Multiple myeloma (MM) is a plasma B-cell hematologic cancer that causes significant skeletal morbidity. Despite improvements in survival, heterogeneity in response remains a major challenge in MM. Cluster of differentiation 38 (CD38) is a type II transmembrane glycoprotein overexpressed in myeloma cells and is implicated in MM cell signaling. Daratumumab is a U.S. Food and Drug Administration-approved high-affinity monoclonal antibody targeting CD38 that is clinically benefiting refractory MM patients. Here, we evaluated [Zr]Zr-desferrioxamine (DFO)-daratumumab PET/CT imaging in MM tumor models. Daratumumab was conjugated to DFO--benzyl-isothiocyanate (DFO-Bz-NCS) for radiolabeling with Zr. Chelator conjugation was confirmed by electrospray ionization-mass spectrometry, and radiolabeling was monitored by instant thin-layer chromatography. Daratumumab was conjugated to Cyanine5 (Cy5) dye for cell microscopy. In vitro and in vivo evaluation of [Zr]Zr-DFO-daratumumab was performed using CD38 human myeloma MM1.S- (MM1.S) cells. Cellular studies determined the affinity, immunoreactivity, and specificity of [Zr]Zr-DFO-daratumumab. A 5TGM1- (5TGM1)/KaLwRij MM mouse model served as control for imaging background noise. [Zr]Zr-DFO-daratumumab PET/CT small-animal imaging was performed in severe combined immunodeficient mice bearing solid and disseminated MM tumors. Tissue biodistribution (7 d after tracer administration, 1.11 MBq/animal, = 4-6/group) was performed in wild-type and MM1.S tumor-bearing mice. A specific activity of 55.5 MBq/nmol (0.37 MBq/μg) was reproducibly obtained with [Zr]Zr-daratumumab-DFO. Flow cytometry confirmed CD38 expression (>99%) on the surface of MM1.S cells. Confocal microscopy with daratumumab-Cy5 demonstrated specific cell binding. Dissociation constant, 3.3 nM (±0.58), and receptor density, 10.1 fmol/mg (±0.64), was obtained with a saturation binding assay. [Zr]Zr-DFO-daratumumab/PET demonstrated specificity and sensitivity for detecting CD38 myeloma tumors of variable sizes (8.5-128 mm) with standardized uptake values ranging from 2.1 to 9.3. Discrete medullar lesions, confirmed by bioluminescence images, were efficiently imaged with [Zr]Zr-DFO-daratumumab/PET. Biodistribution at 7 d after administration of [Zr]Zr-DFO-daratumumab showed prominent tumor uptake (27.7 ± 7.6 percentage injected dose per gram). In vivo blocking was achieved with a 200-fold excess of unlabeled daratumumab. [Zr]Zr-DFO- and Cy5-daratumumab demonstrated superb binding to CD38 human MM cells and significantly low binding to CD38 cells. Daratumumab bioconjugates are being evaluated for image-guided delivery of therapeutic radionuclides.

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The cerebellum is known for its crossed activation pattern with the contralateral cerebral hemisphere during language functional magnetic resonance imaging (fMRI) tasks in healthy patients. Crossed cerebro-cerebellar activation has been previously shown to occur in patients with brain tumors not affecting the activation areas. However, the presence of a tumor in left Broca's area in the inferior frontal gyrus is known to disrupt cerebral activation during language tasks. This study investigated if crossed cerebro-cerebellar activation patterns for language tasks would still occur in such patients. A total of 43 right-handed patients with a glioma affecting left Broca's area were examined for their cerebral and cerebellar activation during an fMRI language task. Only 13 of the 43 patients exhibited crossed cerebro-cerebellar activation patterns. Statistically significant differences of atypical cerebro-cerebellar activation patterns were found between cerebral right-dominant (RD) and cerebral co-dominant (CD) (p < 0.001) as well as cerebral RD and cerebral left-dominant (LD) patients (p < 0.01), while no differences were found when patients were divided based on cerebellar dominance (p > 0.75) or tumor grade (p > 0.5). No relation was found between the cerebellar and cerebral laterality index (LI) values (ρ = - 0.20; p = 0.21). Atypical activation patterns are suspected to have been caused by the tumor, perhaps a result of contralateral reorganization in some cases and false negative activation in left Broca's area from neurovascular uncoupling (NVU) in others. Cerebellar activation may also potentially indicate cerebral false negative behavior and future cerebral contralateral reorganization.

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4β1) is a key player in cell–cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

Purpose: Cluster of differentiation 38 (CD38) is a promising therapeutic target in multiple myeloma (MM) patients and has resulted in the development of several CD38 immunotherapies. Current methods to evaluate CD38 expression in the preclinical setting include ex vivo flow cytometry and immunohistochemistry, which can be cumbersome and do not give whole-body information. In vivo imaging technologies such as positron emission tomography rely on decay of radioisotopes, limiting the number of molecular interactions observed at any given time point. Here, we demonstrate the use of near-infrared (NIR) fluorescence imaging for spatiotemporal monitoring of CD38 expression in preclinical MM using the anti-CD38 daratumumab (DARA) conjugated to the NIR fluorophore IRDye800CW (DARA-IRDye800).Procedures: Stability studies with human serum and binding assays with human myeloma cells were performed with DARA-IRDye800. Immunocompromised mice with intra-and extramedullary tumors (n = 5/group) were administered with DARA-IRDye800 for in vivo imaging up to 7 days after injection. Ex vivo biodistribution and flow cytometry studies were performed to validate in vivo imaging results. A separate therapy study was performed in mice with intramedullary tumors that were treated and not treated with DARA at a therapeutic dose (n = 7/group). DARA-IRDye800 was administered for subsequent in vivo and ex vivo imaging in both cohorts of mice.Results: DARA-IRDye800 maintained stability and had high affinity for CD38 (K D = 3.5 ± 0.05 nM). DARA-IRDye800 demonstrated a 5-and 18-fold increase in contrast in tumor-bearing regions of mice with extra-and intramedullary MM. Finally, mice treated with therapeutic