CD11b Antibodies Research Articles

Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Oxygen Therapy After Spinal Cord Injury Reduces Spinal Microglial Inflammatory Signalling

Spinal cord injury (SCI) interrupts blood flow, causing O2 partial pressure near the site of injury to decrease to very low levels. Spinal hypoxia contributes to tissue necrosis and neuroinflammation, leading to secondary injury. The majority of prior O2 therapy studies after SCI use hyperbaric O2 (HBO), and considerable evidence suggests HBO decreases spinal inflammation and neurodegeneration. HBO consists of 100% O2 (hyperoxia), which is easy to implement, at elevated pressure (hyperbaria), which is challenging to implement. Using an adult rat model of cervical SCI (C2 hemilesion), we compared the impact of daily HBO vs. normobaric hyperoxia therapy on the pro-inflammatory profile of spinal microglia isolated via immunomagnetic cell separation. Rats (n=3 per group) were given C2 hemilesion, followed by 14 days of normobaric hyperoxia (100% O2, 1 hour/day) or HBO (100% O2, 3 ATA, 1 hour/day). The C2-C6 spinal cord was harvested for immunomagnetic microglial isolation using CD11b antibodies. We screened pro-inflammatory gene expression with a PCR array (Rat Toll-Like Receptor Signaling Pathway, Qiagen). Both types of O2 therapy impacted suppressed microglial pro-inflammatory gene expression. In particular, subunit expression of the canonical transcription factor orchestrating microglial inflammatory responses, NFkB2, was significantly reduced. Downregulation of NFkB after HBO has been previously reported, but this is the first evidence for downregulation of NFkB after normobaric O2, and the first evidence for a microglia-specific response. Other pro-inflammatory genes with reduced expression after O2 therapy include: Clec4e, IL1β and CD14. Clec4e is as a transmembrane pattern recognition receptor and “necrotic cell sensor”. Thus, Clec4e downregulation after O2 therapy suggests a shift towards reduced necrosis after SCI. These initial results indicate that both HBO and normobaric hyperoxia treatments reduce the spinal microglia pro-inflammatory responses to cervical SCI, possibly leading to increased cell survival. HL153140 (DDF), HL147554 (GSM), HL149800 (DDF), McKnight Brain Institute (DDF, GSM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

#2709 THE INVOLVEMENT OF NRF2-MEDIATED ANTIOXIDANT SYSTEM IN MYELOPEROXIDASE-POSITIVE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS

Abstract Background and Aims Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs). During AAV, activated neutrophils form NETs via reactive oxygen spices (ROS)s-mediated signaling pathway. The excessive NETs lead to endothelial vascular damage and serve as autoantigens against ANCAs in a vicious cycle. Although the role of NETs in AAV has been revealed, its treatment for targeting NETs remains unestablished. Since nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant proteins against oxidative stress and functions as a defense system, we investigated the role of Nrf2 in the development of AAV. Method In vitro, human neutrophils treated with the Nrf2 activator bardoxolone methyl (Bard) were stimulated with ANCA, and the effects on NETs formation and signaling pathways including intracellular ROS were evaluated by cell imaging and biochemical approaches. The effects of Nrf2 activators on NETs-induced cytotoxicity were examined using human renal glomerular endothelial cells (HRGECs). Nrf2 knockout or WT mouse-derived neutrophils were stimulated with anti-MPO antibody to induce NETs. In vivo studies, Bard was administered to ANCA-transfer AAV model or spontaneous AAV developing mice models (SCG/Kj). Renal function was assessed by biochemical analysis, and the expression of ROS and antioxidant protein NAD(P)H Quinone Dehydrogenase 1 (NQO1) in tissues was evaluated by fluorescent immunostaining and immunoblotting. The formation of NETs in the glomeruli was evaluated by immunostaining and glomerular vascular endothelial damage was assessed by the expression of CD31 and TUNEL positivity. To assess the role of Nrf2 in immunological abnormality during AAV, murine splenocytes were collected and flow cytometry were conducted using CD4, CD8, B220, MCHCII, CD11c, F4/80, Ly 6G, Ly6b, and CD11b antibodies. Moreover, to assess the genetic role of Nrf2 in AAV, we established SCG/Kj mice carrying a germ-line deletion of the Nrf2-genes. A spontaneous mouse model of vasculitis genetically deficient in Nrf2 was analyzed by the same parameters to evaluate the effect of Nrf2. N1 offspring mice were generated by crossing Nrf2-/- mice with SCG/Kj mice. These backcrossed mice were obtained by crossing Nrf2-/- mice to background SCG/Kj mice for three generations. The experimental group was mated to N4 heterozygous mice, and female mice in the experimental group were analyzed at 18 weeks. Results In vitro, pharmacological activation of Nrf2 suppressed ANCA-induced NETs via the upregulation of NQO1 and inhibition of ROS. While, Nrf2-deficienet neutrophils showed the massive NETs in response to ANCA, compared to WT neutrophils. Furthermore, Nrf2 activation directly protected endothelium from cellular injury caused by the exposure of ANCA-mediated NETs. In vivo, the pharmacological activation of Nrf2 ameliorated glomerulonephritis in two kinds of AAV models through the upregulation of antioxidant and inhibition of ROS-mediated NETs. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in spontaneous AAV model. The activation of Nrf2 regulated the expansion of splenocytes in AAV mice. In particular, pan-T cells in spleen and the infiltration of Th17 cells in kidney were suppressed by Nrf2 activation. Conclusion During ANCA-stimulation in neutrophils, the activation of Nrf2 initiates antioxidant system and inhibits intracellular ROS, leading to the suppression of NETs formation. Nrf2 may be a novel therapeutic target for AAV.

Development of flow cytometry analysis on measuring tumour immune microenvironment (TIME) in mice bearing EMT6 tumour model

We presented a flow cytometry protocol to isolate and measure the cellular component of tumour immune microenvironment (TIME) of mice bearing breast cancer models. The immune cells infiltrating the tumour microenvironment (TME) have been getting more attention as the determination of their pro- or/and anti-tumorigenic activity contributes to cancer treatment success. In addition, our group has a strong interest to investigate the infiltration of eosinophils within the TME as their tumorigenic role is still unclear. This study aims to measure the immune cells infiltrating the TME, including eosinophils using flow cytometry with 6-colour detection. The single-cell suspensions derived from tumour sections of mice bearing EMT6 tumour model were harvested (n=4) and treated with CD45, Siglec-F, I-A/I-E, CD 11b, and Ly6G antibodies. A gating protocol was used based on the reported publications. Results showed that immune cells in the tumour section were detected by positive staining to CD45. Neutrophils were isolated based on Ly6G+, while the remaining granulocytes, such as macrophage and dendritic cells (DC), were isolated based on I-A/I-E+SCChi, and eosinophils on the positive expression of Siglec F. Overall, our tumour model presented with the highest percentage of neutrophils (63.05 ± 5.61%), while eosinophils constitute 1.64 ± 0.75% of the total population. In conclusion, our flow cytometric protocol with 6 colour detection are able to isolate and measure several immune cells within the TME, including eosinophils from the tumour section.

Notoginsenoside R1-loaded mesoporous silica nanoparticles targeting the site of injury through inflammatory cells improves heart repair after myocardial infarction

