In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe

Citation: Melemenidis, S.; Knight, J.C.; Kersemans, V.; Perez-Balderas, F.; Zarghami, N.; Soto, M.S.; Cornelissen, B.; Muschel, R.J.; Sibson, N.R. In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe. Int. J. Mol. Sci. 2024, 25, 7160. https://doi.org/10.3390/ ijms25137160 Academic Editor: Mi-Ae Park Received: 28 May 2024 Revised: 21 June 2024 Accepted: 26 June 2024 Published: 28 June 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe Stavros Melemenidis 1 , James C. Knight 2 , Veerle Kersemans 3 , Francisco Perez-Balderas 4 , Niloufar Zarghami 5 , Manuel Sarmiento Soto 6, Bart Cornelissen 7, Ruth J. Muschel 5and Nicola R. Sibson 5,* 1Department of Radiation Oncology, Stanford School of Medicine, Cancer Institute, Stanford University, Stanford, CA 94305, USA; [email protected] 2School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK; [email protected] 3Clinical Nuclear Medicine Imaging, Siemens Healthineers, 2595 BN The Hague, The Netherlands; [email protected] 4CureVac, AG. Paul-Ehrlich-Str. 15, 72076 Tubingen, Germany; franciscoper[email protected] 5Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK; [email protected] (N.Z.); [email protected] (R.J.M.) 6Department of Biochemistry and Molecular Biology, University of Seville, 41004 Seville, Spain; [email protected] 7Department of Nuclear Medicine, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands; [email protected] *Correspondence: [email protected]; Tel.: +44-1865-225836; Fax: +44-1865-857127 Abstract: Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1–2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 µ m diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 ( 89 Zr, t 1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent ( 89 Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 µ m in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAMMPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases. Keywords: micrometastasis; early detection; microparticles of iron oxide (MPIO); vascular cell adhesion molecule-1 (VCAM-1); dual modality (PET/MRI) 1. Introduction Metastasis from a primary tumour site to distant organs is a complex succession of events collectively known as the invasion-metastasis cascade [ 1 ]. Currently metastasis accounts for 90% of human cancer deaths and is one of the major challenges in cancer therapy [2] . Owing to the capacity of cancer cells to spread to the lungs, this is the second most frequent site of metastasis, with an estimated 20 to 54% of tumours originating elsewhere in the body metastasising to this organ [ 3 ]. The most prevalent cancers that metastasise to the lung parenchyma include those originating from the breast, lung, colon, Int. J. Mol. Sci. 2024,25, 7160. https://doi.org/10.3390/ijms25137160 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024,25, 7160 2 of 19 uterine leiomyosarcoma, and head and neck squamous cell carcinomas [ 3 , 4 ]. In children, it is rare to see primary lung cancer, and the majority of lung cancer metastasis cases metastasise from a primary site to the lungs [ 5 ]. The most frequently employed non-invasive imaging modalities for identifying lung metastases are computed tomography (CT) with or without the application of contrast agents and 18 F fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET)/CT scans. Although the improvement in CT technologies allows for spatial resolution detection of lung nodules below 5 mm, it cannot provide criteria to predict the nature of these nodules [ 6 ]. 18 F-FDG PET complements the CT resolution, offering information about uncertain small lesions and their metabolic activity. It is important to note, however, that the detection sensitivity of 18 F-FDG PET relies on the accumulation of highly metabolically active cells (both proliferating tumour cells and inflammatory cells) to allow tumour detection/diagnosis, which typically reflects a relatively late stage of tumour development and leaves micrometastases undetected [7,8] . This resolution limitation results in decreased sensitivity for low-volume diseases, leading to potential false negatives [ 9 , 10 ]. Moreover, CT scans depend on size and density contrast to identify abnormalities and such contrasts may not be evident in early-stage disease, causing micrometastases to be overlooked [ 11 ]. At the same time, 18 F-FDG is not exclusive to tumours and accumulates in any glucose-avid tissue, including those affected by inflammation. This non-specific uptake can obscure the presence of a disease or mimic it, complicating the differentiation between cancerous and non-cancerous conditions [ 12 , 13 ]. Owing to the lack of imaging sensitivity for the early stages of tumour cell extravasation from circulation and micrometastatic formation, clinical therapy is often applied at a time that is beyond the point of effective intervention. Thus, significant advantages may be gained if more sensitive imaging methods can be developed to allow molecular targeting of markers that enable the early detection of micrometastatic colonies. We have previously demonstrated that it is possible to detect micrometastases in mouse brain very early in their development using molecularly targeted magnetic resonance imaging (MRI) via the associated endothelial activation and upregulation of endovascular adhesion molecules [ 14 – 16 ]. These endothelial adhesion molecules are rapidly upregulated in response to disease or injury and mediate leukocyte rolling, adhesion, and transmigration across the vascular wall [ 17 ]. There is now evidence to suggest that tumour cells also use such adhesion molecules to promote their adhesion to and extravasation across the vascular endothelium [ 18 ]. One such adhesion molecule is vascular cell adhesion molecule 1 (VCAM-1), which is upregulated very early in brain metastasis formation [ 19 ] and has also been shown to enhance endothelial cell recruitment for new blood vessel formation, thus supporting tumour growth and spread [ 20 ]. The early and marked upregulation of VCAM- 1 makes this molecule a potential early biomarker for detecting metastatic activity, offering advantages over traditional imaging markers that may not clearly distinguish between tumour stages or identify early metastatic spread. To this end, using an MRI contrast agent based on microparticles of iron oxide (MPIO) and targeted to VCAM-1, we have previously demonstrated that VCAM-1 provides a sensitive and