Julia Jones, Scientific Manager, Cancer Research UK, Cambridge Institute gave an overview of hedgehog signalling, the thymus, T cell differentiation, and how the HiPlex assay works. The HiPlex assay from ACD is a manual method for analysing FFPE and frozen tissue. Jones also decided to focus on the Akoya PhenoImager and HALO AI for image analysis. She then discussed some of the key results from whole thymus analysis and some single cell analysis in the thymic medulla.
A collaborator, Louise O’Brien, mentioned that she conducted a fourplex RNAscope in the thymus for different hedgehog components but there is no spatial data. The antibodies that were tested performed poorly and the targets were very low in abundance. So, O’brien ran multiple multiplex experiments and selected the HiPlex assay.
Hedgehog signalling has key functions in cell-fate regulation throughout embryonic development and adult tissue homeostasis; mice lacking these hedgehog components fail to develop correctly. In the signalling pathway, there are nine different components including three hedgehog ligands, receptors Patch1/2; and transcription factors Gli1-3.
T cell differentiation occurs in the thymus. Jones explained that it is possible to track the differentiation of T cells with various markers such as CD45, CD4, and CD8. T cell differentiation in the thymus is a complex process involving transitions from double-negative to double-positive and finally to single-positive CD4 or CD8 T cells.
Upon reviewing a stained image of the thymus, Jones and her team found that segmenting single cells in dense tissue was very difficult. So, the hi-plex assay relies on label probes to perform hybridization and one can focus on multiple targets at a time. Imaging was conducted on an Akoya PhenoImager and the fluorophores but unfortunately, the autofluorescence didn’t perform well when the images were combined.
However, HALO AI was trained for many hours and performed nuclear segmentation at a good level. Jones analysed a medulla and the markup showed that they managed to find all of the targets. Jones said: “In those single positive and stromal phenotypes, we were able to detect all nine of our hedgehog components.” She added that she was able to detect all of the hedgehog components in both cortex and medulla and single positive T cells.
Visiopharm showed auspicious results in segmenting cells in both the cortex and medulla. Therefore, the team wants to continue with Visiopharm analysis, developing a second panel of HiPlex assays, and exploring HiPlex automation for human breast and kidney cancer tissue.
