Preclinical immunoPET imaging of glioblastoma-infiltrating myeloid cells using Zirconium-89-labeled anti-CD11b antibody

AbstractPurposeGlioblastoma is a lethal brain tumor, heavily infiltrated by tumor-associated myeloid cells (TAMCs). TAMCs are emerging as a promising therapeutic target as they suppress antitumor immune responses and promote tumor cell growth. Quantifying TAMCs using non-invasive immunoPET could facilitate patient stratification for TAMC-targeted treatments and monitoring of treatment efficacy. As TAMCs uniformly express the cell surface marker, integrin CD11b, we evaluated a89Zr-labeled anti-CD11b antibody for non-invasive imaging of TAMCs in a syngeneic orthotopic mouse glioma model.ProceduresA human/mouse cross-reactive anti-CD11b antibody (clone M1/70) was conjugated to a DFO chelator and radiolabeled with Zr-89. PET/CT and biodistribution with or without a blocking dose of anti-CD11b Ab were performed 72 hours post-injection of89Zr-anti-CD11b Ab in mice bearing established orthotopic syngeneic GL261 gliomas. Flow cytometry and immunohistochemistry of dissected GL261 tumors were conducted to confirm the presence of CD11b+TAMCs.ResultsSignificant uptake of89Zr-anti-CD11b Ab was detected at the tumor site (SUVmean = 2.60 ± 0.24) compared with the contralateral hemisphere (SUVmean = 0.6 ± 0.11). Blocking with a 10-fold lower specific activity of89Zr-anti-CD11b Ab markedly reduced the SUV in the right brain (SUVmean = 0.11 ± 0.06), demonstrating specificity. Spleen and lymph nodes (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.ConclusionThese data establish that89Zr-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.

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TMIC-39. DEVELOPMENT OF CD11b TRACER FOR THE IMMUNE PET IMAGING IN GLIOBLASTOMA MODEL - COULD BE A GAME CHANGER FOR IMMUNOTHERAPY APPROACHES

Neuro-Oncology ◽

10.1093/neuonc/noz175.1073 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi256-vi256

Author(s):

Shubhanchi Nigam ◽

Lauren McCarl ◽

Rajeev Kumar ◽

Carolyn Anderson ◽

Barry Edwards ◽

...

Keyword(s):

Flow Cytometry ◽

Clinical Trials ◽

Pet Imaging ◽

Specific Activity ◽

Malignant Gliomas ◽

Myeloid Cells ◽

Cd11b Expression ◽

Left Brain ◽

Right Brain ◽

The Right

Abstract Glioblastoma is a lethal brain tumor, heavily infiltrated by tumor-associated myeloid cells (TAMCs). As up to 30% of a glioma cellular mass may be attributed to immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). TAMCs impede natural and immunotherapy-driven anti-tumor responses, they are a high-priority and promising therapeutic target currently being evaluated in clinical trials. Multiple preclinical and clinical trials have attempted to target these cells, however monitoring of biologic responses to therapy remains a challenge. Quantifying real time status of MDSCs and TAMs at the tumor site using non-invasive immunoPET could improve therapeutic response and allow for better patient stratification and monitoring of targeted treatment responses. TAMCs highly expressed the cell surface marker, integrin CD11b (Mac-1, αMβ2) and may be a highly effective imaging target for immunoPET strategies. The human/mouse cross-reactive anti-CD11b antibody (clone M1/70) was radiolabeled with 89Zr for PET imaging. PET/CT imaging, with or without a blocking dose of anti-CD11b Ab, was performed in mice bearing established orthotopic syngeneic GL261 gliomas. Flow cytometry and histology in tissues collected from post-imaging biodistribution validated targeting of CD11b+ MDSCs and TAMs. There was significant Zr-89-anti-CD11b Ab uptake in the tumor ipsilateral right brain (SUVmean = 2.6 ± 0.24) compared to contralateral left brain (SUVmean = 0.6 ± 0.11). Blocking with 10-fold lower specific activity 89Zr-anti-CD11b Ab reduced the SUV in right brain with (SUVmean = 0.11 ± 0.06). Immune rich organs spleen and lymph nodes showed high uptake. These results correlated with biodistribution analysis. CD11b expression in the right and left brain were validated using flow cytometry, H&E and IHC, showing high CD11b expression in the right brain. Imaging TAMs and MDSCs with 89Zr-labeled anti-CD11b Ab targeting was validated in a mouse model of malignant gliomas, demonstrating the feasibility of monitoring immune response during immunotherapy.

