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Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the “vicious cycle” of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.

PURPOSE. Multiple myeloma (MM) is a bone marrow malignancy that remains mostly incurable. Elotuzumab is an FDA-approved therapeutic monoclonal antibody targeted to the cell surface glycoprotein CS1, which is overexpressed in MM cells. Identifying patients who will respond to CS1 targeted treatments such as elotuzumab requires the development of a companion diagnostic to assess the presence of CS1. Here, we evaluated [(89)Zr]DFO-elotuzumab as a novel PET tracer for imaging CS1 expression in preclinical MM models. METHODS. Conjugation of desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS) to elotuzumab enabled Zirconium-89 radiolabeling. MM.1S-CG cells were intravenously injected in NOD-SCID-Gamma (NSG) mice. Small animal PET imaging with [(89)Zr]DFO-elotuzumab (1.11 MBq/mouse, 7 d post-injection), [(89)Zr]DFO-IgG (1.11 MBq/mouse, 7 d post-injection) and [(18)F]FDG (7–8 MBq, 1 h post-injection) was performed. Additionally, biodistribution of [(89)Zr]DFO-elotuzumab post-imaging at 7 d was also done. In vivo specificity of [(89)Zr]DFO-elotuzumab was further evaluated with a blocking study and ex vivo autoradiography. RESULTS. [(89)Zr]DFO-elotuzumab was produced with high specific activity (56 MBq/nmol ± 0.75), radiochemical purity (99% ± 0.5) and yield (93.3% ± 1.5). Dissociation constant of 40.4 nM and receptor density of 126 fmol/mg was determined in MM.1S-CG cells. Compared to [(89)Zr]DFO-IgG, [(89)Zr]DFO-elotuzumab localized with a significantly higher standard uptake value in tumor-bearing bone tissue (8.59 versus 4.77). Blocking with unlabeled elotuzumab significantly reduced (p3-fold enhanced uptake in bones. CONCLUSION. These data demonstrate the feasibility of [(89)Zr]DFO-elotuzumab as a companion diagnostic for CS1 targeted therapies.

Rapid liver and spleen opsonization of systemically administered nanoparticles (NPs) for in vivo applications remains the Achilles’ heel of nanomedicine, allowing only a small fraction of the materials to reach the intended target tissue. Although focusing on diseases that reside in the natural disposal organs for nanoparticles is a viable option, it limits the plurality of lesions that could benefit from nanomedical interventions. Here we designed a theranostic nanoplatform consisting of reactive oxygen (ROS)-generating titanium dioxide (TiO(2)) NPs, coated with a tumor-targeting agent, transferrin (Tf), and radiolabeled with a radionuclide ((89)Zr) for targeting bone marrow, imaging the distribution of the NPs, and stimulating ROS generation for cell killing. Radiolabeling of TiO(2) NPs with (89)Zr afforded thermodynamically and kinetically stable chelate-free (89)Zr-TiO(2)-Tf NPs without altering the NP morphology. Treatment of multiple myeloma (MM) cells, a disease of plasma cells originating in the bone marrow, with (89)Zr-TiO(2)-Tf generated cytotoxic ROS to induce cancer cell killing via apoptosis pathway. Positron emission tomography/X-ray computed tomography (PET/CT) imaging and tissue biodistribution studies revealed that in vivo administration of (89)Zr-TiO(2)-Tf in mice leveraged the osteotropic effect of (89)Zr to selectively localize about 70% of the injected radioactivity in mouse bone tissue. A combination of small animal PET/CT imaging of NP distribution and bioluminescence imaging of cancer progression showed that a single dose (89)Zr-TiO(2)-Tf treatment in a disseminated MM mouse model completely inhibited cancer growth at euthanasia of untreated mice and at least doubled the survival of treated mice. Treatment of the mice with cold Zr-TiO(2)-Tf, (89)Zr-oxalate, or (89)Zr-Tf had no therapeutic benefit compared to untreated controls. This study reveals an effective radionuclide sensitizing nanophototherapy paradigm for the treatment of MM and possibly other bone-associated malignancies.

