Background: Immune checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4, have dramatically improved clinical outcomes for various classes of cancer patients. However, many questions remain as to why only certain patients respond to such therapies and when combination therapies should be employed. There is growing evidence that the sub-cellular spatial organization of immune receptors and signaling molecules play an important role in regulating immune cell signaling. We are working to better understand the nano- to microscale localization of PD-1 on various classes of immune cells in human melanoma tissue sections.
Materials and Methods: We are labeling human melanoma tissue sections using immunofluorescence. These tissue sections are then imaged using laser scanning confocal microscopy (LSCM) and Stimulated Emission Depletion (STED) microscopy. The resolution of LSCM is limited by diffraction to ~300 nm, allowing us to study both the distribution of PD-1 expression on specific subpopulations of lymphocytes throughout the tumor, as well as the sub-cellular 3D architecture of PD-1. On the other hand, our custom-built STED microscope allows us to surpass the diffraction limit, obtaining a lateral resolution of ~65 nm. This allows us to study the nanoscale localization of PD-1 in single cells. Importantly, the images from both techniques provide high-resolution information about PD-1 and its interactions with other receptors inside the tumor microenvironment.
Results: We have successfully imaged fluorescently labeled PD-1 in human melanoma tissue sections. Preliminary results suggest PD-1 forms microclusters, similar to what has been previously observed for the T cell receptors. At the same time, with our high spatial resolution, PD-1 expression is highly heterogeneous across the tumor. Finally, experiments are in progress to test if PD-1 colocalizes with other immune receptors.
Conclusions: Probing the sub-cellular localization and clustering properties of receptors may prove crucial in understanding and improving immune checkpoint inhibitor therapies. We have successfully imaged PD-1 using 3D fluorescence microscopy in human melanoma tissue sections, showing PD-1 forms microclusters in the tumor microenvironment.