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

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 SIRP alpha 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 SIRP alpha 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 SIRP alpha 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.