Introduction Cardiovascular disease is the leading cause of death in the United States. The identification of vulnerable plaque at risk of rupture has been a major focus of research. Hypoxia has been identified as a potential factor in the formation of vulnerable plaque, and it is clear that decreased oxygen plays a role in the development of plaque angiogenesis leading to plaque destabilization. The purpose of this study is to demonstrate the feasibility of copper-64 labeled diacetyl-bis (N 4 -methylthiosemicarbazone) ( 64 Cu-ATSM), a positron-emitting radiopharmaceutical taken up in low-oxygen-tension cells, for the identification of hypoxic and potentially unstable atherosclerotic plaque in a mouse model. Methods 64 Cu-ATSM PET was performed in 21 atherosclerotic apolipoprotein E knockout (ApoE −/− ) mice, 6 of which were fed high-fat diet (HFD) while the others received standard-chow diet (SCD), and 13 control wild type mice fed SCD. 4 SCD ApoE −/− mice and 4 SCD wild type mice also underwent 18 F-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET) imaging one day prior to 64 Cu-ATSM PET. Results 64 Cu-ATSM uptake was increased in the aortic arch in SCD ApoE −/− mice (average aortic arch/muscle (A/M) standardized uptake value ratio 7.5–30min post injection: (5.66±0.23) compared to control mice (A/M SUV ratio 7.5–30min post injection (3.87±0.22), p −/− mice also showed similarly increased aortic arch uptake on PET imaging in comparison to control mice. Immunohistochemistry in both HFD and SCD ApoE −/− mice revealed noticeable hypoxia by pimonidazole stain in atherosclerosis which was co-localized to macrophage by CD68 staining. Autoradiography assessment demonstrated the presence of hypoxia by 64 Cu-ATSM uptake correlated with pimonidazole uptake within the ex vivo atherosclerotic aortic arch specimens. A significant increase in 18 F-FDG uptake in the SCD ApoE −/− mice in comparison to controls was also observed at delayed time points. Conclusion This pre-clinical study suggests that 64 Cu-ATSM is a potential PET tracer for hypoxia imaging in atherosclerosis. Advances in Knowledge and Implications for Patient Care While studies in humans are necessary for conclusive data, in the long term, a 64 Cu-ATSM PET imaging strategy could help facilitate the study of plaque biology in human patients.

Multiple myeloma (MM) is an age-related hematological malignancy with an estimated 30,000 new cases and 13,000 deaths per year. A disease of antibody-secreting malignant plasma B-cells that grow primarily in the bone marrow (BM), MM causes debilitating fractures, anemia, renal failure, and hypercalcemia. In addition to the abnormal genetic profile of MM cells, the permissive BM microenvironment (BMM) supports MM pathogenesis. Although advances in treatment options have significantly enhanced survival in MM patients, transient perfusion of small-molecule drugs in the BM does not provide sufficient residence to enhance MM cell-drug interaction, thus allowing some myeloma cells to escape the first line of treatment. As such, there remains a crucial need to develop advanced drug delivery systems that can navigate the complex BMM and effectively reach the myeloma cells. The high vascular density and spongy nature of bone structure suggest that nanoparticles (NPs) can serve as smart drug-delivery systems capable of extravasation and retention in various BM compartments to exert a durable therapeutic effect. In this focus article, we first summarize the pathophysiology of MM, emphasizing how the BM niche presents serious challenges for effective treatment of MM with small-molecule drugs. We then pivot to current efforts to develop NP-based drug carriers and intrinsically therapeutic nanotherapeutics. The article concludes with a brief perspective on the opportunities and challenges in developing and translating nanotherapeutics to improve the treatment outcomes of MM patients. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Despite over 20 years of clinical use, IL-2 has not fulfilled expectations as a safe and effective form of tumour immunotherapy. Expression of the high affinity IL-2Rα chain on regulatory T cells mitigates the anti-tumour immune response and its expression on vascular endothelium is responsible for life threatening complications such as diffuse capillary leak and pulmonary oedema. Here we describe the development of a recombinant fusion protein comprised of a cowpox virus encoded NKG2D binding protein (OMCP) and a mutated form of IL-2 with poor affinity for IL-2Rα. This fusion protein (OMCP-mutIL-2) potently and selectively activates IL-2 signalling only on NKG2D-bearing cells, such as natural killer (NK) cells, without broadly activating IL-2Rα-bearing cells. OMCP-mutIL-2 provides superior tumour control in several mouse models of malignancy and is not limited by mouse strain-specific variability of NK function. In addition, OMCP-mutIL-2 lacks the toxicity and vascular complications associated with parental wild-type IL-2., High-affinity IL-2Rα expressed by Tregs mitigates the potential of IL-2 use in cancer therapy. Here, the authors fuse IL-2 with an NKDG2 binding domain, and show that it induces IL-2 signalling selectively in NKG2D+ cells, delaying tumour growth in mice without the side effects of conventional IL-2 therapy.

