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

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.

Autoradiographic analysis of the effect of cytokinin on protein and RNA syntheses in the Ceratodon purpureus protonema

In the protonema of <em>Ceratodon purpureus</em> (Hedw.) Brid., apical parts of the protonemal filaments (apical cells, initials of protonemal side branches and of gametophore buds) proved to be preferential sites of [¹⁴C]-leucine incorporation into proteins. In some filaments, a similar preference for [³H]-uridine incorporation into RNA was observed, whereas in others there was a rather uniform distribution of label over all cells. A short (0.5-2 h) treatment with cytokinin (N⁶-2-isopentenyladenine) enhanced [¹⁴C]-leucine incorporation, without changing the relative distribution of label. No such enhancement, as well as no change in label distribution could be observed in [³H]-uridine incorporation. No direct relationship seems to exist between the early promotion of protein synthesis by cytokinin in the protonema and cytokinin induction of gametophore buds.

Effect of concentration on the accuracy of convective imaging distribution of a gadolinium-based surrogate tracer

Object Accurate real-time imaging of coinfused surrogate tracers can be used to determine the convective distribution of therapeutic agents. To assess the effect that a concentration of a Gd-based surrogate tracer has on the accuracy of determining the convective distribution, the authors infused different concentrations of Gd-diethylenetriamine pentaacetic acid (DTPA) in primates during MR imaging. Methods Five nonhuman primates underwent convective infusion (1 or 5 mM, 21–65 μl) of Gd-DTPA alone, Gd-DTPA and 14C-sucrose, or Gd-DTPA and 14C-dextran into the bilateral striata. Animals underwent real-time MR imaging during infusion (5 animals) and autoradiographic analysis (2 animals). Results Gadolinium-DTPA could be seen filling the striata at either concentration (1 or 5 mM) on real-time MR imaging. While the volume of distribution (Vd) increased linearly with the volume of infusion (Vi) for both concentrations of tracer (1 mM: R2 = 0.83; 5 mM: R2 = 0.96), the Vd/Vi ratio was significantly (p < 0.0001) less for the 1-mM (2.3 ± 1.0) as compared with the 5-mM (7.4 ± 1.9) concentration. Autoradiographic and MR volumetric analysis revealed that the 5-mM concentration most accurately estimated the Vd for both small (sucrose [359 D], 12% difference between imaging and autoradiographic distribution) and large (dextran [70 kD], 0.2% difference) molecules compared with the 1-mM concentration (sucrose, 65% difference; dextran, 68% difference). Conclusions The concentration of infused Gd-DTPA plays a critical role in accurately assessing the distribution of molecules delivered by CED. A 5-mM concentration of Gd-DTPA most accurately estimated the Vd over a wide range of molecular sizes.

Image-guided convection-enhanced delivery of muscimol to the primate brain

Object Muscimol is a potent γ-aminobutyric acid-A receptor agonist that temporarily and selectively suppresses neurons. Targeted muscimol suppression of neuronal structures could provide insight into the pathophysiological processes and treatment of a variety of neurological disorders. To determine if muscimol delivered to the brain by convection-enhanced delivery could be monitored using a coinfused surrogate MR imaging tracer, the authors perfused the striata of primates with tritiated muscimol and Gd–diethylenetriamine pentaacetic acid (DTPA). Methods Three primates underwent convective coinfusion of 3H-muscimol (0.8 μM) and Gd-DTPA (5 mM) into the bilateral striata. Primates underwent serial MR imaging during infusion, and the animals were killed immediately after infusion. Postmortem quantitative autoradiography and histological analysis was performed. Results Real-time MR imaging revealed that infusate (tritiated muscimol and Gd-DTPA) distribution was clearly discernible from the noninfused parenchyma. Real-time MR imaging of the infusion revealed the precise region of anatomical perfusion in each animal. Imaging analysis during infusion revealed that the distribution volume (Vd) of infusate linearly increased (R = 0.92) with volume of infusion (Vi). Overall, the mean (± SD) Vd/Vi ratio was 8.2 ± 1.3. Autoradiographic analysis revealed that MR imaging of Gd-DTPA closely correlated with the distribution of 3H-muscimol, and precisely estimated its Vd (mean difference in Vd, 7.4%). Quantitative autoradiograms revealed that muscimol was homogeneously distributed over the perfused region in a square-shaped concentration profile. Conclusions Muscimol can be effectively delivered to clinically relevant volumes of the primate brain. Moreover, the distribution of muscimol can be tracked using coinfusion of Gd-DTPA and MR imaging. The ability to perform accurate monitoring and to control the anatomical extent of muscimol distribution during its convection-enhanced delivery will enhance safety, permit correlations of muscimol distribution with clinical effect, and should lead to an improved understanding of the pathophysiological processes underlying a variety of neurological disorders.