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CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Blood ◽

10.1182/blood.v116.21.1452.1452 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1452-1452

Author(s):

Tiffany Simms-Waldrip ◽

Michelle Yoonha Cho ◽

Kenneth Dorshkind ◽

Kathleen M Sakamoto

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Cell Proliferation ◽

Research Funding ◽

Myeloid Cells ◽

Myeloid Cell ◽

Wild Type ◽

In Vivo Models ◽

Hematopoietic Stem

Abstract Abstract 1452 The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment. The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 106) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 106) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations. At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 106 cells/mL and 1.3 × 106 cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics. Disclosures: Sakamoto: Abbott Laboratories, Inc.: Research Funding; Genentech, Inc.: Research Funding.

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Surface APRIL Is Elevated on Myeloid Cells and Is Associated with Disease Activity in Patients with Rheumatoid Arthritis

The Journal of Rheumatology ◽

10.3899/jrheum.140630 ◽

2015 ◽

Vol 42 (5) ◽

pp. 749-759 ◽

Cited By ~ 9

Author(s):

Abby Jones Weldon ◽

Ioana Moldovan ◽

Marven G. Cabling ◽

Elvin A. Hernandez ◽

Sheri Hsu ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Flow Cytometry ◽

Disease Activity ◽

Mononuclear Cells ◽

Myeloid Cells ◽

Myeloid Cell ◽

Monocyte Subsets ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Highly Correlated

Objective.To assess surface APRIL (a proliferation-inducing ligand; CD256) expression by circulating myeloid cells in rheumatoid arthritis (RA) and to determine its relationship to disease activity.Methods.Peripheral blood mononuclear cells (PBMC) and plasma were obtained from patients with RA and healthy donors. PBMC were stained for flow cytometry to detect surface APRIL and blood cell markers to identify circulating myeloid cell subsets. Based on CD14 and CD16 phenotypes, monocyte subsets described as classical (CD14+CD16−), intermediate (CD14+CD16+), and nonclassical (CD14loCD16+) were identified. Levels of surface APRIL expression were measured by flow cytometry and median fluorescence intensity was used for comparisons. Levels of soluble APRIL in the plasma were determined by ELISA. Disease activity was measured by the Disease Activity Score in 28 joints.Results.In patients with RA, total myeloid cells showed expression of surface APRIL that correlated with disease activity and with plasma APRIL levels observed in these patients. In healthy donors, classical monocytes were composed of > 80% of circulating monocytes. However, in patients with RA, the intermediate and nonclassical subsets were elevated and made up the majority of circulating monocytes. In contrast to healthy donors, where high levels of surface APRIL were only observed in nonclassical monocytes, patients with RA showed high levels of surface APRIL expression by all circulating monocyte subsets.Conclusion.Surface APRIL is elevated in circulating myeloid cells in patients with RA where it is highly correlated with disease activity. Patients with RA also showed skewing of monocytes toward subsets associated with secretion of tumor necrosis factor-α and/or interleukin 1β.

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A Rapid Automated Method for the Sequential Isolation of CD19, CD3 and Myeloid Cells from One Tube of Whole Blood.

Blood ◽

10.1182/blood.v110.11.4867.4867 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4867-4867

Author(s):

Karina L. McQueen ◽

Maureen Fairhurst ◽

Melany Nauer ◽

Jenna L. Warren ◽

Allen C. Eaves ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Magnetic Nanoparticles ◽

