Biology subjects

Low, P. S.

Publications and source records attributed to Low, P. S..

7 recordsLinked to original sources

bioRxiv2025

Targeting Macrophage Folate Receptor-β for Diagnosis and Treatment of Autoimmune Myocarditis

We investigated folate receptor-{beta} (FR-{beta}) expressed on activated macrophages as a therapeutic and imaging target in autoimmune myocarditis (AIM). Treatment with a folate-aminopterin conjugate (EC2319) targeting FR-{beta}-positive macrophages significantly reduced macrophage infiltration compared to saline (5.23 {+/-} 2.84% vs. 1.24 {+/-} 0.66%, P < 0.001) in rat experimental AIM. After immunization, FR-{beta} targeting positron emission tomography (PET) tracer [18F]FOL detected myocardial inflammatory lesions and revealed reduction in inflammation following EC2319 treatment relative to saline or tumor necrosis factor-alpha inhibitor Etanercept [standardized uptake value (SUV) 1.07 {+/-} 0.22 vs. 2.38 {+/-} 0.90 vs. 2.59 {+/-} 0.56, P < 0.001]. [18F]FOL PET signal correlated with myocardial CD68-positive macrophage content (r = 0.720, P < 0.001). These findings demonstrate that EC2319 attenuates myocardial inflammation in AIM and underscore the pathogenic role of FR-{beta}-positive macrophages. [18F]FOL PET provides a noninvasive means of detecting myocardial inflammatory activity and therapeutic response. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/686077v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@18e9296org.highwire.dtl.DTLVardef@139b8b3org.highwire.dtl.DTLVardef@8d1a74org.highwire.dtl.DTLVardef@1c3f06d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

CAR T cell cytotoxic responses are rapidly generated and sensitive to the unligated TCR

Chimeric antigen receptor (CAR) T cells expressing tumor-targeting engineered receptors can robustly eliminate cancer cells through secretion of cytotoxic factors. Durable remission in leukemia and lymphoma treatment has not been matched in solid tumors. Efforts to maximize tumor destruction and minimize toxicities have driven efforts to tune CAR signaling. However, the molecular mechanisms for CAR triggering and thresholds for activation are incompletely understood. Here, we measured the collection of CAR binding interactions that culminate in polarized delivery of lytic granules to the junction with the target. CAR T cells binarized cytotoxic activities in response to a few binding events and population outcomes were dominated by a subset of cells. Activation at the single molecule level matches the sensitivity of the native T cell receptor (TCR) and points to potent downstream signal propagation. Disruption of the unligated TCR with a transmembrane-targeting inhibitory peptide strongly dampened CAR T cell activation, indicating a critical crosstalk between the two receptors. Harnessing CAR T cell efficacy and reduction of toxicity will require new approaches to modify integration of the binding events, collected stochastically, that are rapidly digitized. These sensitive CAR T cell responses provide new insights into driving cytotoxic signaling through surface interaction engineering.

immunology↗

bioRxiv2025

A mechanistic model for dynamics between CAR T cells and target cells captures features that determine killing profiles

Chimeric antigen receptor (CAR) T cells represent a potent, programmable therapeutic that repurposes T cell cytotoxicity toward target cell elimination. Direct killing of tumor cells has been demonstrated in several cancer contexts but deficits in destroying solid tumors have been attributed to molecular and mechanical complexity. Understanding and predicting the efficacy of CAR T cell therapy requires a rigorous framework to capture the mechanistic interactions between immune cells and tumor targets. Toward enumerating such features and to quantitatively describe tumor cell survival in response to CAR T cell visitation, we propose a parametric hazard rate model for the right-censored conditional lifetime of a tumor cell, having the form of a time-discounted integral of cell:cell engagement. The model, conditioned using data on the numbers, durations, and modes of contacts made during the stochastic encounters between the two cell types, extracts mechanistic details from the experiments. We suggest two types of parameters to encapsulate features of CAR T cell killing potency ({kappa}) and the resilience of the tumor ({gamma}), respectively. These parsimonious parameters, nevertheless, generate substantial insights. Firstly, phenotypic heterogeneity from interactions mediated by substrate-engaged CAR T cells dominate killing outcomes, while those initiated in suspension contribute minimally. This emphasizes that a subset of the CAR T cell population is dominating the population outcome. Secondly, the model can be used to predict the killing as a function of time upon perturbation of the system. In this case, biasing the arrival process, toward more engagement in the adherent mode, can tune the rate of killing. Collectively, insights from the model framework imply that CAR T cell accumulation and killing efficiency depend not on the sum of individual T cell interactions, but rather on the weighted, time-dependent sum of heterogeneous interactions that are environmentally modulated. The model facilitates predictive insights into how contact sequences modulate CAR T cell responses and may provide strategies to alter therapeutic regimens.

biophysics↗

bioRxiv2022

Preclinical assessment of Fibroblast Activation Protein-specific MRI as an alternative to PSMA for image-guided focal therapy of prostate cancer

