Cancer immunotherapy is rapidly changing the landscape of cancer treatment, and monitoring the immune status of a patient is a critical aspect of research in this field. One particular area of interest in immuno-oncology is the role of regulatory T cells. Beginning with the discovery of CD4+/CD25+/FOXP3+ positive cells in 1995, the list of T cell subsets and their function in tumours and other tissues has grown considerably. While quantifying specific subsets of immune cells is routine using flow cytometry, monitoring the numbers and distribution of this same subset of immune cells in FFPE biopsies of solid tumours remains difficult with standard methods.
To address this we present a methodology for staining, imaging and analysing the CD4+/CD25+/FOXP3+ phenotype of regulatory T cells in head and neck squamous cell carcinoma FFPE specimens.
A sequential multiplex staining technique using tyramide signal amplification conjugated fluorophores with DAPI nuclear counterstain was used. Imaging was performed using a multispectral imaging system, which enabled the quantitative separation of the fluorophores and elimination of autofluorescence. Image analysis was executed using an automated image analysis program. Each 20x image underwent a morphologic segmentation into tumour or stromal. The CD4+/CD25+/FOXP3+ phenotype cells were identified and enumerated in the tumour and in the stroma of the sample, therefore providing not just their cell counts, but also the context of their locations. Results from this study showed that counts of Cytotoxic T cells or Helper T cells could be automated and measured what percentages were in the tumour as well as their densities.
This study shows the utility of automating the assessment of the various phenotypes of T regulatory cells in FFPE tissue sections. This methodology can be expanded to 7 or 8 markers across various cell and tissue types, to provide a context-rich images.