GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation

Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo, and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro compared to unsorted and GP2- fractions, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminated the formation of teratoma in vivo. This study establishes that the GP2-enriched PPs represent a safe option for T1D treatment.

The Molecular Basis of Different Approaches for the Study of Cancer Stem Cells and the Advantages and Disadvantages of a Three-Dimensional Culture

Cancer stem cells (CSCs) are a rare tumor subpopulation with high differentiation, proliferative and tumorigenic potential compared to the remaining tumor population. CSCs were first discovered by Bonnet and Dick in 1997 in acute myeloid leukemia. The identification and isolation of these cells in this pioneering study were carried out through the flow cytometry, exploiting the presence of specific cell surface molecular markers (CD34+/CD38−). In the following years, different strategies and projects have been developed for the study of CSCs, which are basically divided into surface markers assays and functional assays; some of these techniques also allow working with a cellular model that better mimics the tumor architecture. The purpose of this mini review is to summarize and briefly describe all the current methods used for the identification, isolation and enrichment of CSCs, describing, where possible, the molecular basis, the advantages and disadvantages of each technique with a particular focus on those that offer a three-dimensional culture.

Tolerogenic Immunotherapy: Targeting DC Surface Receptors to Induce Antigen-Specific Tolerance

Dendritic cells (DCs) are well-established as major players in the regulation of immune responses. They either induce inflammatory or tolerogenic responses, depending on the DC-subtype and stimuli they receive from the local environment. This dual capacity of DCs has raised therapeutic interest for their use to modify immune-activation via the generation of tolerogenic DCs (tolDCs). Several compounds such as vitamin D3, retinoic acid, dexamethasone, or IL-10 and TGF-β have shown potency in the induction of tolDCs. However, an increasing interest exists in defining tolerance inducing receptors on DCs for new targeting strategies aimed to develop tolerance inducing immunotherapies, on which we focus particular in this review. Ligation of specific cell surface molecules on DCs can result in antigen presentation to T cells in the presence of inhibitory costimulatory molecules and tolerogenic cytokines, giving rise to regulatory T cells. The combination of factors such as antigen structure and conformation, delivery method, and receptor specificity is of paramount importance. During the last decades, research provided many tools that can specifically target various receptors on DCs to induce a tolerogenic phenotype. Based on advances in the knowledge of pathogen recognition receptor expression profiles in human DC subsets, the most promising cell surface receptors that are currently being explored as possible targets for the induction of tolerance in DCs will be discussed. We also review the different strategies that are being tested to target DC receptors such as antigen-carbohydrate conjugates, antibody-antigen fusion proteins and antigen-adjuvant conjugates.

Abstract 16226: Single-cell RNA Sequencing Coupled With Optical Imaging for Targeted Real-time Visualization of the Cardiac Conduction System

Introduction: Optical imaging has the potential to revolutionize cardiothoracic surgery by allowing for the real-time visualization of structures often inadvertently damaged due to inadequate visibility. The cardiac conduction system (CCS) consists of specialized cells embedded within the heart that are essential for cardiac function yet indistinguishable from heart muscle tissue. Intraoperative CCS injury is a major complication in cardiac surgery, representing a significant source of morbidity and mortality. To date, there exists no intraoperative method to visualize the CCS. Hypothesis: We hypothesized that unique, CCS-specific cell surface markers could be used for the in vivo labelling of the CCS. Objectives: Use single-cell RNA sequencing (scRNAseq) to discover cell surface markers that may serve as the basis for generating optical imaging agents for real-time CCS visualization. Methods/Results: Gene expression analysis of a comprehensive scRNAseq dataset of the entire murine CCS revealed significant enrichment of a host of CCS-specific cell surface genes. A subset of genes were subsequently validated in the CCS of mice and/or human tissue. In total, 7 novel cell surface markers were confirmed to have unique expression patterns throughout or within distinct components of the CCS. Next, optical imaging agents were created consisting of a near-infrared (NIR) dye conjugated to antibodies directed against two distinct CCS-specific cell surface markers. Each optical imaging agent demonstrated high sensitivity and specificity in labeling the entire CCS in vivo following a single intravenous injection in mice. Specificity was confirmed within intact, whole hearts using both closed-field NIR imaging and whole mount immunolabeling with volume imaging (iDISCO+). Dosage, timecourse and biodistribution analyses were performed as well as safety validation by surface ECG. Conclusions: In summary, we coupled scRNAseq with optical imaging to create novel tools for the real-time visualization of a complex tissue substructure. We provide a proof-of-principle for broadening the scope of optical imaging but also address a significant unmet clinical need, laying the foundation for translational opportunities in cardiac intervention and imaging.

Wiring the Brain by Clustered Protocadherin Neural Codes

There are more than a thousand trillion specific synaptic connections in the human brain and over a million new specific connections are formed every second during the early years of life. The assembly of these staggeringly complex neuronal circuits requires specific cell-surface molecular tags to endow each neuron with a unique identity code to discriminate self from non-self. The clustered protocadherin (Pcdh) genes, which encode a tremendous diversity of cell-surface assemblies, are candidates for neuronal identity tags. We describe the adaptive evolution, genomic structure, and regulation of expression of the clustered Pcdhs. We specifically focus on the emerging 3-D architectural and biophysical mechanisms that generate an enormous number of diverse cell-surface Pcdhs as neural codes in the brain.

Disulfide-Linked Peptides for Blocking BTLA/HVEM Binding

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14–39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.

68Ga PSMA PET-CT: New Hope in Prostate Cancer Imaging and Therapy

Prostate cancer is the second most common cancer in men. Its diagnosis and management are still challenging as both PSA levels and imaging have had their limitations. Prostate specific membrane antigen (PSMA) is aprostate specific cell surface protein and has been emerging as a novel target for PET imaging of prostate cancer. 68Ga PSMA PET-CT imaging have been shown as a better imaging technology in primary prostate cancer and at the same time detecting lymph node and bone metastases even in low PSA levels. Moreover, its counterpart 177 Lu PSMA has a great prospectto be used for the treatment of castration resistantprostate cancer. Bangladesh J. Nuclear Med. 22(1): 53-57, Jan 2019

Neutrophil plasticity in the tumor microenvironment

Neutrophils act as the body’s first line of defense against infection and respond to diverse inflammatory cues, including cancer. Neutrophils display plasticity, with the ability to adapt their function in different inflammatory contexts. In the tumor microenvironment, neutrophils have varied functions and have been classified using different terms, including N1/N2 neutrophils, tumor-associated neutrophils, and polymorphonuclear neutrophil myeloid–derived suppressor cells (PMN-MDSCs). These populations of neutrophils are primarily defined by their functional phenotype, because few specific cell surface markers have been identified. In this review, we will discuss neutrophil polarization and plasticity and the function of proinflammatory/anti-inflammatory and protumor/antitumor neutrophils in the tumor microenvironment. We will also discuss how neutrophils with the ability to suppress T-cell activation, referred to by some as PMN-MDSCs, fit into this paradigm.