Accurate and precise delineation of gross tumour volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumours, such as prostate cancer. Magnetic resonance imaging of tumour molecular targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-LINAC. Fibroblast activation protein (FAP) is a transmembrane protein overexpressed in stromal components of >90% of epithelial carcinomas. Herein we compare targeted MRI of gold standard PSMA with FAP in the delineation of orthotopic tumours in a mouse model of prostate cancer. Control (no ligand), FAP and PSMA-targeting iron oxide nanoparticles were prepared with modification of an MRI agent (FerroTrace). Mice with orthotopic LNCaP tumours underwent T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles, which was most pronounced on the tumour periphery. FAP-targeted MRI increased the proportion of tumour contrast enhancing black pixels by 13.37% when compared to PSMA. Furthermore, analysis of changes in R2 values between healthy prostates and LNCaP tumours indicated an increase in contrast enhancing pixels in the tumour border of 15%, when targeting FAP, in contrast to PSMA This study demonstrates preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.

bioengineering↗

bioRxiv2020

Radiosynthesis and Preclinical Evaluation of Ga-NOTA-Folate for PET Imaging of Folate Receptor β Positive Macrophages

Folate receptor {beta} (FR-{beta}) is one of the markers expressed on macrophages and a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [68Ga]Ga-NOTA-folate (68Ga-FOL). First, we determined the affinity of 68Ga-FOL using human FR-{beta} expressing cells. Then, we studied atherosclerotic mice with 68Ga-FOL and 18F-FDG PET/CT. After sacrifice, the tissues excised were measured with a {gamma}-counter for ex vivo biodistribution. Further, the tracer distribution and co-localization with macrophages in aorta cryosections were studied using autoradiography, hematoxylin-eosin staining and immunostaining with anti-Mac-3 antibody. Specificity of 68Ga-FOL was assessed in a blocking study with excess of folate glucosamine. As a last step, human radiation doses were extrapolated from rat PET data. We were able to produce 68Ga-FOL at high radioactivity concentration, with high molar activity and radiochemical purity. The cell binding studies showed high (5.1 {+/-} 1.1 nM) affinity of 68Ga-FOL to FR-{beta}. The myocardial uptake of 68Ga-FOL (SUV 0.43 {+/-} 0.06) was 20-folds lower compared to 18F-FDG (SUV 10.6 {+/-} 1.8, P = 0.001). The autoradiography and immunohistochemistry of aorta revealed that 68Ga-FOL radioactivity co-localized with Mac-3-positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of 68Ga-FOL (2.44 {+/-} 0.15) was significantly higher than that of 18F-FDG (1.93 {+/-} 0.22, P = 0.005). Blocking studies verified 68Ga-FOL specificity to FR. As estimated from rat data the human effective dose was 0.0105 mSv/MBq. The organ with highest absorbed dose was kidney (0.1420 mSv/MBq). In conclusion, 68Ga-FOL is a promising new FR-{beta}-targeted tracer for imaging macrophage-associated inflammation. TABLE OF CONTENT/ABSTRACT GRAPHIC O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biochemistry↗

bioRxiv2020

Fluorescence Labeling of Circulating Tumor Cells with Folate Receptor Targeted Molecular Probes for Diffuse In Vivo Flow Cytometry

PurposeWe recently developed a new instrument called diffuse in vivo flow cytometry (DiFC) for enumeration of rare fluorescently-labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins, or were pre-labeled with a fluorescent dye ex-vivo. In this work, we investigated the use of two folate receptor (FR)-targeted fluorescence molecular probes for in vivo labeling of FR+ CTCs for DiFC. MethodsWe used EC-17 and Cy5-PEG-FR fluorescent probes. We studied the affinity of these probes for L1210A and KB cancer cells, both of which over-express FR. We tested the labeling specificity in cells in culture in vitro, in whole blood, and in mice in vivo. We also studied detectability of labeled cells with DiFC. ResultsBoth EC-17 and Cy5-PEG-FR probes had high affinity for FR+ CTCs in cell culture in vitro. However, only EC-17 had sufficient specificity for CTCs in whole blood. EC-17 labeled CTCs were also readily detectable in circulation in mice with DiFC. ConclusionsThis work demonstrates the feasibility of labeling CTCs for DiFC with a cell surface receptor targeted probe, greatly expanding the utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.

bioengineering↗

bioRxiv2018

Enhancing microRNA activity through increased endosomal release mediated by nigericin

The therapeutic promise of small RNA therapeutics (siRNAs, miRNAs) is not only limited by the lack of delivery vehicles, but also by the inability of the small RNAs to reach intracellular compartments where they can be biologically active. We previously reported successful delivery of functionally active miRNAs via receptor-mediated endocytosis1. This type of targeted therapy still faces one of the major challenges in the delivery field, endosomal sequestration. Here, a new method has been developed to promote endosomal escape of delivered miRNA. The strategy relies on the difference in solute contents between nascent endosomes and the cytoplasm: early endosomes are rich in sodium ions (Na+) while the intracellular fluid is rich is potassium ions (K+). Exploiting this difference through favoring the influx of K+ into the endosomes without the exchange for a osmotically active ion (Na+), results in an osmotic differential leading to endosome swelling and bursting. One molecule that is able to exchange K+ for an osmotically inactive hydrogen ion is the ionophore nigericin. Via generating an intramolecular miRNA delivery vehicle, containing a ligand, in this case folate, and nigericin we achieve escape of folate-RNA conjugates (e.g. FolamiRs) from their entrapping endosomes into the cytoplasm where they bind the RNA Induced Silencing complex (RISC) and activate the RNAi response.

molecular biology↗