specific endothelial biomarker of early brain metastasis [14,15], as well as improving detection of the tumour-brain interface [21]. Previous studies have also shown that VCAM-1 is markedly upregulated in both lung and liver metastases [ 22 , 23 ], with little expression in the normal lung endothelium. Thus, VCAM-1 potentially represents a target for the early detection of micrometastasis in the lungs. However, owing to the low proton density of the lung tissue, it is not possible to effectively detect the presence of MPIO by MRI in the lung, as this relies on dephasing existing proton signals to achieve binding-specific contrast. Alternatively, positron emission tomography (PET), which is typically combined with CT, offers an attractive solution as it provides high sensitivity (10 −12 M), enabling the location and distribution of radiolabelled molecules in the lung to be tracked in vivo . The prevailing PET imaging agent for identifying pulmonary metastases is 18 F-FDG, which accumulates in regions with elevated glucose metabolism. This functional imaging method, however, is size-limited to tumours greater than 1 cm in diameter [ 24 , 25 ]. In the current study, Int. J. Mol. Sci. 2024,25, 7160 3 of 19 therefore, we propose that labelling the VCAM-MPIO contrast agent with a positronemitting radioisotope such as 89 Zr will enable PET detection of micrometastases in the lung. In prior studies, PET probes featuring different radioisotopes directed towards VCAM-1 have been reported, utilising either nanobodies derived from unique heavy-chain-only antibodies [ 26 ] or antibodies alone [ 27 ]. However, these probes have very long circulation times in the blood and are also likely to extravasate from the vasculature. Our approach maintains all the advantages of the MPIO-based strategy, including short circulatory halflife and high valency for targeting ligands, while enabling detection in an organ that would be insensitive to the molecular MRI approach. Further, this novel agent would enable sequential or concurrent MRI and PET detection of micrometastases in different organs (e.g., the brain and lungs) within the same subject. The aim of this study, therefore, was to develop a dual-contrast PET/MRI probe targeting VCAM-1 to enable the early detection of micrometastases in both the brain and lungs using a single contrast agent. This dual-modality approach not only promises to improve the accuracy of metastasis detection but may also facilitate the early initiation of targeted therapies, potentially improving patient outcomes significantly. 2. Results 2.1. Non-Radiolabelled VCAM-, IgG- or BSA-MPIO Conjugates 2.1.1. Synthesis and Loading of Conjugates Both VCAM-MPIO and IgG-MPIO were successfully synthesised, and the antibody loading was calculated to be ca. 3350 antibodies per µ m 2 of the MPIO surface (11,650 per MPIO). Following the 89 Zr radiolabelling reactions, approximately 250 89 Zr atoms were associated with each MPIO (MA = 1.06 ×10−4MBq/µg of MPIO). 2.1.2. Ex Vivo Quantitation of VCAM-MPIO Binding in Pulmonary Metastasis Model Adherent VCAM-MPIO were evident in metastasis-associated microvasculature and nearby larger vessels in the lungs of mice injected with VCAM-MPIO (Figure 1A,B; Figure S1 A), while few or no MPIO were found in the lungs of naïve mice injected with VCAM-MPIO. Specific binding of VCAM-MPIO on marked VCAM-1 expressing lung vasculature was confirmed immunohistochemically (Figure 1A,B) and, again, was absent from the lungs of naïve mice. Studies in an initial cohort of animals (n = 6) demonstrated the reproducibility of the lung micrometastasis model used, validating subsequent comparisons between groups; no significant differences were found in either tumour number ( 4.50 ±1.23 ; mean number of metastases per lung section analysed ± standard deviation [SD]) or area-derived diameter (137.01 ± 45.17 µ m; mean ± SD) between animals (Figure S1B,C). A significantly greater number of VCAM-MPIO than IgG-MPIO (quantified as the number of adherent MPIO per µ m of inflamed endothelium, excluding capillaries) were found associated with micrometastatic colonies at 10 min post-injection [VCAM-MPIO vs. IgG-MPIO, (9.80 ± 1.35) × 10 −3 vs. (4.45 ± 4.08) × 10 −4 MPIO/ µ m endothelium; mean ± SD; unpaired ttest p< 0.005; Figure 1C]. VCAM-MPIO retention in metastasisbearing mice was significantly reduced at 3 h compared to metastasis-bearing mice at 10 min post-injection [(1.54 ± 1.82) × 10 −3 vs. (9.8 ± 1.35) × 10 −3 MPIO/ µ m 2 tumour area; mean ±SD ;p< 0.01; Figure 1C], most likely owing to immune cell (neutrophil) phagocytosis (Figure S2A,B). Quantitative analysis of non-specific MPIO retention assessed from normal tissue (i.e., not containing metastases) in the lungs of 4T1-GFP injected animals showed substantially lower levels than for metastasis-associated retention at 10 min post-injection ((2.51 ± 1.07) × 10 −5 MPIO/ µ m 2 vs. (0.12 ± 0.09) × 10 −2 MPIO/ µ m 2 ,p< 0.0001; Figure 1D). Similarly, low levels of retention were found in the naïve mice injected with either VCAM-MPIO ((1.22 ±0.05) ×10−5MPIO/µm2) or IgG-MPIO ((0.64 ±0.21) ×10−5MPIO/µm2). Int. J. Mol. Sci. 2024,25, 7160 4 of 19 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 of 19 Figure 1. Quantitative and spatial analysis of VCAM-MPIO and IgG-MPIO binding in lung tissues of in metastasis-bearing and naïve mice. (A,B) Representative images of lung tissue acquired from the metastatic mouse model (day 10), 10 min post-intravenous injection of VCAM-MPIO, stained for VCAM-1 (brown) with a nuclear counterstain (blue). (A) Substantial numbers of MPIO are associated with the metastasis microvasculature (red arrows and higher magnification areas), and also on nearby larger vessels showing high VCAM-1 expression (B). (C,D) Immunohistochemical quantitation of MPIO binding in metastasis-bearing or naïve lungs (10 min or 3 h after i.v. injection). (C) Bar graphs represent the number of MPIO per µm vessel circumference and (D) the number of MPIO per µm 2 in areas of metastasis (met), areas of no apparent metastasis in the metastasis model (no met), or healthy lung tissue (naïve mice) 10 min after i.v. injection. At 10 min post-administration VCAM-MPIO showed significantly greater retention on VCAM-1 expressing vessels associated with micrometastases compared to IgG-MPIO control). Similar levels of targeted and control agent retention at 3 h indicate rapid clearance of MPIO from the tissue. * p < 0.01; ** p < 0.005 compared to