Phenotyping of the Tumor Microenvironment Using DNA Barcoded Multiplex Technology (OLC)

Course overview

Multiplex immunohistochemistry and immunofluorescence offer powerful ways to understand target expression in the tumor microenvironment. However, there are challenges with the development of traditional multiplex methods due to the limitations of chromogenic dyes and secondary antibody conjugated fluorophores. DNA barcoded InSituPlex technology provides us with an innovative way to detect multiple markers simultaneously without new capital investment or the use of antibody stripping methods. Using this technology, we successfully combined UltiVues InSituPlex and DNA exchange in a custom 7 plex assay that was applied to sections of human NSCLC and bladder cancer. This method used two separate panels that were designed using barcoded primary antibodies against CD8/CD68/PD-L1 and Ki67/Granzyme B/CD3/PanCK. Following the application of the first round of staining, slides were temporarily coverslipped then imaged, then processed using the DNA exchange protocol and application of the second antibody panel then imaged again. For each section, two whole slide images were acquired, fused into one single image and analyzed using HALO image analysis. Using this technology we were able to detect 7 markers simultaneously using the same tissue section, which significantly expanded our capability to understand the expression and distribution of the immune cells in these tumors.