Non-invasive assessment of murine PD-L1 levels in syngeneic tumor models by nuclear imaging with nanobody tracers

Blockade of the inhibitory PD-1/PD-L1 immune checkpoint axis is a promising cancer treatment. Nonetheless, a significant number of patients and malignancies do not respond to this therapy. To develop a screen for response to PD-1/PD-L1 inhibition, it is critical to develop a non-invasive tool to accurately assess dynamic immune checkpoint expression. Here we evaluated non-invasive SPECT/CT imaging of PD-L1 expression, in murine tumor models with varying PD-L1 expression, using high affinity PD-L1-specific nanobodies (Nbs). We generated and characterized 37 Nbs recognizing mouse PD-L1. Among those, four Nbs C3, C7, E2 and E4 were selected and evaluated for preclinical imaging of PD-L1 in syngeneic mice. We performed SPECT/CT imaging in wild type versus PD-L1 knock-out mice, using Technetium-99m (99mTc) labeled Nbs. Nb C3 and E2 showed specific antigen binding and beneficial biodistribution. Through the use of CRISPR/Cas9 PD-L1 knock-out TC-1 lung epithelial cell lines, we demonstrate that SPECT/CT imaging using Nb C3 and E2 identifies PD-L1 expressing tumors, but not PD-L1 non-expressing tumors, thereby confirming the diagnostic potential of the selected Nbs. In conclusion, these data show that Nbs C3 and E2 can be used to non-invasively image PD-L1 levels in the tumor, with the strength of the signal correlating with PD-L1 levels. These findings warrant further research into the use of Nbs as a tool to image inhibitory signals in the tumor environment

Strong vacuum squeezing from bichromatically driven Kerrlike cavities: from optomechanics to superconducting circuits

Squeezed light, displaying less fluctuation than vacuum in some observable, is key in the flourishing field of quantum technologies. Optical or microwave cavities containing a Kerr nonlinearity are known to potentially yield large levels of squeezing, which have been recently observed in optomechanics and nonlinear superconducting circuit platforms. Such Kerr-cavity squeezing however suffers from two fundamental drawbacks. First, optimal squeezing requires working close to turning points of a bistable cycle, which are highly unstable against noise thus rendering optimal squeezing inaccessible. Second, the light field has a macroscopic coherent component corresponding to the pump, making it less versatile than the so-called squeezed vacuum, characterised by a null mean field. Here we prove analytically and numerically that the bichromatic pumping of optomechanical and superconducting circuit cavities removes both limitations. This finding should boost the development of a new generation of robust vacuum squeezers in the microwave and optical domains with current technology

Single Cell Kinetics of Intracellular, Nonviral, Nucleic Acid Delivery Vehicle Acidification and Trafficking

Mechanistic understanding of the intracellular trafficking of nonviral nucleic acid delivery vehicles remains elusive. A live, single cell-based assay is described here that is used to investigate and quantitate the spatiotemporal, intracellular pH microenvironment of polymeric-based nucleic acid delivery vehicles. Polycations such as polyethylenimine (PEI), poly-L-lysine (PLL), β-cyclodextrin-containing polymers lacking or possessing imidazole termini (CDP or CDP-imid), and cyclodextrin-grafted PEI (CD-PEI) are used to deliver an oligonucleotide containing a single fluorophore with two emission lines that can be employed to measure the pH. Delivery vehicles were also sterically stabilized by addition of poly(ethylene glycol) (PEG) and investigated. The intracellular trafficking data obtained via this new methodology show that vectors such as PEI and CDP-imid can buffer the endocytic vesicles while PLL and CDP do not. Additionally, the PEGylated vectors reveal the same buffering capacity as their unstabilized variants. Here, the live cell, spatiotemporal mapping of these behaviors is demonstrated and, when combined with cell uptake and luciferase expression data, shows that there is not a correlation between buffering capacity and gene expression