Related Experiment Video
Updated: Jul 12, 2025
High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy
Published on: July 25, 2014
Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with
Yurong Wei1, Min Zhao1, Tianpei He1
1College of Chemistry and Molecular Sciences, Key Laboratory of Biomedical Polymers of Ministry of Education, Institute of Molecular Medicine, Renmin Hospital of Wuhan University, School of Microelectronics, Wuhan University, Wuhan 430072, P. R. China.
Insights
Researchers visualized immune checkpoints CD47 and SIRPα using quantitative direct stochastic optical reconstruction microscopy (QdSTORM). This method revealed how these checkpoints cluster on cell membranes, offering insights into cancer immunotherapy mechanisms.
Area of Science:
- Immunology
- Cancer Biology
- Biophysics
Background:
- Cancer immunotherapy, particularly immune checkpoint inhibition, has transformed cancer treatment.
- Understanding immune checkpoint interactions is crucial for developing effective immunotherapies.
- Current imaging techniques lack the resolution to visualize single-molecule interactions of immune checkpoints in situ.
Purpose of the Study:
- To visualize and quantify the spatial distribution and organization of CD47 on bladder tumor cells and SIRPα on macrophages.
- To investigate the molecular mechanisms of immune checkpoint interactions at the single-molecule level.
- To demonstrate the utility of quantitative direct stochastic optical reconstruction microscopy (QdSTORM) in studying immunotherapy.
Main Methods:
- Utilized quantitative direct stochastic optical reconstruction microscopy (QdSTORM) for super-resolution imaging.
- Quantified the spatial distribution and clustering of CD47 and SIRPα on cell membranes.
- Employed theoretical simulations to analyze molecular reorganization upon binding.
- Investigated the impact of blocking agents (small-molecule inhibitors and antibodies) on spatial clustering.
Main Results:
- CD47 and SIRPα were observed as heterogeneous clusters on cell membranes before activation.
- Binding of CD47 and SIRPα led to their reorganization into larger clusters.
- Blocking immune checkpoint interactions significantly altered CD47 spatial clustering on tumor cells.
- QdSTORM successfully visualized and quantified these dynamic molecular changes.
Conclusions:
- QdSTORM provides a powerful tool for visualizing and quantifying immune checkpoint interactions at the single-molecule level.
- The study elucidates the spatial reorganization of CD47 and SIRPα upon binding, contributing to understanding immunotherapy mechanisms.
- This approach holds promise for advancing research in signal regulation, cancer therapy, and immunotherapy development.
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.
More Related Videos
06:05Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment
Published on: June 2, 2023
09:01Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking
Published on: October 29, 2018