VCAM-MPIO retention at 10 min post-injection. Non-specific retention was negligible compared to specific binding. A significantly greater number of VCAM-MPIO than IgG-MPIO (quantified as the number of adherent MPIO per µm of inflamed endothelium, excluding capillaries) were found associated with micrometastatic colonies at 10 min post-injection [VCAM-MPIO vs. IgG-MPIO, (9.80 ± 1.35) × 10 −3 vs. (4.45 ± 4.08) × 10 −4 MPIO/µm endothelium; mean ± SD; unpaired t test p < 0.005; Figure 1C]. VCAM-MPIO retention in metastasis-bearing mice was significantly reduced at 3 h compared to metastasis-bearing mice at 10 min post-in- jection [(1.54 ± 1.82) × 10 −3 vs. (9.8 ± 1.35) × 10 −3 MPIO/µm 2 tumour area; mean ± SD; p < Figure 1. Quantitative and spatial analysis of VCAM-MPIO and IgG-MPIO binding in lung tissues of in metastasis-bearing and naïve mice. (A,B) Representative images of lung tissue acquired from the metastatic mouse model (day 10), 10 min post-intravenous injection of VCAM-MPIO, stained for VCAM-1 (brown) with a nuclear counterstain (blue). (A) Substantial numbers of MPIO are associated with the metastasis microvasculature (red arrows and higher magnification areas), and also on nearby larger vessels showing high VCAM-1 expression (B). (C,D) Immunohistochemical quantitation of MPIO binding in metastasis-bearing or naïve lungs (10 min or 3 h after i.v. injection). (C) Bar graphs represent the number of MPIO per µ m vessel circumference and (D) the number of MPIO per µ m 2 in areas of metastasis (met), areas of no apparent metastasis in the metastasis model (no met), or healthy lung tissue (naïve mice) 10 min after i.v. injection. At 10 min postadministration VCAM-MPIO showed significantly greater retention on VCAM-1 expressing vessels associated with micrometastases compared to IgG-MPIO control). Similar levels of targeted and control agent retention at 3 h indicate rapid clearance of MPIO from the tissue. * p< 0.01; ** p< 0.005 compared to VCAM-MPIO retention at 10 min post-injection. Non-specific retention was negligible compared to specific binding. 2.2. Radiolabelled VCAM-, IgG- or BSA-MPIO Conjugates In Vitro Binding of [89Zr]-DFO-VCAM-MPIO Radioimmunoassays were performed to confirm that the binding characteristics of VCAM-MPIO were not affected by radiolabelling, and indicated significantly greater binding of [ 89 Zr]-DFO-VCAM-MPIO to recombinant mouse VCAM-1 (% binding ± SD, 29.51 ± 7.03; Figure 2A) than both of the negative controls [ 89 Zr]-DFO-IgG-MPIO (10.34 ± 0.57; Int. J. Mol. Sci. 2024,25, 7160 5 of 19 p< 0.001) and [ 89 Zr]-DFO (13.41 ± 1.20; p< 0.005). No significant difference was found between the binding of [ 89 Zr]-DFO-VCAM-MPIO and the positive control [ 89 Zr]-DFO- VCAM (22.78 ± 2.51). A significant reduction in binding was observed following partial blocking with VCAM-1 antibody (% cpm ± SD, 14.30 ± 2.28; p< 0.001 compared to unblocked binding; Figure 2A). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 5 of 19 0.01; Figure 1C], most likely owing to immune cell (neutrophil) phagocytosis (Figure S2A,B). Quantitative analysis of non-specific MPIO retention assessed from normal tissue (i.e., not containing metastases) in the lungs of 4T1-GFP injected animals showed substantially lower levels than for metastasis-associated retention at 10 min post-injection ((2.51 ± 1.07) × 10 −5 MPIO/µm 2 vs. (0.12 ± 0.09) × 10 −2 MPIO/µm 2 , p < 0.0001; Figure 1D). Similarly, low levels of retention were found in the naïve mice injected with either VCAM-MPIO ((1.22 ± 0.05) × 10 −5 MPIO/µm 2 ) or IgG-MPIO ((0.64 ± 0.21) × 10 −5 MPIO/µm 2 ). 2.2. Radiolabelled VCAM-, IgG- or BSA-MPIO Conjugates In Vitro Binding of [ 89 Zr]-DFO-VCAM-MPIO Radioimmunoassays were performed to confirm that the binding characteristics of VCAM-MPIO were not affected by radiolabelling, and indicated significantly greater binding of [ 89 Zr]-DFO-VCAM-MPIO to recombinant mouse VCAM-1 (% binding ± SD, 29.51 ± 7.03; Figure 2A) than both of the negative controls [ 89 Zr]-DFO-IgG-MPIO (10.34 ± 0.57; p < 0.001) and [ 89 Zr]-DFO (13.41 ± 1.20; p < 0.005). No significant difference was found between the binding of [ 89 Zr]-DFO-VCAM-MPIO and the positive control [ 89 Zr]-DFO- VCAM (22.78 ± 2.51). A significant reduction in binding was observed following partial blocking with VCAM-1 antibody (% cpm ± SD, 14.30 ± 2.28; p < 0.001 compared to unblocked binding; Figure 2A). Figure 2. Specificity and competitive inhibition of [ 89 Zr]-DFO-VCAM-MPIO binding to VCAM-1 and TNF-stimulated HUVECs: radioimmunoassay evaluation. (A) Radioimmunoassay plots showing the % of bound radiolabelled agents against recombinant VCAM-1 or (B) TNF-stimulated HU- VECs with 30 min static incubation, +/− competition with VCAM-1 antibody (blocking). (A) [ 89 Zr]- DFO-VCAM-MPIO showed significantly greater % binding compared to both negative controls [ 89 Zr]-DFO-IgG-MPIO and [ 89 Zr]-DFO (p < 0.005). Following blockade with VCAM-1 antibody, [ 89 Zr]-DFO-VCAM-MPIO binding was significantly reduced (p < 0.001). (B) [ 89 Zr]-DFO-VCAM- MPIO showed significantly greater % binding to TNF-stimulated HUVECs compared to both negative controls [ 89 Zr]-DFO-IgG-MPIO and [ 89 Zr]-DFO (p < 0.001). Binding was substantially reduced with the inclusion of the blocking antibody and was negligible on unstimulated cells. One-way ANOVA with Tukey post-hoc tests; * p < 0.01; ** p < 0.005; *** p < 0.001; all comparisons are against [ 89 Zr]-DFO-VCAM-MPIO. Subsequently, binding to endothelial VCAM-1 was assessed on TNF-stimulated HU- VECs and, again, [ 89 Zr]-DFO-VCAM-MPIO showed significantly greater binding (% ± SD, 14.23 ± 4.43; p < 0.01; Figure 2B) than either of the negative controls [ 89 Zr]-DFO-IgG-MPIO (4.22 ± 0.92) and [ 89 Zr]-DFO (3.27 ± 0.72). In contrast, the positive control [ 89 Zr]-DFO- VCAM showed similar levels of binding (17.69 ± 4.71) to [ 89 Zr]-DFO-VCAM-MPIO. Preincubation with an excess of unlabelled VCAM-1 antibody effectively out-competed binding of both [ 89 Zr]-DFO-VCAM-MPIO and [ 89 Zr]-DFO-VCAM to TNF-stimulated HUVECs (% cpm ± SD, 4.45 ± 0.94 and 6.93 ± 0.99, respectively; Figure 2B). The binding of [ 89 Zr]- DFO-VCAM-MPIO to naïve unstimulated HUVECs was negligible (1.59 ± 0.41; Figure 2B). Figure 2. Specificity and competitive inhibition of [ 89 Zr]-DFO-VCAM-MPIO binding to VCAM-1 and TNF-stimulated HUVECs: radioimmunoassay evaluation. (A) Radioimmunoassay plots showing the % of bound radiolabelled agents against recombinant VCAM-1 