Bombesin is a 14–amino-acid amphibian peptide that binds with high affinity to the gastrin-releasing peptide receptor (GRPR), which is overexpressed on a variety of solid tumors. It has been demonstrated that bombesin analogs can be radiolabeled with a variety of radiometals for potential diagnosis and treatment of GRPR-positive tumors. In this regard, several studies have used different chelators conjugated to the 8 C-terminal amino acids of bombesin(7-14) for radiolabeling with 64 Cu. These analogs have demonstrated GRPR-specific small-animal PET of tumors but have various advantages and disadvantages. The objective of this study was to conjugate the previously described (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) (SarAr) chelator to bombesin (7-14), radiolabel the conjugate with 64 Cu, and evaluate in vitro and in vivo. Methods: SarAr was synthesized as previously published and conjugated to bombesin(7-14) by solid-phase peptide synthesis using standard Fmoc chemistry. Succinic acid (SA), 8-aminooctanoic acid (Aoc), and Gly-Ser-Gly (GSG) were used as linkers between SarAr and bombesin(7-14) to yield the resulting SarAr-SA-Aoc-bombesin(7-14) and SarArSA-Aoc-GSG-bombesin(7-14) peptides. The unlabeled peptides were evaluated in a competitive binding assay using PC-3 prostate cancer cells and 125 I-Tyr 4 -bombesin to determine the inhibitory concentration of 50%. The peptides were radiolabeled with 64 Cu and evaluated for internalization into PC-3 cells in vitro and for in vivo tumor uptake in mice bearing PC-3 xenografts using biodistribution and small-animal PET/CT studies. Results: The competitive binding assay demonstrated that both SarAr-SA-Aoc-bombesin(7-14) and SarAr-SA-Aoc-GSGbombesin(7-14) bound with high affinity to GRPR with an inhibitory concentration of 50% of 3.5 and 4.5 nM, respectively. Both peptides were radiolabeled with 64 Cu at room temperature without further purification and demonstrated similar internalization into PC-3 cells. In vivo, the radiolabeled peptides demonstrated tumor-specific uptake (13.0 and 8.5 percentage injected dose per gram for 64 Cu-SarAr-SA-Aoc-bombesin(714) and 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14), respectively, at 1 h) and imaging that was comparable to, or better than, that of the previously reported 64Cu-labeled bombesin analogs. The 64Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) had more rapid blood clearance and lower tumor and normal-tissue uptake than 64Cu-SarAr-SA-Aoc-bombesin(7-14), resulting in similar tumor-to-blood ratios for each analog (15.1 vs. 11.3 for 64Cu-SarAr-SA-Aoc-bombesin(7-14) and 64Cu-SarAr-SA-AocGSG-bombesin(7-14), respectively, at 1 h). Conclusion: These studies demonstrate that 64 Cu-SarAr-SA-Aoc-bombesin(7-14) and 64 Cu-SarAr-SA-Aoc-GSG-bombesin(7-14) bound with high affinity to GRPR-expressing cells and that these peptides can be used for PET of GRPR-expressing prostate cancer.

A series of multivalent, functional polymer nanoparticles with diagnostic/imaging units and targeting ligands for molecular targeting were synthesized with the loading of the chain-end-functionalized GRGDS peptide targeting sequence (model system based on integrin α(v)β(3)) ranging from 0 to 50%. Accurate structural and functional group control in these systems was achieved through a modular approach involving the use of multiple functionalized macromonomer/monomer units combined with living free radical polymerization. In cellulo results show an increase in uptake in α(v)β(3) integrin-positive U87MG glioblastoma cells with increasing RGD loading and a possible upper limit on the effectiveness of the number of RGD peptides for targeting α(v)β(3) integrin. Significantly, this increased targeting efficiency is coupled with in vivo biodistribution results, which show decreased blood circulation and increased liver uptake with increasing RGD loading. The results demonstrate the importance of controlling ligand loading in order to achieve optimal performance for therapeutic and imaging applications for multivalent nanoparticle-based systems.

The goal of this work was to study the efficacy of the positron emission tomography (PET) tracers 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 64Cu-diacetyl-bis(N4-methylthiosemicarbazone) ([64Cu]ATSM) and in monitoring placental and fetal functional response to acute hyperoxia in late-term pregnant mice subjected to experimentally induced chronic hypoxia. E15 mice were maintained at 12% inspired oxygen for 72 hours and then imaged during oxygen inhalation with either [18F]FDG to monitor nutrient transport or 64Cu-ATSM to establish the presence of hypoxia. Computed tomography (CT) with contrast allowed clear visualization of both placentas and fetuses. The average ratio of fetal to placental [18F]FDG uptake was 0.45 ± 0.1 for the hypoxic animals and 0.55 ± 0.1 for the normoxic animals, demonstrating a significant decrease (p = .0002) in placental function in dams exposed to chronic hypoxic conditions. Hypoxic placentas and fetuses retained more 64Cu-ATSM compared to normoxic placentas and fetuses. Herein we report first-in-mouse PET imaging of fetuses employing both tracers [18F]FDG (metabolism) and 64Cu-ATSM (hypoxia). [18F]FDG PET/CT imaging allowed clear visualization of placental-fetal structures and supported quantification of tracer uptake, making this a sensitive tool for monitoring placental function in preclinical rodent models. These measurements illustrate the potentially irreversible damage generated by chronic exposure to hypoxia, which cannot be corrected by acute exposure to hyperoxia.

Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2-specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with (89)Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of (89)Zr-pertuzumab in HER2-expressing BT-474 and HER2-nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2-expressing cells. In vivo evaluation of (89)Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. (89)Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of (89)Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that (89)Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin–cytoskeleton interactions of β2integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin–cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of αIIbβ3-integrin, in which activation results in a large conformational change. Direct activation of αIIbβ3by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin–cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both α- and β-cytoplasmic domains were required for these links. This suggests that 1) both β2- and β3-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin–cytoskeleton interactions; and 3) the role of the α-subunit in integrin–cytoskeleton interactions in at least some circumstances is more direct than generally supposed.

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxy-terminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named proteins linking IAP with cytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

CD47 functions as a marker of “self” by inhibiting phagocytosis of autologous cells. CD47 has been shown to be overexpressed by various tumor types as a means of escaping the antitumor immune response. The goal of this research was to investigate the utility of CD47 imaging using positron emission tomography (PET) in both human xenograft and murine allograft tumor models. Anti-CD47 antibodies were conjugated with p -isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89 Zr. We employed xenograft and allograft small-animal models of cancer in biodistribution and PET imaging studies to investigate the specificity and PET imaging robustness of CD47. Ab-Df-Bz-NCS conjugates were labeled with 89 Zr with specific activity of 0.9 to 1.6 μCi/μg. Biodistribution studies in the xenograft and allograft model showed similar specific tumor uptake of the antihuman and antimouse CD47 antibodies. However, the tracer retention in the liver, spleen, and kidneys was significantly higher in the allograft-bearing animals, suggesting uptake mediated by the CD47 normally expressed throughout the reticular endothelial system. CD47, a marker of “self,” was evaluated as a diagnostic PET biomarker in xenograft and allograft cancer animal models. CD47 imaging is feasible, warranting further studies and immunoPET tracer development.

The goal of this study was to determine the specificity of ⁶⁴Cu-CB-TE2A-c(RGDyK) (⁶⁴Cu-RGD) for osteoclast-related diseases, such as Paget's disease or rheumatoid arthritis.C57BL/6 mice were treated systemically with osteoprotegerin (OPG) for 15 days or RANKL for 11 days to suppress and stimulate osteoclastogenesis, respectively. The mice were then imaged by positron emission tomography/computed tomography using ⁶⁴Cu-RGD, followed by determination of serum TRAP5b and bone histology. Standard uptake values were determined to quantify ⁶⁴Cu-RGD in bones and other tissues.Mice treated with OPG showed decreased bone uptake of ⁶⁴Cu-RGD at 1, 2, and 24 h post-injection of the tracer (p 0.01 for all time points) compared to vehicle controls, which correlated with a post-treatment decrease in serum TRAP5b. In contrast, mice treated with RANKL showed significantly increased bone uptake at 2 h post-injection of (⁶⁴Cu-RGD (p 0.05) compared to the vehicle control group, corresponding to increased serum TRAP5b and OC numbers as determined by bone histology.These data demonstrate that ⁶⁴Cu-RGD localizes to areas in bone with increased osteoclast numbers and support the use of ⁶⁴Cu-RGD as an imaging biomarker for osteoclast number that could be used to monitor osteoclast-related pathologies and their treatments.

