Search

For the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII-animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate decorate all mammalian cell surfaces. These mucopolysaccharides act as coreceptors for extracellular ligands, regulating cell signaling, growth, proliferation, and adhesion. In glioblastoma, the most common type of primary malignant brain tumor, dysregulated GAG biosynthesis results in altered chain length, sulfation patterns, and the ratio of contributing monosaccharides. These events contribute to the loss of normal cellular function, initiating and sustaining malignant growth. Disruption of the aberrant cell surface GAGs with small molecule inhibitors of GAG biosynthetic enzymes is a potential therapeutic approach to blocking the rogue signaling and proliferation in glioma, including glioblastoma. Previously, 4-azido-xylose-α-UDP sugar inhibited both xylosyltransferase (XYLT-1) and β−1,4-galactosyltransferase-7 (β-GALT-7)—the first and second enzymes of GAG biosynthesis—when microinjected into a cell. In another study, 4-deoxy-4-fluoro-β-xylosides inhibited β-GALT-7 at 1 mM concentration in vitro. In this work, we seek to solve the enduring problem of drug delivery to human glioma cells at low concentrations. We developed a library of hydrophobic, presumed prodrugs 4-deoxy-4-fluoro-2,3-dibenzoyl-(α- or β-) xylosides and their corresponding hydrophilic inhibitors of XYLT-1 and β-GALT-7 enzymes. The prodrugs were designed to be activatable by carboxylesterase enzymes overexpressed in glioblastoma. Using a colorimetric MTT assay in human glioblastoma cell lines, we identified a prodrug–drug pair (4-nitrophenyl-α-xylosides) as lead drug candidates. The candidates arrest U251 cell growth at an IC(50) = 380 nM (prodrug), 122 μM (drug), and U87 cells at IC(50) = 10.57 μM (prodrug). Molecular docking studies were consistent with preferred binding of the α- versus β-nitro xyloside conformer to XYLT-1 and β-GALT-7 enzymes.

Chronic neuroinflammation and microglial dysfunction are key features of many neurological diseases, including Alzheimer's Disease and multiple sclerosis. While there is unfortunately a dearth of highly selective molecular imaging biomarkers/probes for studying microglia in vivo, P2Y12R has emerged as an attractive candidate PET biomarker being explored for this purpose. Importantly, P2Y12R is selectively expressed on microglia in the CNS and undergoes dynamic changes in expression according to inflammatory context (e.g., toxic versus beneficial/healing states), thus having the potential to reveal functional information about microglia in living subjects. Herein, we identified a high affinity, small molecule P2Y12R antagonist (AZD1283) to radiolabel and assess as a candidate radiotracer through in vitro assays and in vivo positron emission tomography (PET) imaging of both wild-type and total knockout mice and a non-human primate.First, we evaluated the metabolic stability and passive permeability of non-radioactive AZD1283 in vitro. Next, we radiolabeled [We determined the half-life of AZD1283 in mouse and human liver microsomes to be 37 and 160 min, respectively, and predicted passive CNS uptake with a small amount of active efflux, using a Caco-2 assay. Our radiolabeling efforts afforded [Despite our initial encouraging liver microsome and Caco-2 monolayer data, in addition to the observed high stability of [

Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases. Several GAG cleaving enzymes are widely used for studying GAG glycobiology: heparitinases, chondroitinases, heparanases, hyaluronidases, and keratanases. Herein, we describe a method to synthesize four distinct nanometal surface energy transfer (NSET)-based gold-GAG-dye conjugates (nanosensors). Heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid are covalently linked with distinct fluorescent dyes and then immobilized on gold nanoparticles (AuNPs) to build nanosensors that serve as excellent substrates for GAG cleaving enzymes. Upon treatment of nanosensors with their respective GAG cleaving enzymes, dye-labeled oligosaccharides/disaccharides are released from AuNPs resulting in enhanced fluorescence recovery. These nanosensors have a great promise as diagnostic tools in various human pathophysiological conditions for detecting dysregulated expression of GAG cleaving enzymes and also as a sensitive analytical tool for assessing the quality control of pharmaceutical grade heparin polysaccharides that are produced in millions of small- and medium-sized animal slaughter houses worldwide.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4 h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.

Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful. This study demonstrates that small molecules, termed click-xylosides, can promote angiogenesis in the in vitro matrigel tube formation assay and the ex ovo chick chorioallantoic membrane assay, depending on their aglycone moieties. Xyloside treatment enhances network connectivity and cell survivability, thereby, maintaining the network structures on matrigel culture for an extended period of time. These effects were achieved via the secreted xyloside-primed glycosaminoglycans (GAG) chains that in part, act through an ERK1/2 mediated signaling pathway. Through the remodeling of GAGs in the extracellular matrix of endothelial cells, the glycan approach, involving xylosides, offers great potential to effectively promote therapeutic angiogenesis.

We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.