrán Hernández et al 2019 ). In short, his-tagged nanobodies were expressed in E. coli and purified from the periplasmic fraction by TALON purification and reconstituted in PBS.…”

“…Moreover, our experiments reveal that isotope-enrichment of amino acids in spheroids is readily achieved using a standard culture medium. Finally, we adapted our earlier procedures (Beltrán Hernández et al 2019 ) to study the [ 13 C, 15 N]-labelled anti-EGFR nanobody 7D12 (Roovers et al 2011 ) after penetration into the spheroids using sensitivity-enhanced DNP-ssNMR. Our data highlight the potential of conducting ssNMR-based studies on 3D cell cultures that in the future may reveal structure–function relationship of proteins in their native 3D cell microenvironment.…”