Notoginsenoside R1 (NGR1) is the main monomeric component extracted from the dried roots and rhizomes of Panax notoginseng, and exerts pharmacological action against myocardial infarction (MI). Owing to the differences in compound distribution, absorption, and metabolism in vivo, exploring a more effective drug delivery system with a high therapeutic targeting effect is crucial. In the early stages of MI, CD11b-expressing monocytes and neutrophils accumulate at infarct sites. Thus, we designed a mesoporous silica nanoparticle-conjugated CD11b antibody with loaded NGR1 (MSN-NGR1-CD11b antibody), which allowed NGR1 precise targeted delivery to the heart in a noninvasively manner. By increasing targeting to the injured myocardium, intravenous injection of MSN-NGR1-CD11b antibody nanoparticle in MI mice improved cardiac function and angiogenesis, reduced cell apoptosis, and regulate macrophage phenotype and inflammatory factors and chemokines. In order to further explore the mechanism of NGR1 protecting myocardium, cell oxidative stress model and oxygen-glucose deprivation (OGD) model were established. NGR1 protected H9C2 cells and primary cardiomyocytes against oxidative injury induced by H2O2 and OGD treatment. Further network pharmacology and molecular docking analyses suggested that the AKT, MAPK and Hippo signaling pathways were involved in the regulation of NGR1 in myocardial protection. Indeed, NGR1 could elevate the levels of p-Akt and p-ERK, and promote the nuclear translocation of YAP. Furthermore, LY294002 (AKT inhibitor), U0126 (ERK1/2 inhibitor) and Verteporfin (YAP inhibitor) administration in H9C2 cells indicated the involvement of AKT, MAPK and Hippo signaling pathways in NGR1 effects. Meanwhile, MSN-NGR1-CD11b antibody nanoparticles enhanced the activation of AKT and MAPK signaling pathways and the nuclear translocation of YAP at the infarcted site. Our research demonstrated that MSN-NGR1-CD11b antibody nanoparticle injection after MI enhanced the targeting of NGR1 to the infarcted myocardium and improved cardiac function. More importantly, our pioneering research provides a new strategy for targeting drug delivery systems to the ischemic niche.

M-CSF-stimulated myeloid cells can convert into epithelial cells to participate in re-epithelialization and hair follicle regeneration during dermal wound healing.

Dermal wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells which compose of a significant proportion of the inflammatory cells infiltrate to the to a wound site where they play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have the capacity to be converted into fibroblast-like cells and endothelial cells during wound healing process. However, whether myeloid cells in wounds can convert into epithelial cells where they contribute to healing process is not clear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result showed that during the healing process, some of the CD45-positive hematopoietic cells also expressed keratin 14, a marker for keratinocytes. Further, double immunofluorescent staining in dermal wounds, using CD11b and K14 antibodies indicated that CD11b-positive myeloid cells were the origin of newly generated epithelial cells. Through tracing injected labeled splenocyte-derived myeloid cells in skin, we confirmed that myeloid cells were able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provided new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration through conversion of M-CSF-stimulated CD11b-positive myeloid cells into epithelial cells in a murine model.

Abstract 5985: Marrow immune landscape participates dormancy and activation of disseminated tumor cells in a breast cancer bone metastasis model

