Front Physiol 9:170C170

Front Physiol 9:170C170. 2.60 0.24) compared with the contralateral hemisphere (SUVmean = 0.6 0.11). Blocking with a 10-fold lower specific activity of 89Zr-anti-CD11b Ab markedly reduced the SUV in the right brain (SUVmean = 0.11 0.06), demonstrating specificity. Spleen and lymph nodes BAY 61-3606 dihydrochloride (myeloid cell rich organs) also showed high uptake of the tracer, and biodistribution analysis correlated with the imaging results. CD11b expression within the tumor was validated using flow cytometry and immunohistochemistry, which showed high CD11b expression primarily in the tumoral hemisphere compared to the contralateral hemisphere with very little accumulation in normal brain. Conclusion These data establish that 89Zr-anti-CD11b Ab immunoPET targets CD11b+ cells (TAMCs) with high specificity in a mouse model of GBM, demonstrating the potential for non-invasive quantification of tumor infiltrating CD11b+ immune cells during disease progression and immunotherapy in patients with GBM. BAY 61-3606 dihydrochloride single cell sequencing and mass cytometry), and histology on surgically removed tumors [15C18]. While these approaches directly measure TAMC levels, they lack the temporal and spatial resolution of non-invasive diagnostic imaging. Although MRI is usually a powerful diagnostic tool for GBM, it lacks the capacity to directly quantify the status of immune cell populations throughout the tumor microenvironment [19]. Positron-emission tomography (PET) provides the real-time status of tumor-infiltrating immune cells during disease progression and treatment. Using appropriate radiotracers, PET may be able to improve therapeutic responses by selecting early-phase responders and guiding the adjustment of treatment strategies. There is currently an unmet need for PET tracers to quantify the highly dynamic populations of tumor-infiltrating immune cells. To overcome this limitation, we have validated a 89Zr-labeled anti-human/mouse CD11b antibody for targeting both MDSCs and TAMCs in an orthotopic mouse GBM model by immunoPET. The same strategy as presented here in mouse models will ultimately be applied with anti-human CD11b antibodies in patients with GBM. Materials and Methods Reagents All chemicals were purchased from Sigma-Aldrich Chemicals (St. Louis, MO) unless otherwise specified. Aqueous solutions were prepared using ultrapure water (resistivity, 18.2 M?cm). Para-NCS-Bz-DFO was purchased from CheMatech (Dijon, France). All antibodies for flow cytometry were purchased from BioLegend (San Diego, CA). Zirconium-89 oxalate was purchased from Washington University (St. Louis, MO). Instrumentation Radiochemistry reaction progress and BAY 61-3606 dihydrochloride purity were monitored using BIOSCAN ITLC (Eckert & Ziegler, Germany) and an Agilent 1260 infinity HPLC (Agilent Technologies, Santa Clara, CA) using Superose? 12 10/300 GL SEC column (GE Healthcare, Chicago, IL). MRI data were acquired on a Bruker ClinScan 7 T MRI (Billerica, MA). Biodistribution samples were counted using a PerkinElmer 2470 WIZARD2 Automatic Gamma Counter (Waltham, MA). PET/CT data were acquired on an Inveon Preclinical Imaging Station (Siemens Medical Solutions, Knoxville, TN). Flow cytometry studies were performed on a BD LSRII (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (BD Biosciences). IHC and hematoxylin and eosin (H&E) images were acquired using a Leica DFC7000T microscope (Leica Microsystems Inc). Anti-CD11b Ab conjugation with a p-NCS-Bz-DFO chelator A para-NCS-Bz-DFO stock solution (10 mM) was prepared in dry DMSO. A portion of the para-NCS-Bz-DFO stock (4.5 l, 45 nmole) was added to anti-CD11b Ab (clone M1/70, 0.5 mg/500 l, 3 nanomoles). The alkalinity was adjusted to pH~9 by the addition of sodium carbonate (5 l, 0.1 M Na2CO3). After 18 h (at 4C), the reaction mixture was applied to a desalting column (MWCO 50,000) and eluted with buffer (0.5 M HEPES, pH 7.4). The fractions containing the DFO-anti-CD11b conjugate were pooled, concentrated, and stored BAY 61-3606 dihydrochloride at ?20C. Size exclusion chromatography (SEC) was performed using PBS as a mobile phase to assess the purity of the conjugate. Radiolabeling Zirconium-89 oxalate (74 MBq) was added to a labeling buffer (equal volumes of 1 1 M HEPES (100 L) and 1 M Na2CO3 (100 L). Then, DFO-anti-CD11b Ab (0.2 nmol in 1M HEPES) was added to the mixture for radiolabeling (1 h, 37C). The reaction was monitored by instant thin-layer chromatography (ITLC; BIODEX, green Ab strips, New York, NY) and MMP15 purified with a PD-10 desalting column.