or (B) TNF-stimulated HUVECs with 30 min static incubation, +/ − competition with VCAM-1 antibody (blocking). (A) [ 89 Zr]- DFO-VCAM-MPIO showed significantly greater % binding compared to both negative controls [ 89 Zr]-DFO-IgG-MPIO and [ 89 Zr]-DFO (p< 0.005). Following blockade with VCAM-1 antibody, [ 89 Zr]-DFO-VCAM-MPIO binding was significantly reduced (p< 0.001). (B) [ 89 Zr]-DFO-VCAM- MPIO showed significantly greater % binding to TNF-stimulated HUVECs compared to both negative controls [ 89 Zr]-DFO-IgG-MPIO and [ 89 Zr]-DFO (p< 0.001). Binding was substantially reduced with the inclusion of the blocking antibody and was negligible on unstimulated cells. One-way ANOVA with Tukey post-hoc tests; * p< 0.01; ** p< 0.005; *** p< 0.001; all comparisons are against [ 89 Zr]-DFO- VCAM-MPIO. Subsequently, binding to endothelial VCAM-1 was assessed on TNF-stimulated HU- VECs and, again, [ 89 Zr]-DFO-VCAM-MPIO showed significantly greater binding (% ± SD, 14.23 ± 4.43; p< 0.01; Figure 2B) than either of the negative controls [ 89 Zr]-DFO-IgG- MPIO (4.22 ± 0.92) and [ 89 Zr]-DFO (3.27 ± 0.72). In contrast, the positive control [ 89 Zr]- DFO-VCAM showed similar levels of binding (17.69 ± 4.71) to [ 89 Zr]-DFO-VCAM-MPIO. Pre-incubation with an excess of unlabelled VCAM-1 antibody effectively out-competed binding of both [ 89 Zr]-DFO-VCAM-MPIO and [ 89 Zr]-DFO-VCAM to TNF-stimulated HU- VECs (% cpm ± SD, 4.45 ± 0.94 and 6.93 ± 0.99, respectively; Figure 2B). The binding of [ 89 Zr]-DFO-VCAM-MPIO to naïve unstimulated HUVECs was negligible (1.59 ± 0.41; Figure 2B). 2.3. In Vivo Imaging 2.3.1. In Vivo PET Imaging of [89Zr]-DFO-VCAM-MPIO in Pulmonary Metastasis Model The binding of [ 89 Zr]-DFO-VCAM-MPIO in vivo in mice injected with 4T1-GFP cells was compared to that of both [ 89 Zr]-DFO-IgG-MPIO and the agent without MPIO, [ 89 Zr]-DFO-VCAM, at 10 min post-administration using PET. In accord with the known route of MPIO clearance, the highest levels of activity for both [ 89 Zr]-DFO-VCAM-MPIO ( Figures 3A and S3A ) and [ 89 Zr]-DFO-IgG-MPIO (Figure 3B and Figure S3B) were found in the liver and spleen. Although radioactivity within the lungs was not visually apparent on individual single-slice static PET images from pulmonary metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (Figure S2A,B), maximum intensity plots obtained from the full 3D dataset revealed marked radioactive uptake in the lungs (Figure 3A) compared to metastasis-bearing mice injected with the isotype control agent [ 89 Zr]-DFO-IgG-MPIO (Figure 3B). Although radioactivity was clearly evident in the 3D maximum intensity plots Int. J. Mol. Sci. 2024,25, 7160 6 of 19 from the diseased lungs, this could not be resolved into individual micrometastatic colonies (Figure 3A), likely owing to a combination of movement and the relatively low spatial resolution of the PET images. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 19 2.3. In Vivo Imaging 2.3.1. In Vivo PET Imaging of [ 89 Zr]-DFO-VCAM-MPIO in Pulmonary Metastasis Model The binding of [ 89 Zr]-DFO-VCAM-MPIO in vivo in mice injected with 4T1-GFP cells was compared to that of both [ 89 Zr]-DFO-IgG-MPIO and the agent without MPIO, [ 89 Zr]- DFO-VCAM, at 10 min post-administration using PET. In accord with the known route of MPIO clearance, the highest levels of activity for both [ 89 Zr]-DFO-VCAM-MPIO (Figures 3A and S3A) and [ 89 Zr]-DFO-IgG-MPIO (Figures 3B and S3B) were found in the liver and spleen. Although radioactivity within the lungs was not visually apparent on individual single-slice static PET images from pulmonary metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (Figure S2A,B), maximum intensity plots obtained from the full 3D dataset revealed marked radioactive uptake in the lungs (Figure 3A) compared to metastasis-bearing mice injected with the isotype control agent [ 89 Zr]-DFO-IgG-MPIO (Figure 3B). Although radioactivity was clearly evident in the 3D maximum intensity plots from the diseased lungs, this could not be resolved into individual micrometastatic colonies (Figure 3A), likely owing to a combination of movement and the relatively low spatial resolution of the PET images. Figure 3. Differential uptake of radiolabelled VCAM-MPIO and IgG-MPIO in metastasis-bearing and naïve mice. (A,B) In vivo, non-gated maximum intensity plots obtained from 3D PET datasets from mice with pulmonary metastases and injected with (A) [ 89 Zr]-DFO-VCAM-MPIO or (B) [ 89 Zr]- DFO-IgG-MPIO. (A) Marked radioactivity is evident in the lungs, liver, and spleen in the metastasisbearing mouse injected with [ 89 Zr]-DFO-VCAM-MPIO, but (B) only from the liver and spleen in the metastasis-bearing mouse injected with the isotype control agent [ 89 Zr]-DFO-IgG-MPIO. (C,D) Representative average activity uptake curves for (C) [ 89 Zr]-DFO-VCAM-MPIO and (D) [ 89 Zr]-DFO- IgG-MPIO in liver, blood, and lungs from metastasis-bearing mice (n = 4 per group). The liver shows rapid and high accumulation of radioactivity in both targeted and control groups. (C) Lungs showed a higher accumulation of activity for the [ 89 Zr]-DFO-VCAM-MPIO injected mice than (D) the [ 89 Zr]- DFO-IgG-MPIO group. (E–G) Graphs showing average activity uptake over a duration of 55 min for liver, blood and lung from metastasis-bearing mice injected with (E) [ 89 Zr]-DFO-VCAM-MPIO or (F) [ 89 Zr]-DFO-IgG-MPIO, or (G) from naïve mice injected with [ 89 Zr]-DFO-VCAM-MPIO. In all cases, the liver showed significantly higher uptake than other tissues (p < 0.0001), reflecting the role of the liver in scavenging unbound MPIO from the blood. (E) Lungs from metastasis-bearing mice injected with the targeted agent showed significantly (p < 0.0005) higher uptake than (F) lungs from mice injected with the control agent or (G) lungs from naïve mice injected with the targeted agent (p < 0.0005). Figure 3. Differential uptake of radiolabelled VCAM-MPIO and IgG-MPIO in metastasis-bearing and naïve mice. (A,B) In vivo , non-gated maximum intensity plots obtained from 3D PET datasets from mice with pulmonary metastases and injected with (A) [ 89 Zr]-DFO-VCAM-MPIO or (B) [ 89 Zr]- DFO-IgG-MPIO. (A) Marked radioactivity is evident in the lungs, liver, and spleen in the metastasisbearing mouse injected with [ 89 Zr]-DFO-VCAM-MPIO, but (B) only from the liver and spleen in the metastasis-bearing mouse injected with the isotype control agent [ 89 Zr]-DFO-IgG-MPIO. (C,D) Representative average activity uptake curves for (C) [ 