Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival

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.

In Vivo Quantitative Assessment of Therapeutic Response to Bortezomib Therapy in Disseminated Animal Models of Multiple Myeloma With [18F]FDG and [64Cu]LLP2A PET

BackgroundMultiple myeloma (MM) is a disease of cancerous plasma cells. Current treatments have improved the survival rate; however, most MM patients relapse. Imaging based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4) is over expressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]LLP2A for quantitative PET imaging in MM models of variable VLA4 expression, and following bortezomib therapy.MethodsIn vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (CG: luciferase and green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no-tumor) were longitudinally imaged with [64Cu]LLP2A (2-3 MBq; Molar Activity: 44.14±1.40 MBq/nmol) and [18F]FDG (7.4-8.0 MBq) PET respectively.ResultsFlow cytometry confirmed high expression of CD49d in U266 cells (>99%) and moderate expression in MM.1S cells (~52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31) and femur (0.72 versus 0.39) at week 4 post tumor inoculation. In U266-CG treated versus untreated mice, there was a 4-time percent [64Cu]LLP2A increase in spine at week 3. Compared to [64Cu]LLP2A, [18F]FDG PET detected treatment related changes at later time points.Conclusion[64Cu]LLP2A is a promising tracer for in vivo assessment of therapeutic response in disseminated models of MM.

Genetically engineered cell membrane–coated nanoparticles for targeted delivery of dexamethasone to inflamed lungs

As numerous diseases are associated with increased local inflammation, directing drugs to the inflamed sites can be a powerful therapeutic strategy. One of the common characteristics of inflamed endothelial cells is the up-regulation of vascular cell adhesion molecule–1 (VCAM-1). Here, the specific affinity between very late antigen–4 (VLA-4) and VCAM-1 is exploited to produce a biomimetic nanoparticle formulation capable of targeting inflammation. The plasma membrane from cells genetically modified to constitutively express VLA-4 is coated onto polymeric nanoparticle cores, and the resulting cell membrane–coated nanoparticles exhibit enhanced affinity to target cells that overexpress VCAM-1 in vitro. A model anti-inflammatory drug, dexamethasone, is encapsulated into the nanoformulation, enabling improved delivery of the payload to inflamed lungs and significant therapeutic efficacy in vivo. Overall, this work leverages the unique advantages of biological membrane coatings to engineer additional targeting specificities using naturally occurring target-ligand interactions.

Detection of Human Cytomegalovirus Proteins in Paraffin-Embedded Breast Cancer Tissue Specimens—A Novel, Automated Immunohistochemical Staining Protocol

Emerging evidence supports a significant association between human cytomegalovirus (HCMV) and human malignancies, suggesting HCMV as a human oncomodulatory virus. HCMV gene products are found in >90% of breast cancer tumors and seem to be correlated with more aggressive disease. The definitive diagnosis of HCMV relies on identification of virus inclusions and/or viral proteins by different techniques including immunohistochemical staining. In order to reduce biases and improve clinical value of HCMV diagnostics in oncological pathology, automation of the procedure is needed and this was the purpose of this study. Tumor specimens from 115 patients treated for primary breast cancer at Akershus University Hospital in Norway were available for the validation of the staining method in this retrospective study. We demonstrate that our method is highly sensitive and delivers excellent reproducibility for staining of HCMV late antigen (LA), which makes this method useful for future routine diagnostics and scientific applications.

Virus sensing receptors in cellular infectivity of influenza A virus

An innate immune response is essential to mobilize protective immunity upon the infection of respiratory epithelial cells with influenza A virus (IAV). The response is classified as early (nonspecific effectors), local systematic (effector cells recruitment) and late (antigen to lymphoid organ transport, naive B and T cells recognition, effector cells clonal expansion and differentiation). Virus particles are detected by the host cells as non-self by various sensors that are present on the cell surface, endosomes and cytosol. These sensors are collectively termed as pattern recognition receptors (PRRs). The PRRs distinguish unique molecular signatures known as pathogen-associated molecular pattern, which are present either on the cell surface or within intracellular compartments. PRRs have been classified into five major groups: C-Type Lectin Receptor (CLR), Toll-like receptor (TLR), Nod-like receptor (NLR), Retinoic acid-inducible gene-I-like receptor (RLR), which play a role in innate immunity to IAV infection, and the pyrin and hematopoietic interferon-inducible nuclear (PYHIN) domain protein. Here, we discuss the role of PRRs in cellular infectivity of IAV and highlight the recent progress.

Integrin VLA-4 as a PET imaging biomarker of hyper-adhesion in transgenic sickle mice

In sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion–associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4–positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4–mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.