Key Points: BMSCs and MM cells mutually communicate through exosomes, which carry selective cytokines. BMSC-derived exosomes favor MM cell proliferation, migration, and survival and induce drug resistance to bortezomib.

A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb). Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution. Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection. In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti–epidermal growth factor receptor (anti-EGFR) Nanobodies, ^99mTc-7C12 and ^99mTc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with ^99mTc-tricarbonyl (^99mTc(CO)₃) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with ^99mTc-7C12 and ^99mTc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six ⁵⁷Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm³) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either ^99mTc-7C12 or ^99mTc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both ^99mTc-7C12 and ^99mTc-7D12 (4.55 ± 0.24 %IA/cm³ and 4.62 ± 0.36 %IA/cm³, respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 ± 0.14 for ^99mTc-7C12 and 1.49 ± 0.60 for ^99mTc-7D12). ^99mTc-7C12 showed significantly higher kidney uptake (63.48 ± 2.36 vs. 56.25 ± 2.46 %IA/cm³) and lower liver uptake (2.55 ± 0.26 vs. 4.88 ± 0.86 %IA/cm³) than did ^99mTc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, ^99mTc-7C12 showed higher tumor uptake (9.11 ± 1.12 %IA/g) than did ^99mTc-7D12 (6.09 ± 0.77 %IA/g). ^99mTc-7D12 demonstrated significantly higher blood activity than did ^99mTc-7C12, but both showed short plasma half-lives (<10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, ^99mTc-7C12 was selected for further studies.

Rationale:: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective:: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results:: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE ^−/− ) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) ( P <0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE ^−/− mice were successfully identified by single-photon emission computed tomography imaging. ^99m Tc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions:: The ^99m Tc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.