89 Zr]-DFO-VCAM-MPIO and (D) [ 89 Zr]- DFO-IgG-MPIO in liver, blood, and lungs from metastasis-bearing mice (n = 4 per group). The liver shows rapid and high accumulation of radioactivity in both targeted and control groups. (C) Lungs showed a higher accumulation of activity for the [ 89 Zr]-DFO-VCAM-MPIO injected mice than (D) the [ 89 Zr]-DFO-IgG-MPIO group. (E–G) Graphs showing average activity uptake over a duration of 55 min for liver, blood and lung from metastasis-bearing mice injected with (E) [ 89 Zr]-DFO-VCAM- MPIO or (F) [ 89 Zr]-DFO-IgG-MPIO, or (G) from naïve mice injected with [ 89 Zr]-DFO-VCAM-MPIO. In all cases, the liver showed significantly higher uptake than other tissues (p< 0.0001), reflecting the role of the liver in scavenging unbound MPIO from the blood. (E) Lungs from metastasis-bearing mice injected with the targeted agent showed significantly (p< 0.0005) higher uptake than (F) lungs from mice injected with the control agent or (G) lungs from naïve mice injected with the targeted agent (p< 0.0005). Naïve (control) mice injected with [ 89 Zr]-DFO-VCAM-MPIO also showed uptake, primarily in the liver and spleen (Figure S2C). Metastasis-bearing animals injected with [ 89 Zr]-DFO-VCAM (antibody alone) showed high levels of the agent in the blood for the first hour (Figure S2D,E) and high levels of radioactivity in the heart. The liver and bladder also showed high uptake of radioactivity, whereas lower levels of radioactivity were evident in the spleen and bones (Figure S2D). Despite the lack of visible focal signal in the lungs of metastasis-bearing animals injected with [ 89 Zr]-DFO-VCAM-MPIO, time-activity curves for the lungs showed clear activity uptake (equilibrating to 0.53 ± 0.28 g/mL; mean ± SD; Figure 3C,E). This uptake was significantly higher than the activity uptake of either [ 89 Zr]-DFO-IgG-MPIO in lungs from metastasis-bearing mice (0.12 ± 0.04 g/mL; mean ± SD; p< 0.0001; Figure 3D,F) or [ 89 Zr]-DFO-VCAM-MPIO uptake in naïve mouse lungs (0.10 ± 0.02 g/mL; mean ± SD; Int. J. Mol. Sci. 2024,25, 7160 7 of 19 p< 0.0001; Figure 3G). The blood activity in all groups was consistently lower than that of lung uptake of [ 89 Zr]-DFO-VCAM-MPIO in metastasis-bearing animals ( Figure 3C–G ; p< 0.0001). Time-activity curves for the livers of metastasis-bearing animals injected with [ 89 Zr]- DFO-VCAM-MPIO (Figure 3C) or [ 89 Zr]-DFO-IgG-MPIO (Figure 3D) showed a rapid increase in uptake, which plateaued at 5 min post-administration. Metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO showed comparable levels of liver uptake, over 50 min, to those injected with either [ 89 Zr]-DFO-IgG-MPIO (2.65 ± 0.28 vs. 2.38 ± 0.12 g/mL; mean ± SD; Figure 3E,F) or naïve mice injected with [ 89 Zr]-DFO-VCAM-MPIO (2.52 ± 0.32 g/mL; mean ±SD; Figure 3E,G). Activity uptake curves for [ 89 Zr]-DFO-VCAM in metastasis-bearing mice showed a rapid increase in uptake into the heart, liver, and bladder (Figure S2E), reaching a plateau at 5 min post-administration. The half-life of [ 89 Zr]-DFO-VCAM in the circulation was determined from the heart measurements. Heart measurements were also used to determine the half-life of [ 89 Zr]-DFO-VCAM-MPIO or [ 89 Zr]-DFO-IgG-MPIO in the circulation, and findings were consistent with the values calculated from the biological clearance study; circulatory half-life PET vs. MRI, 36.6 vs. 39.9 s (Figure S3). Static analysis of lung activity was also performed at two time points, 10 and 55 min post-administration, on the 1 h in vivo dynamic dataset. Lungs from the metastasis-bearing group injected with [ 89 Zr]-DFO-VCAM-MPIO showed significantly greater (p< 0.0005) activity compared to both metastasis-bearing animals injected with [ 89 Zr]-DFO-IgG-MPIO and naïve animals injected with [ 89 Zr]-DFO-VCAM-MPIO at both time points (Figure 4A,B). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 19 Naïve (control) mice injected with [ 89 Zr]-DFO-VCAM-MPIO also showed uptake, primarily in the liver and spleen (Figure S2C). Metastasis-bearing animals injected with [ 89 Zr]-DFO-VCAM (antibody alone) showed high levels of the agent in the blood for the first hour (Figure S2D,E) and high levels of radioactivity in the heart. The liver and bladder also showed high uptake of radioactivity, whereas lower levels of radioactivity were evident in the spleen and bones (Figure S2D). Despite the lack of visible focal signal in the lungs of metastasis-bearing animals injected with [ 89 Zr]-DFO-VCAM-MPIO, time-activity curves for the lungs showed clear activity uptake (equilibrating to 0.53 ± 0.28 g/mL; mean ± SD; Figure 3C,E). This uptake was significantly higher than the activity uptake of either [ 89 Zr]-DFO-IgG-MPIO in lungs from metastasis-bearing mice (0.12 ± 0.04 g/mL; mean ± SD; p < 0.0001; Figure 3D,F) or [ 89 Zr]- DFO-VCAM-MPIO uptake in naïve mouse lungs (0.10 ± 0.02 g/mL; mean ± SD; p< 0.0001; Figure 3G). The blood activity in all groups was consistently lower than that of lung uptake of [ 89 Zr]-DFO-VCAM-MPIO in metastasis-bearing animals (Figure 3C–G; p < 0.0001). Time-activity curves for the livers of metastasis-bearing animals injected with [ 89 Zr]- DFO-VCAM-MPIO (Figure 3C) or [ 89 Zr]-DFO-IgG-MPIO (Figure 3D) showed a rapid increase in uptake, which plateaued at 5 min post-administration. Metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO showed comparable levels of liver uptake, over 50 min, to those injected with either [ 89 Zr]-DFO-IgG-MPIO (2.65 ± 0.28 vs. 2.38 ± 0.12 g/mL; mean ± SD; Figure 3E,F) or naïve mice injected with [ 89 Zr]-DFO-VCAM-MPIO (2.52 ± 0.32 g/mL; mean ± SD; Figure 3E,G). Activity uptake curves for [ 89 Zr]-DFO-VCAM in metastasis-bearing mice showed a rapid increase in uptake into the heart, liver, and bladder (Figure S2E), reaching a plateau at 5 min post-administration. The half-life of [ 89 Zr]-DFO-VCAM in the circulation was determined from the heart measurements. Heart measurements were also used to determine the half-life of [ 89 Zr]-DFO-VCAM-MPIO or [ 89 Zr]-DFO-IgG-MPIO in the circulation, and findings were consistent with the values calculated from the biological clearance study; circulatory half-life PET vs. MRI, 36.6 vs. 39.9 s (Figure S3). Static analysis of lung activity was also performed at two time points, 10 and 55 min post-administration, on the 1 h in vivo dynamic dataset. Lungs from the metastasis-bear- ing group injected with [ 89 Zr]-DFO-VCAM-MPIO showed significantly greater (p < 0.0005) activity