Despite advances in cancer treatment over the past few decades, metastatic disease remains the primary cause of morbidity and mortality. Recent reports suggest the formation of a “premetastatic niche” before the metastatic cascade, where niche is defined as the microenvironment for tumor cells to be able to engraft and proliferate at secondary sites. Bone marrow–derived (BMD) cells that express vascular endothelial growth factor receptor–1 and very late antigen–4 (VLA-4) have been shown to arrive at sites of metastasis to form a receptive environment for tumor cells. Here we describe experiments toward imaging of VLA-4–positive BMD cells using a high-affinity PET probe, (64)Cu-labeled 11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (CB-TE2A)-LLP2A. METHODS: VLA-4–negative MDA-MB-231/firefly luciferase (fluc) human breast tumor cells were injected intraarterially in the left ventricle in nude mice. Tumor metastasis in mice was monitored for 30 d by bioluminescence imaging and small-animal PET/CT. Small-animal PET images were collected 2 h after mice were injected in the tail vein with (64)Cu-CB-TE2A-LLP2A (5.6–11.1 MBq [150–300 μCi; specific activity, 400 μCi/μg]). Cellular uptake of (64)Cu-CB-TE2A-LLP2A was determined in VLA-4–positive B16F10 mouse melanoma cells and VLA-4–negative MDA-MB-231/fluc human breast cancer tumor cells. Biodistribution experiments in nude mice bearing VLA-4–positive B16F10 subcutaneous tumors in the flank were conducted to validate targeting of VLA-4–positive cells in vivo. RESULTS: Uptake of (64)Cu-CB-TE2A-LLP2A was higher in VLA-4–positive human melanoma B16F10 cells than in VLA-4–negative MDA-MB-231 cells (P < 0.05). In B16F10 tumor–bearing mice, (64)Cu-CB-TE2A-LLP2A had high uptake in the VLA-4–rich organs marrow, spleen, and tumor (11.26% ± 2.59%, 8.36% ± 2.15%, and 3.09% ± 0.58% injected dose/g, respectively). Cumulative standardized uptake value data from 2 independent studies (n = 7 and 8 mice) on nude mice implanted with VLA-4–negative MDA-MB-231/fluc human breast tumor cells suggested an influx of VLA-4–positive BMD cells that corresponded to metastasis (P < 0.05). Immunohistochemical analysis and flow cytometry also showed upregulation of VLA-4–positive cell clusters and BMD cells at the metastatic sites, providing evidence for noninvasive imaging of BMD cells in the premetastatic niche. CONCLUSION: The results of the study demonstrated the potential of PET with VLA-4–targeted (64)Cu-CB-TE2A-LLP2A to visualize BMD cell reorganization and expansion noninvasively in vivo.

This article describes the evaluation of the radiopharmaceutical (64)Cu-CB-TE2A-c(RGDyK) ((64)Cu-RGD) as an imaging agent for osteolytic bone metastases and their associated inflammation by targeting of the alpha(v)beta(3) integrin on osteoclasts and the proinflammatory cells involved at the bone metastatic site.The (64)Cu-RGD radiotracer was evaluated in the transgenic mouse expressing Tax (Tax(+)), which spontaneously develops osteolytic tumors throughout the vertebrae and hind limbs, using biodistribution studies and small-animal PET/CT. Histologic analysis was also performed on Tax(+) mouse tails, using hematoxylin and eosin and tartrate-resistant acid phosphatase to confirm the presence of osteolytic bone lesions and the presence of osteoclasts, respectively. Additionally, a proof-of-principle study was conducted with a small group of Tax(+) animals presenting with osteolytic lesions. These animals were treated with the bisphosphonate zoledronic acid and imaged with (64)Cu-RGD to determine whether this radiopharmaceutical was sensitive enough to detect a response to the bisphosphonate therapy.Biodistribution studies using (64)Cu-RGD demonstrated that Tax(+) mice between the ages of 6 and 12 mo had a greater accumulation of activity in their tail vertebrae than did the wild-type (WT) cohort (P = 0.013). Additionally, Tax(+) mice between the ages of 6 and 12 mo had significantly more tracer activity associated with their tail vertebrae than did Tax(+) mice older than 12 mo (P = 0.003), suggesting that earlier bone metastases cause an increased recruitment of alpha(v)beta(3)-expressing cells. Small-animal PET/CT with (64)Cu-RGD was conducted on Tax(+) and WT mice. On the basis of standardized uptake value analysis, Tax(+) mice had approximately 2-fold more tail-associated activity than did WT animals (P = 0.0157). Additionally, decreases in uptake were observed in the tails of Tax(+) mice after treatment with the osteoclast inhibitor zoledronic acid, and histologic analysis of Tax(+) mouse-tail vertebrae revealed the presence of Tax(+) tumor cells, osteoclasts, and proinflammatory cells within the bone microenvironment.Together, these data suggest that (64)Cu-RGD has the potential to effectively image osteolytic bone metastases and monitor the physiologic changes in the bone metastatic microenvironment after osteoclast-inhibiting bisphosphonate therapy.

Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed. Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 ± 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. Conclusion: The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor–positive tumors.

Biomedical imaging techniques such as skeletal survey and (18)F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30-50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α(4)β(1) integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, (64)Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P