T Cell ◽

B Cell ◽

Whole Blood ◽

Genomic Dna ◽

Myeloid Cells ◽

Myeloid Cell ◽

Post Transplantation

Abstract Immune ablation followed by allogeneic hematopoietic cell transplantation in humans necessitates hematopoietic cell reconstitution and immune re-education. All blood cell lineages are affected and post-transplantation immune restoration depends upon the graft’s ability to generate both lymphoid and myeloid lineage cells. Decisions regarding immunomodulation treatment post-transplantation are often made on the basis of chimerism testing. Chimerism analysis is typically performed on small blood samples, especially with pediatric patients. Since lymphoid and myeloid engraftment is asynchronous the determination of lineage-specific chimerism is needed. Analysis of purified cell subsets requires techniques which can isolate >1 cell type from a single small starting sample. This avoids dividing the sample. Performing flow cytometry as well as isolation of DNA from the purified subsets means that high cell recovery is essential. Preparation of the sample using a ficoll-based method often results in cell loss of 50% while certain lysis and wash steps can affect granulocyte content. We describe a method of sequential selections to isolate B cells, then T cells and finally myeloid lineages (CD33+ and/or CD66b+) using a fully automated pipetting robot called RoboSep®. RoboSep® can process sample sizes that range from 0.5 to 4.25 ml of human whole blood. CD19 (B cell) positive and CD3 (T cell) positive and myeloid cell fractions are isolated using immunomagnetic, column-free positive selection (EasySep®). Briefly, cells are first labeled with antibody targeting CD19 positive cells. These are then coupled to magnetic nanoparticles and the sample is placed in a magnet. The supernatant with unlabeled cells is removed to a new tube, leaving isolated CD19 positive cells in the magnet. The supernatant is then labeled with anti-CD3 antibody, magnetic nanoparticles, placed in a magnet and the supernatant is removed to a new tube leaving isolated CD3 positive cells. Finally, a cocktail of antibodies (anti-CD33, anti-CD66b) is used to label and select the myeloid cells from the supernatant. The resultant positive cells are collected in the magnet. Assessment by flow cytometry yields average purities over 90% for all cell types. Cell isolation using this method produces highly purified cells in quantities sufficient to generate genomic DNA for chimerism testing, even from very small amounts of starting sample. For example, 2.0 ml of whole blood yields on average 1.3ug of B cell genomic DNA, 10.2ug of T cell genomic DNA and 6.1ug of myeloid cell genomic DNA. In conclusion, we have developed a rapid, fully automated RoboSep® method to sequentially isolate highly purified B cells, then T cells and finally myeloid cells from a single starting sample of whole blood. The number of cells (x106) and amount of total genomic DNA (range) obtained from 2.0 ml of whole blood starting sample (n=3). No. Enriched Cells Total DNA (ug) CD19+ 0.12 – 0.34 1.1 – 1.6 CD3+ 1.8 – 3.2 7.9 – 11.9 Myeloid 2.2 – 2.9 4 – 7.2

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Targeting CSF-1 ameliorates experimental autoimmune encephalomyelitis by depleting inflammatory monocytes and microglia in the central nervous system without affecting quiescent microglia

10.1101/2020.10.23.352534 ◽

2020 ◽

Author(s):

Daniel Hwang ◽

Larissa Lumi Watanabe Ishikawa ◽

Alexandra Boehm ◽

Ziver Sahin ◽

Giacomo Casella ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Experimental Autoimmune Encephalomyelitis ◽

Myeloid Cells ◽

Myeloid Cell ◽

Autoimmune Encephalomyelitis ◽

Inflammatory Monocytes ◽

The Central Nervous System ◽

Spleen And Lymph Nodes ◽

Experimental Autoimmune

ABSTRACTMultiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by extensive infiltration of myeloid cells into the central nervous system (CNS). Although myeloid cells are essential to MS/EAE pathology, none of the current MS therapies specifically target them. A promising strategy for bridging this gap may be targeting the biological activity of CSF-1R, a receptor tyrosine kinase important for survival and functioning of certain myeloid cells, such as monocytes and macrophages. It has been shown that CSF-1R inhibitors suppress EAE, but it is not known whether targeting CSF-1R ligands, CSF-1 and IL-34, could be a viable therapeutic strategy. We found that neutralization of CSF-1 with Ab attenuates ongoing EAE, similar to CSF-1R inhibitor BLZ945, whereas neutralization of IL-34 had no effect. Both anti-CSF-1- and BLZ945-treated mice with EAE had greatly diminished numbers of monocyte-derived dendritic cells and microglia in the CNS. However, anti-CSF-1 antibody selectively depleted inflammatory microglia, whereas BLZ945 depleted virtually all microglia, including quiescent microglia. We also found depletion of myeloid cells in the spleen and lymph nodes of anti-CSF-1- and BLZ945-treated mice, but only a modest decrease in encephalitogenic T cell responses, suggesting that the depletion of CNS myeloid cells is more relevant to EAE suppression. Decreased myeloid cell populations in treated mice resulted in reduced production of IL-1β, a key inflammatory mediator in EAE. The treatments also reduced the frequencies of CCL2- and CCR2-expressing cells in the CNS, suggesting that CSF-1/CSF-1R inhibition may hinder recruitment of immune cells to the CNS. Our findings suggest that targeting CSF-1 may be effective in ameliorating myeloid cell-mediated MS pathology, while preserving homeostatic functions of microglia and decreasing risks that might arise from their ablation with small molecule inhibitors of CSF-1R.

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Non-Invasive Molecular Survey of Sarcoptic Mange in Wildlife: Diagnostic Performance in Wolf Faecal Samples Evaluated by Multi-Event Capture–Recapture Models

Pathogens ◽

10.3390/pathogens10020243 ◽

2021 ◽

Vol 10 (2) ◽

pp. 243

Author(s):

Julieta Rousseau ◽

Mónia Nakamura ◽

Helena Rio-Maior ◽

Francisco Álvares ◽

Rémi Choquet ◽

...