compared to both metastasis-bearing animals injected with [ 89 Zr]-DFO-IgG-MPIO and naïve animals injected with [ 89 Zr]-DFO-VCAM-MPIO at both time points (Figure 4A,B). Figure 4. Temporal and spatial analysis of radiolabelled VCAM-MPIO or IgG-MPIO uptake in metastasis-bearing and naïve mice. (A,B) In vivo average activity uptake in the lungs of mice injected with (A) [ 89 Zr]-DFO-VCAM-MPIO at 10 and (B) 50 min post-administration (n = 4 per group) showed significantly greater retention in metastasis-bearing lungs compared to naïve lungs or to metastasisbearing mice injected with [ 89 Zr]-DFO-IgG-MPIO (*** p< 0.0001). (C,D) Post-mortem (10 min post i.v.) 2 h acquisition whole-body PET images of mice injected with (C) [ 89 Zr]-DFO-VCAM-MPIO or (D) [ 89 Zr]-DFO-IgG-MPIO showing marked radioactivity in the liver and spleen. (C) Lungs from the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO also show notable radioactivity (D), whereas signals from the lungs of the mouse injected with [ 89 Zr]-DFO-IgG-MPIO do not. (E) The mouse injected with [ 89 Zr]-DFO-VCAM-MPIO in image (C) was further examined using a long PET acquisition (60 h) after removing the body and organs below the diaphragm, to confirm that the apparent lung signal, as seen in image (C), originated from the lungs and was not a partial volume effect from liver and spleen. Int. J. Mol. Sci. 2024,25, 7160 8 of 19 2.3.2. Post Mortem PET Imaging of [89Zr]-DFO-VCAM-MPIO in Pulmonary Metastasis Model To circumvent issues of motion in the in vivo PET images, two metastasis-bearing mice injected with either [ 89 Zr]-DFO-VCAM-MPIO (Figure 4C) or [ 89 Zr]-DFO-IgG-MPIO (Figure 4D) were terminated 10 min post-administration and imaged over a 2 h time frame with PET. Both images showed high levels of radioactivity in the liver, while only the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO showed significant radioactivity in the lungs. Subsequently, the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO was further investigated with 60 h PET acquisition after excision of all organs below the diaphragm to verify that the signal originated from the lungs and was not a partial volume effect from the liver and spleen (Figure 4E). 2.4. Ex Vivo Analysis 2.4.1. Ex Vivo Biodistribution of [89Zr]-DFO-VCAM-MPIO Biodistribution of all agents was further assessed ex vivo, and the highest uptake across all of the organs studied was found in the lungs from metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (68.9 ± 22.9% ID/g; mean ± SD); lung uptake was significantly higher than that in all other experimental groups (one-way ANOVA, p< 0.05; Figure 5). The spleen and liver exhibited the second highest activity uptake in metastasisbearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (49.6 ± 37.6% ID/g; mean ± SD), and the levels were similar in all groups. The heart, kidney, bone, and blood samples showed relatively low levels of uptake, while the stomach, small and large intestine, pancreas, and samples from muscle, skin, and fat showed negligible uptake in all groups (Figure 5). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 19 Figure 4. Temporal and spatial analysis of radiolabelled VCAM-MPIO or IgG-MPIO uptake in metastasis-bearing and naïve mice. (A,B) In vivo average activity uptake in the lungs of mice injected with (A) [ 89 Zr]-DFO-VCAM-MPIO at 10 and (B) 50 min post-administration (n = 4 per group) showed significantly greater retention in metastasis-bearing lungs compared to naïve lungs or to metastasis-bearing mice injected with [ 89 Zr]-DFO-IgG-MPIO (*** p < 0.0001). (C,D) Post-mortem (10 min post i.v.) 2 h acquisition whole-body PET images of mice injected with (C) [ 89 Zr]-DFO-VCAM- MPIO or (D) [ 89 Zr]-DFO-IgG-MPIO showing marked radioactivity in the liver and spleen. (C) Lungs from the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO also show notable radioactivity (D), whereas signals from the lungs of the mouse injected with [ 89 Zr]-DFO-IgG-MPIO do not. (E) The mouse injected with [ 89 Zr]-DFO-VCAM-MPIO in image (C) was further examined using a long PET acquisition (60 h) after removing the body and organs below the diaphragm, to confirm that the apparent lung signal, as seen in image (C), originated from the lungs and was not a partial volume effect from liver and spleen. 2.3.2. Post Mortem PET Imaging of [ 89 Zr]-DFO-VCAM-MPIO in Pulmonary Metastasis Model To circumvent issues of motion in the in vivo PET images, two metastasis-bearing mice injected with either [ 89 Zr]-DFO-VCAM-MPIO (Figure 4C) or [ 89 Zr]-DFO-IgG-MPIO (Figure 4D) were terminated 10 min post-administration and imaged over a 2 h time frame with PET. Both images showed high levels of radioactivity in the liver, while only the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO showed significant radioactivity in the lungs. Subsequently, the mouse injected with [ 89 Zr]-DFO-VCAM-MPIO was further investigated with 60 h PET acquisition after excision of all organs below the diaphragm to verify that the signal originated from the lungs and was not a partial volume effect from the liver and spleen (Figure 4E). 2.4. Ex Vivo Analysis 2.4.1. Ex Vivo Biodistribution of [ 89 Zr]-DFO-VCAM-MPIO Biodistribution of all agents was further assessed ex vivo, and the highest uptake across all of the organs studied was found in the lungs from metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (68.9 ± 22.9% ID/g; mean ± SD); lung uptake was significantly higher than that in all other experimental groups (one-way ANOVA, p < 0.05; Figure 5). The spleen and liver exhibited the second highest activity uptake in metastasisbearing mice injected with [ 89 Zr]-DFO-VCAM-MPIO (49.6 ± 37.6% ID/g; mean ± SD), and the levels were similar in all groups. The heart, kidney, bone, and blood samples showed relatively low levels of uptake, while the stomach, small and large intestine, pancreas, and samples from muscle, skin, and fat showed negligible uptake in all groups (Figure 5). Figure 5. Organ-specific biodistribution of radiolabelled VCAM-MPIO or IgG-MPIO in metastasisbearing and naïve mice. Bar graph showing the average % of injected dose per mass of organ for all groups (n = 8 per group). In the lungs, metastasis-bearing mice injected with [ 89 Zr]-DFO-VCAM- MPIO (red) showed significantly (p < 0.05) greater radiation yield than either the control [ 89 Zr]-DFO- IgG-MPIO injected groups (blue and cyan) or naïve mice injected with [ 89 Zr]-DFO-VCAM-MPIO (orange). The liver and spleen showed the next highest levels of radioactivity uptake, supporting Figure 5. Organ-specific biodistribution of radiolabelled VCAM-MPIO or IgG-MPIO in metastasisbearing and naïve mice. Bar graph showing the average % of injected dose per mass of organ for all groups (n = 8 per group). In the lungs, metastasis-bearing mice injected with [ 89 Zr]-DFO- VCAM-MPIO (red) showed significantly (p< 0.05) greater radiation yield than either the control [ 89 Zr]-DFO-IgG-MPIO injected groups (blue and cyan) or naïve mice injected with [ 89 Zr]-DFO- VCAM-MPIO (orange). The liver and spleen showed the next highest levels of radioactivity uptake, supporting their role in the scavenging of non-bound particles in circulation. The kidney and bone showed comparable levels of %ID/g to the blood and heart, and thus, they are likely to be background circulating levels. One-way ANOVA; * p< 0.05; ** p< 0.01. 