Abstract Breast cancer has the highest incidence rate among women and the second-highest mortality rate in cancer-related death in the world. Although the research on breast cancer treatment has made some progress, metastatic breast cancer is still incurable. Bone is the most common site for breast cancer metastasis. This work is dedicated to the use of mass cytometry and single cell analysis to explore the molecular alterations in the marrow immune microenvironment which participates the dormancy and activation of disseminated tumor cells (DTCs). Firstly, four Balb/c mouse breast cancer bone metastasis groups were established. Animals were divided into intracardiac injection group (n=10), subcutaneous injection group (n=10), tumor resection and suture group (n=10), and control group (n=10). In the intracardiac injection group, a breast cancer bone metastatic model was constructed by directly injecting mCherry-labeled 4T1 cells into the left ventricle. After detecting obvious bone metastasis by bioluminescence imaging (BLI), femur and tibia bone marrow were excised for flow cytometry cell sorting, mass cytometry and single cell analysis. Both the subcutaneous and the resection and suture groups were injected with mCherry-labeled 4T1 cells into the right-back of Balb/c mice. In the tumor resection and suture group, the tumor was removed at the time when the subcutaneous tumor was just palpable. In the subcutaneous injection group, the animals were sacrificed when tumor volume reached 1 cubic centimeter. The classification of bone marrow cells was determined by using fluorescent dye labeled CD45, CD4, CD8, CD19, NK1.1, CD11b, CD11c, IA/IE, Ly6C, Ly6G and other antibodies. Constructed as a local immune landscape in the marrow, certain numbers of B cells and T cells, in the bone marrow of mice with breast cancer bone metastasis were significantly less than those in control mice, indicating that the acquired immunity of the mice with bone metastasis is diminished. In addition, the proportions of CD11b+ myeloid cells and neutrophils in the marrow of mice with bone metastasis were significantly higher than that in the control group, whereas the proportion of macrophages and eosinophils were all significantly decreased indicating that the bone marrow immune environment has changed in the process of tumor occurrence and metastasis. Research is ongoing, including ex-marrow spleen cell analysis in those animals, to explore the mechanism of the interaction between the immune microenvironment and tumor cells that determines tumor heterogeneity and sensitivity to therapy. This work was supported by NSFC projects 81773146, 81972766, 81972420; Shenzhen Science and Technology Commission Project JCYJ20180302174235893; and College students' innovative entrepreneurial training program (S202114325028) Citation Format: Yingxuan Gong, Yinghao Wang, Xinyu Jiang, Xiuzhi Li, Yueming Li, Ming Chang, Yi Lu, Jian Zhang. Marrow immune landscape participates dormancy and activation of disseminated tumor cells in a breast cancer bone metastasis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5985.

Abstract WMP23: Increased Central And Peripheral Monocytes/ Macrophages In Female Mice With Cerebral Amyloid Angiopathy

Introduction: Cerebral amyloid angiopathy (CAA) is a known cause of cognitive impairment and intracerebral hemorrhage in the elderly. Cerebral microbleeds (CMBs) detected on MRI are the diagnostic hallmarks of this disease. Recent studies have shown increased CMBs and worst cognition in female CAA mice as compared to males. The underlying mechanisms for this sexual dichotomy are unclear. We hypothesized that increased inflammatory response in females contributes to neuronal damage and cognitive decline in CAA. Methods: C57BL6 Tg-SwDI male and female mice (12 months) were used as a mouse model for CAA. Peripheral blood and brain tissues were collected and single cell suspensions were acquired for immunophenotyping using flow cytometry. We used CD45, CD11b, Ly6C and CD3 antibodies to gate for monocytes/macrophages (CD45 high CD11b + Ly6C + ) and T cells (CD45 high CD11b - CD3 + ) respectively. Data was analyzed using two sample t-test on GraphPad PRISM. Results: Our results demonstrated that in the peripheral blood, Tg-SwDI female mice had significantly increased frequency of monocytes/macrophages (CD45 + CD11b + Ly6C + ) as compared to males (females, 64±8.1%, n=10 vs. males, 33.9±6.9%, n=8, p=0.01). Similar to peripheral blood, there were significantly increased monocytes/macrophages (CD45 high CD11b + Ly6C + ) in the female brain (females, 36.5±6.3%, n=10, vs. males, 20.9±2.7%, n=8, p= 0.04). No significant differences were seen in T cells in the blood (females, 27±4.3% vs. males, 26.9±3.3%, p= 0.9) or brain (57.2±6.8% vs. 65.3±5.4%, p= 0.3). Conclusion: Recent studies suggest that vascular Aβ accumulation in CAA causes an innate inflammatory response, leading to further cognitive decline and CMB formation. Our results highlight a monocyte/macrophage driven pro-inflammatory milieu in female CAA mice which may contribute to worse outcomes. Regulation of monocytes/ macrophages and their cytokine-mediated inflammatory pathways may be a potential sex specific therapeutic target in CAA.