Keyword(s):

Molecular Detection ◽

High Specificity ◽

Sarcoptes Scabiei ◽

Sarcoptic Mange ◽

Diagnostic Specificity ◽

Non Invasive ◽

Faecal Samples ◽

Capture Recapture ◽

The Mean ◽

Event Capture

Sarcoptic mange is globally enzootic, and non-invasive methods with high diagnostic specificity for its surveillance in wildlife are lacking. We describe the molecular detection of Sarcoptes scabiei in non-invasively collected faecal samples, targeting the 16S rDNA gene. We applied this method to 843 Iberian wolf Canis lupus signatus faecal samples collected in north-western Portugal (2006–2018). We further integrated this with serological data (61 samples from wolf and 20 from red fox Vulpes vulpes, 1997–2019) in multi-event capture–recapture models. The mean predicted prevalence by the molecular analysis of wolf faecal samples from 2006–2018 was 7.2% (CI95 5.0–9.4%; range: 2.6–11.7%), highest in 2009. The mean predicted seroprevalence in wolves was 24.5% (CI95 18.5–30.6%; range: 13.0–55.0%), peaking in 2006–2009. Multi-event capture–recapture models estimated 100% diagnostic specificity and moderate diagnostic sensitivity (30.0%, CI95 14.0–53.0%) for the molecular method. Mange-infected individually identified wolves showed a tendency for higher mortality versus uninfected wolves (ΔMortality 0.150, CI95 −0.165–0.458). Long-term serology data highlights the endemicity of sarcoptic mange in wild canids but uncovers multi-year epidemics. This study developed and evaluated a novel method for surveying sarcoptic mange in wildlife populations by the molecular detection of S. scabiei in faecal samples, which stands out for its high specificity and non-invasive character.

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Identification of Tumor-Specific MRI Biomarkers Using Machine Learning (ML)

Diagnostics ◽

10.3390/diagnostics11050742 ◽

2021 ◽

Vol 11 (5) ◽

pp. 742

Author(s):

Rima Hajjo ◽

Dima A. Sabbah ◽

Sanaa K. Bardaweel ◽

Alexander Tropsha

Keyword(s):

Machine Learning ◽

High Specificity ◽

Cancer Biomarkers ◽

Cancer Prognosis ◽

Current Status ◽

Imaging Data ◽

Non Invasive ◽

Machine Learning Methods ◽

Cancer Types ◽

Magnetic Resonance Imaging Mri

The identification of reliable and non-invasive oncology biomarkers remains a main priority in healthcare. There are only a few biomarkers that have been approved as diagnostic for cancer. The most frequently used cancer biomarkers are derived from either biological materials or imaging data. Most cancer biomarkers suffer from a lack of high specificity. However, the latest advancements in machine learning (ML) and artificial intelligence (AI) have enabled the identification of highly predictive, disease-specific biomarkers. Such biomarkers can be used to diagnose cancer patients, to predict cancer prognosis, or even to predict treatment efficacy. Herein, we provide a summary of the current status of developing and applying Magnetic resonance imaging (MRI) biomarkers in cancer care. We focus on all aspects of MRI biomarkers, starting from MRI data collection, preprocessing and machine learning methods, and ending with summarizing the types of existing biomarkers and their clinical applications in different cancer types.

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A novel transcript encoding an N-terminally truncated AML1/PEBP2 alphaB protein interferes with transactivation and blocks granulocytic differentiation of 32Dcl3 myeloid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4133 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4133-4145 ◽

Cited By ~ 50

Author(s):

Y W Zhang ◽

S C Bae ◽

G Huang ◽

Y X Fu ◽

J Lu ◽

...

Keyword(s):

Fetal Liver ◽

Myeloid Cells ◽

Myeloid Cell ◽

Protein Product ◽

Terminal Region ◽

Human Leukemia ◽

Granulocytic Differentiation ◽

Runt Domain ◽

Rnase Protection ◽

Novel Transcript

The gene AML1/PEBP2 alphaB encodes the alpha subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. We have identified a new isoform of the AML1 transcript, termed AML1 deltaN, in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1 deltaN transcript was detected in various hematopoietic cell lines of lymphoid to myeloid cell origin, as revealed by RNase protection and reverse transcriptase PCR analyses. The protein product of AML1 deltaN lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the beta subunit. However, AML1 deltaN was found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1 deltaN in 32Dcl3 myeloid cells blocked granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1 deltaN acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1.

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