2.4.2. Lung Autoradiography of [89Zr]-DFO-VCAM-MPIO Accumulation in Lungs The binding of [ 89 Zr]-labelled MPIO was further confirmed by comparison of autoradiography with immunohistochemical assessment of VCAM-1 expression and the presence of metastases in the same sections (Figure 6). In all sections from metastasis-bearing mouse injected with [ 89 Zr]-DFO-VCAM-MPIO, a low level of activity was evident across the whole lung, in addition to more intense dark foci in discrete areas of the lungs (Figure 6A). The negligible activity was evident in sections from either metastasis-bearing or naïve mice injected with [ 89 Zr]-DFO-IgG-MPIO (Figure 6C,D) and from the naïve mouse injected with [ 89 Zr]-DFO-VCAM-MPIO (Figure 6B). Lung tissue sections were also used for longer Int. J. Mol. Sci. 2024,25, 7160 9 of 19 exposure autoradiography and subsequent VCAM-1 immunohistochemistry, and the presence of metastases at the location of the intense dark spots on the autoradiography film was confirmed; these further correlated with the presence of [ 89 Zr]-DFO-VCAM-MPIO (Figure 6E–G ; Figure S4B(1–3)). At longer exposures, it was apparent that some dark spots on the film were not correlated with metastases but were correlated with the presence of MPIO (Figure S5B; small red dots on the tissue). These MPIO may be associated with metastases in subsequent tissue sections or may reflect non-specific retention owing to vessel/tissue architecture. It was also found that occasional metastases with a very low MPIO score did not correlate with foci (Figure S4B(4)), most likely owing to low 89 Zr levels. Conversely, in a small number of metastases showing high expression of VCAM-1, it appeared that no MPIO were present, but these still correlated with moderate activity foci (Figure S5B(5)). In this case, it is possible that the MPIO were dislodged during the staining process or obscured by the very intense VCAM-1 staining. The autoradiography in Figure 6E was performed a day earlier than that in Figure 6A and was exposed for 24 h compared to 10 h for the sections in Figure 6A. Because the exposure time was not proportional to the decay of the isotope, the autoradiograph in Figure 6E appears darker than the samples in Figure 6A, due to overexposure. Prior to autoradiography, the lungs were not perfused with saline to remove the blood from the vasculature, and it is likely, therefore, that the uncorrelated dark spots reflect an unknown amount of free 89 Zr in the blood. This notion is supported by the higher signal observed in the heart, which contains a greater blood volume than lung tissue. It should also be noted that in some areas where the tissue is folded, a higher saturated signal is evident that does not reflect the presence of metastases (Figure 6E,F; Figure S5). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 10 of 19 Figure 6. Comparative autoradiographic and immunohistochemical analyses and colocalisation of foci with radiolabelled VCAM-MPIO and micrometastases. (A–D) Paired images of lung tissue autoradiographic data (left; 10 h exposure) and bright-field micrographs of the associated lung sections (right) stained immunohistochemically for VCAM-1 expression (brown) for (A) metastasis-bearing or (B) naïve lungs treated with [ 89 Zr]-DFO-VCAM-MPIO; (C) metastasis-bearing or (D) naïve lungs treated with [ 89 Zr]-DFO-IgG-MPIO. (A) Metastasis-bearing lungs + [ 89 Zr]-DFO-VCAM-MPIO showed sparse basal activity in all areas of the lung sections and localised intense signals (dark spots). (B–D) All other groups showed no or minimal activity. (E,F) Autoradiographic images from the lung tissue section after 24 h exposure and immunohistochemical images from the same lung section manually inspected under magnification. Foci of high activity on the autoradiographic plate (e.g., red dashed square and red circles) colocalise with VCAM-1 expressing micrometastatic areas (black dashed square and red circles). The high activity foci on the autoradiographic plate that do not correlate with apparent metastases do correlate with areas where MPIO were manually scored ([F]; hard to distinguish small red marks). Dotted scale bar = 5 mm. (G) Black dashed square in (F) is shown at a higher resolution; arrows indicate the presence of MPIO. Nuclei are stained blue. Scale bar = 50 µm. 3. Discussion To date, there is no standard non-invasive clinical or preclinical screening method that provides a diagnosis for early micrometastatic development in the lung. In this study, we show that [ 89 Zr]-DFO-VCAM-MPIO specifically binds to activated pulmonary endothelium at sites of micrometastases, enabling very early detection of lung metastases by PET imaging. The spread of metastatic cells to the lymphatic system is common in many types of cancer. Consequently, imaging of lymph nodes has become one of the standard clinical screening methods for secondary tumour spread, and both structural MRI [28] and nuclear imaging with functional tracers [29,30] have enabled the detection of malignant lymph nodes for many years. Contrast-enhanced MRI using superparamagnetic particles of iron oxide (SPIO; 40– 150 nm) and ultrasmall SPIO (USPIO; <50 nm) have been utilised in the clinic for their properties to preferentially phagocytosed by cells in normal lymphoid tissues [31] and facilitated a more accurate assessment of lymph node malignancy [32–36]. However, these methods are limited to the detection of macro- and micrometastatic lesions within the lymphatic system and do not enable site-specific identification of spread to other tissues/organs [16]. Targeting of VCAM-1 with VCAM-MPIO and in vivo MRI has previously been demonstrated in preclinical models of numerous diseases, including neuroinflammation Figure 6. Comparative autoradiographic and immunohistochemical analyses and colocalisation of foci with radiolabelled VCAM-MPIO and micrometastases. (A–D) Paired images of lung tissue autoradiographic data (left; 10 h exposure) and bright-field micrographs of the associated lung sections (right) stained immunohistochemically for VCAM-1 expression (brown) for (A) metastasisbearing or (B) naïve lungs treated with [ 89 Zr]-DFO-VCAM-MPIO; (C) metastasis-bearing or (D) naïve lungs treated with [ 89 Zr]-DFO-IgG-MPIO. (A) Metastasis-bearing lungs + [ 89 Zr]-DFO-VCAM-MPIO showed sparse basal activity in all areas of the lung sections and localised intense signals (dark spots). (B–D) All other groups showed no or minimal activity. (E,F) Autoradiographic images from the lung tissue section after 24 h exposure and immunohistochemical images from the same lung section manually inspected under magnification. Foci of high activity on the autoradiographic plate (e.g., red Int. J. Mol. Sci. 2024,25, 7160 16 of 19 MPIO or [ 89 Zr]-DFO-IgG-MPIO (0.5 MBq per injection) and terminated with lethal injection (200 µ L pentobarbital) 10 min later (n = 8 per group). Blood samples were collected from each animal immediately after incision into the diaphragm by cutting the inferior vena cava and allowing the blood to pool within the diaphragm-constrained area. Subsequently, the heart, lungs, liver, spleen, stomach, large intestine, small intestine, pancreas, kidneys, and samples of bone (right ulna), skin (abdomen), and visceral fat (abdomen) were excised, washed in PBS (to eliminate blood), and left to dry on high-absorbency paper. Selected organs, tissues, and blood were placed in 5 mL tubes of known weight (Thermo Fisher Scientific, Waltham, MA, USA), and the tubes were placed in a gamma counter ( 1470 WIZARD gamma counter, Perkin Elmer, Waltham, MA, USA) to determine radioactivity per sample. Tubes containing the organs were weighed to calculate the mass of the organ, and subsequently, the percentage of injected dose per g (%ID/g) of the organ was calculated. 4.5.2. Ex Vivo Autoradiography of MPIO Accumulation in Lungs Groups equivalent to those described for the biodistribution study were used for ex vivo autoradiography (n = 2 per group; 8 mice in total). Lungs were excised and treated as described above. The lungs were kept in sucrose for 48 h and then cryosectioned as described above. After cryosectioning, the mounted lung sections were placed on a Cylcone ® storage phosphor screen and exposed for 12 or 24 h at room temperature. Subsequently, the same lung sections were stained immunohistochemically for VCAM-1, as described above. 5. Conclusions In conclusion, we present here a novel imaging method for the detection of pulmonary micrometastases in mice by targeting activated pulmonary endothelium using a PET- detectable molecularly targeted contrast agent, [ 89 Zr]-DFO-VCAM-MPIO. By keeping the microparticle platform used for our molecular MRI contrast agents, we both retain their clearance characteristics, yielding highly specific target signal changes and providing a dual-modality agent that can be used to detect micrometastases in different tissue beds with the optimal imaging approach for each. The agent is in itself a platform, as the basic MPIO component can be conjugated to a range of different targeting and contrast molecules to create varied and multifunctional agents. Looking ahead, the next steps for research should focus on further refining the probe’s specificity and efficiency, leading to clinical trials. Downstream, exploring the integration of this imaging technology into routine clinical practice will be crucial. At the research level, expanding the application of this probe to other types of cancer could provide comprehensive insights into its versatility and effectiveness across different oncological contexts. These steps will be pivotal in moving from experimental application to a standard component of oncological imaging and treatment planning, paving the way for advances in personalised medicine and improved prognosis in patients with cancer. Supplementary Materials: The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/ijms25137160/s1. Author Contributions: N.R.S. and S.M. designed the experiments and prepared the manuscript. S.M. and N.Z. performed the in vivo work. J.C.K., B.C. and F.P.-B. designed and synthesised the imaging agent. S.M., J.C.K. and V.K. performed the PET imaging and analysis. R.J.M. and N.R.S. supervised the study, and M.S.S. provided essential supervision and assistance with histology. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by Cancer Research UK (grant number C5255/A15935) and the CR-UK & EPSRC Oxford Cancer Imaging Center (grant number C5255/A1646). SM was funded by a CR-UK & EPSRC Oxford Cancer Imaging Centre Studentship. Institutional Review Board Statement: All animal experiments were approved by the University of Oxford Clinical Medicine Ethics Review Committee and the UK Home Office (Animals [Scientific Procedures] Act, 1986). Int. J. Mol. Sci. 2024,25, 7160 17 of 19 Informed Consent Statement: Not applicable. Data Availability Statement: The datasets supporting the findings of the current study are available from the corresponding author upon request. Acknowledgments: The authors thank the staff of the BMS facility for assistance with animal husbandry and welfare and members of the Oxford InstituteImaging Core (Sean Smart, Paul Kincesch, and Danny Allen) for imaging and analysis support. Conflicts of Interest: The work has no relevance to CureVac and they have no interest in the outcome of the paper. References 1. Valastyan, S.; Weinberg, R.A. Tumor Metastasis: Molecular Insights and Evolving Paradigms. Cell 2011,147, 275–292. [CrossRef] [PubMed] 2. Weigelt, B.; Peterse, J.L.; van’t Veer, L.J. Breast Cancer Metastasis: Markers and Models. Nat. Rev. Cancer 2005,5, 591–602. [CrossRef] [PubMed] 3. Zhao, X.; Wen, X.; Wei, W.; Chen, Y.; Zhu, J.; Wang, C. Clinical Characteristics and Prognoses of Patients Treated Surgically for Metastatic Lung Tumors. Oncotarget 2017,8, 46491–46497. [CrossRef] [PubMed] 4. Stella, G.M.; Kolling, S.; Benvenuti, S.; Bortolotto, C. Lung-Seeking Metastases. Cancers 2019,11, 1010. [CrossRef] [PubMed] 5. 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