Bionic Microbubble Neutrophil Composite for Inflammation-Responsive Atherosclerotic Vulnerable Plaque Pluripotent Intervention

Rupture or erosion of inflammatory atherosclerotic vulnerable plaque is essential to acute coronary events, while the target intervene of vulnerable plaque is very challenging, due to the relatively small volume, high hemodynamic shear stress, and multifactorial nature of the lesion foci. Herein, we utilize the biological functionality of neutrophil and the versatility of microbubble in the acoustic field, to form Neu-balloon through CD11b antibody binding. The Neu-balloon inherits the advantage of neutrophils on firming the endothelium adhesion even at shear stress up to 16 dyne/cm2 and also maintains the acoustic enhancement property from the microbubble, to accumulate at atherosclerotic lesions under acoustic in an atherosclerotic Apo E−/− mice model. Interestingly, Neo-balloon also has high and broad drug loading capacity, which enables the delivery of indocyanine green and miR-126a-5p into vulnerable plagues in vivo. Overall, the bionic Neu-balloon holds great potential to boost on-demand drug transportation into plaques in vivo.

Deficiency of Leukocyte Surface Glycoproteins Mol, LFA-1, and Leu M5 in a Dog With Recurrent Bacterial Infections: An Animal Model

A dog with severe recurrent bacterial infections, impaired pus formation, delayed wound healing, and severe persistent leukocytosis was the result of a mother-son mating. Assessment of leukocyte function revealed profound abnormalities in adherence-dependent activities including impaired granulocyte adhesion to glass/plastic surfaces or nylon wool, decreased granulocyte aggregation and chemotaxis, and diminished lymphocyte blastogenesis, but normal neutrophil oxidative activity, serum immunoglobulin, and complement levels. By immunofluorescence analysis, CD11b and CD18 monoclonal antibodies specific for the 155-kd alpha polypeptide of Mo1 (gp 155, 94) and the 94 kd beta peptide common to Mo1, LFA-1 (gp 170, 94), and Leu M5 (p 150, 94) (surface molecules that promote leukocyte adhesion) failed to bind to unstimulated and A23187 calcium ionophore-stimulated granulocytes or mononuclear cells of the affected dog as compared with strong specific binding to canine control cells. The Mo1 glycoproteins were only barely detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of immunoprecipitates from lysates of 125I surface-labeled neutrophils from the affected dog as compared with intense bands seen with canine control cell precipitates. We conclude that this dog has a severe leukocyte surface glycoprotein deficiency syndrome that is similar, if not identical, to that recently recognized in humans. Dogs with deficiency of leukocyte Mo1, LFA-1, and Leu M5 expression may represent a useful animal model to characterize further the molecular basis for an inherited disorder in leukocyte effector function.

Assessment of 18F-PBR-111 in the Cuprizone Mouse Model of Multiple Sclerosis

The study aims to assess site assessment of the performance of 18F-PBR-111 as a neuroinflammation marker in the cuprizone mouse model of multiple sclerosis (MS). 18F-PBR-111 PET imaging has not been well evaluated in multiple sclerosis applications both in preclinical and clinical research. This study will help establish the potential utility of 18F-PBR-111 PET in preclinical MS research and future animal and future human applications. 18F-PBR-111 PET/CT was conducted at 3.5 weeks (n = 7) and 5.0 weeks (n = 7) after cuprizone treatment or sham control (n = 3) in the mouse model. A subgroup of mice underwent autoradiography with cryosectioned brain tissue. T2 weighted MRI was performed to obtain the brain structural data of each mouse. 18F-PBR-111 uptake was assessed in multiple brain regions with PET and autoradiography images. The correlation between autoradiography and immunofluorescence staining of neuroinflammation (F4/80 and CD11b) was measured. Compared to control mice, significant 18F-PBR-111 uptake in the corpus callosum (p < 0.001), striatum (caudate and internal capsule, p < 0.001), and hippocampus (p < 0.05) was identified with PET images at both 3.5 weeks and 5.0 weeks, and validated with autoradiography. No significant uptake differences were detected between 3.5 weeks and 5.0 weeks assessing these regions as a whole, although there was a trend of increased uptake at 5.0 weeks compared to 3.5 weeks in the CC. High 18F-PBR-111 uptake regions correlated with microglial/macrophage locations by immunofluorescence staining with F4/80 and CD11b antibodies. 18F-PBR-111 uptake in anatomic locations correlated with activated microglia at histology in the cuprizone mouse model of MS suggests that 18F-PBR-111 has potential for in vivo evaluation of therapy response and potential for use in MS patients and animal studies.

