Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy

Abstract

Immunotherapy including immune checkpoint inhibition has reinvigorated the current cancer treatment field. The development of efficient cancer immunotherapies depends on a thorough understanding of the status of immune checkpoints and how they interact. However, the distribution and spatial organization changes of immune checkpoints during their interactions at the single-molecule level remain difficult to directly visualize due to the lack of in situ imaging techniques with appropriate spatial and stoichiometric resolution. Herein, we report the direct visualization and quantification of the spatial distribution and organization of CD47 on the bladder tumor cell membrane and SIRPα on the macrophage membrane by using a single-molecule localization imaging technique called quantitative direct stochastic optical reconstruction microscopy (QdSTORM). Results showed that a portion of CD47 and SIRPα was present on cell membranes as heterogeneous clusters of varying sizes and densities prior to activation. Quantitative analyses of the reconstructed super-resolution images and theoretical simulation revealed that CD47 and SIRPα were reorganized into larger clusters upon binding to each other. Furthermore, we found that blocking the immune checkpoint interaction with small-molecule inhibitors or antibodies significantly impacted the spatial clustering behavior of CD47 on bladder tumor cells, demonstrating the promise of our QdSTORM strategy in elucidating the molecular mechanisms underlying immunotherapy. This work offers a promising strategy to advance our understanding of immune checkpoint state and interactions while also contributing to the fields including signal regulation and cancer therapy.

The Effects of the Carrier and Ligand Spatial Conformation on RNA Nanodrug Cell Delivery

“…RGD is known to preferentially target α V β 3 integrins, and its spatial conformation significantly affect its binding ability. , To evaluate the effect of the spatial conformation on the binding ability of RGD to α V β 3 integrins, we initially analyzed the distribution of cRGD/lRGD-α V β 3 integrin on the cell membrane by using the super-resolution dSTORM technique with a single-molecule localization capability and a spatial resolution down to 30 nm, which enables visualization and quantification of ligand–receptor interactions at a subdiffraction resolution . Herein, Alexa Fluor 647 was used to label cRGD and lRGD followed by incubation of the cells with lRGD or cRGD for 10 min at varying concentrations (0.4–2.4 μM).…”