Investigation of neuro-inflammatory parameters in a cuprizone induced mouse model of multiple sclerosis.

Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.

Analysis of canine myeloid-derived suppressor cells (MDSCs) utilizing fluorescence-activated cell sorting, RNA protection mediums to yield quality RNA for single-cell RNA sequencing

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.

Tumor-associated macrophages derived from cancer stem cells

Macrophages are the most abundant immune cells in the microenvironment of solid tumors. The present study displayed histological and immunohistochemical analyses of a malignant tumor model developed from cancer stem cells (CSCs) converted from human induced pluripotent stem cells (hiPSCs) in a cancer microenvironment prepared from the conditioned medium (CM) of a pancreatic cancer cell line. We focused on the localization and the origin of tumor-associated macrophages (TAMs), To the best of our knowledge this may be the first study to suggest the potential differentiation of CSCs to TAMs.hiPSCs were converted into CSCs in the presence of CM from PK8 cells. CSCs were then transplanted in vivo and formed primary tumors. Primary cultures for these tumors were serially transplanted again to obtain secondary tumors. Secondary tumors exhibited histopathological features of malignancy. Cells derived from tumors maintained the expression of endogenous stemness markers and pancreatic CSCs markers. Simultaneously, high immunoreactivity to anti-mouse CD68, anti-human CD68, CD206 and CD11b antibodies were detected revealing that the tumor tissue derived from CSCs was enriched for macrophages which can originate from both human and mouse cells.The model of CSCs highlighted the possibility of CSCs to differentiate into TAMs.

Abstract 3421: Identifying mechanisms of immune evasion in microsatellite instable endometrial cancers

Abstract Inherited or sporadic defects in mismatch repair genes (MLH1, MSH2, MSH6, and PSM2) can lead to microsatellite instable (MSI) tumors, most commonly colon, endometrial or gastric tumors. High mutation rates in MSI tumors have been associated with greater immunogenicity, but these tumors can evade immune response. Recently developed mouse models of MSI endometrial cancer (EC) were used to evaluate immune surveillance mechanisms. Uterine-targeted MSH2 knockout (PR-Cre+MSH2flox/flox) mice were characterized and determined that 22% of mice develop spontaneous EC by 12-16 months of age. Microsatellite instability was evaluated by PCR. Immune infiltration was quantified by immunohistochemistry (IHC) using FoxP3 (Tregs), CD11b (myeloid cells) and CD8 (tumor infiltrating lymphocytes, TILs) antibodies. Tumors were divided into TILHigh and TILLow (&amp;lt;5 TIL/mm2). Comprehensive transcriptome analysis of tumor and normal controls was performed using the Affymetrix Clariom D assay. A subset of candidate gene changes were validated by RT-PCR and at the protein level by IHC. Cell lines were generated from spontaneous tumors and used for orthotopic syngeneic tumor studies of advanced EC. Immune infiltration in orthotopic tumors was characterized using flow cytometry. All endometrial tumors from PR-Cre+MSH2flox/flox mice were MSI and included endometrioid, serous, and mixed histologies. Tumors showed varying degrees of immune infiltrate, irrespective of histology. Of 7 tumors, 4 (57%) were TILHigh and 3 (43%) were TILLow. Transcriptome analysis showed TILHigh tumors upregulated innate immunity related pathways including, pattern recognition-associated factors, Type I IFN signaling molecules and CXCL5. Activation of adaptive immunity was observed by an increase in T cell chemoattractants, CXCL9 and CCL21. TILHigh tumors displayed at least a 2-fold increase in MHC class I molecules and CD86 needed for T cell activation. IL1A, CSF1, and TGFB2 (cytokines involved in immunosuppressive cell recruitment and induction) were also highly expressed. IHC confirmed co-localization of TILs with myeloid cells and Tregs in TILHigh tumors. TILLow tumors had decreased expression of several members in the IFN family responsible for initiating innate responses. Primary cell lines from TILHigh and TILLow tumors used in an orthotopic syngeneic mouse model retained the immune profile of the original tumor. Understanding mechanisms of immune evasion is critical for improving immunotherapy. This model of MSI EC reflects the spectrum of immunogenicity observed in clinical studies. TILHigh tumors express a wide range of factors involved in innate and adaptive anti-tumor responses, while TILLow tumors lack key mediators needed for immune activation. These new models for MSI EC will be essential to test the efficacy of immunotherapies and study mechanisms of resistance. Citation Format: Brenda Melendez, Emily Hinchcliff, Nisha Gokul, Elizabeth Whitley, Russell R. Broaddus, Rosemarie E. Schmandt, Karen H. Lu, Melinda S. Yates. Identifying mechanisms of immune evasion in microsatellite instable endometrial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3421.

GEFITINIB EFFECTS IN A NPM1-MUTATED ACUTE MYELOID LEUKEMIA CELL LINE

Objective In 2018, leukemias were among the 10 most common cancers in Brazil. The main oral manifestation of leukemias is gingival enlargement. Current evidence supports that gefitinib, an epidermal growth factor receptor inhibitor, is able to revert the undifferentiated phenotype of leukemia cell lines and to inhibit cell proliferation. However, the possible effects of gefitinib in the treatment of patients with mutation in the NPM1 (nucleophosmin) gene, the most frequent genetic alteration in acute myeloid leukemia (AML), were not previously reported. Therefore, in the present work, we evaluate the effects of gefitinib on the differentiation, proliferation, and death rates of NPM1-mutated OCI-AML3 cell line. Study Design The flow cytometric analysis of CD11b, CD11c, Ki-67, and annexin-V antibodies were performed in OCI-AML3 cells treated with gefitinib. Results Crescent doses of gefitinib are able to efficiently trigger apoptosis and decrease the proliferation index of OCI-AML3 cells after 24, 48, and 72 hours of treatment, but it did not induce the CD11b and CD11c expression. Conclusions Our results demonstrate that gefitinib treatment sensitizes OCI-AML cells in a dose- and time-dependent manner. Future studies are necessary to define if gefitinib can be helpful in the treatment of the patients with AML. FAPESP (2016/02713-2 and 2017/00775-3).

Systematic Identification of Cell-Cell Communication Networks in the Developing Brain.

SummarySince the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in ∼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable “Brain interactome explorer”, available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.

Isolation and RNA purification of macrophages/microglia from the adult mouse spinal cord

The isolation of a highly pure and healthy population of macrophages from the CNS in a short time is essential for studying the role of these cells in injury and disease. Current methods rely either on the use of gradients and/or sorting, processes that result either in impure, reduced viability and/or altered populations of cells. Furthermore, RNA extraction after immunopanning is often difficult. Here, a technique combining both gradient isolation and immunopanning to generate highly pure cultures of primary macrophages isolated from the injured adult CNS in <2 h is described. An optimized protocol of a commercially-available RNA extraction kit is also outlined for the isolation of highly pure RNA. A hybridoma cell-line producing CD11b antibody was used to recover activated (CD11b+) macrophages/microglia from the CNS after injury in <2 h with >95% purity.