A novel flow cytometry-based method to study lymphocytes present in low cell numbers infiltrating non-functional and growth-hormone secreting pituitary adenomas

O. Mazzitelli*^a (Dr), JP. Ebejer^a (Dr), NP. Pace^a (Dr), M. Gruppetta^a (Dr), J. Vassallo^a (Prof), D. Saliba^a (Prof)

^a University of Malta, Msida, MALTA

* oriana.mazzitelli@um.edu.mt

As interest in cancer immunotherapy is growing, understanding the diversity of immune cells infiltrating the microenvironments of various tumours is warranted. Nonetheless, although the immune landscape in pituitary adenomas has been investigated in a couple of previous studies, knowledge is still lacking.

We validated an acoustic-assisted flow cytometer based method to identify immune cell populations in 5 fresh Non-Functional Pituitary Adenomas (NFPA) and 4 fresh Growth Hormone-Secreting Pituitary Adenomas (GHPA) obtained from patients after transphenoidal surgery. The tumour tissue was digested enzymatically to single cell suspensions. Using a multi-colour panel of antibodies against cell surface and intracellular antigens, we were able to reveal and quantify cellular populations of lymphocytic and myeloid origin present in the tumours. We then looked into the histology data to check for any correlations.

In all specimens, leucocytic infiltrates, especially lymphocytes, were detected. The CD8:CD4 ratio was equal to or higher than 1 in all GHPAs while 2 out of 5 NFPAs scored a lower ratio. However, no evident correlation was found between CD8:CD4 ratios and Ki-67 values.

Interestingly, most of the CD8+ cells also expressed the myeloid marker CD11b with GHPAs recording a higher percentage (> 30%) of the total lymphocytes than NFPAs. Expression of CD11b seemed to be restricted only to activated CD8+ cells as the double positive cells expressed the activation marker CD44 in both tumour types (> 93%).

Other markers that characterise M1/M2 macrophages, folliculostellate cells, Th/Treg cells as well as immune checkpoint PD-1 and exhaustion marker TIM-3 were also analysed in some specimens however sample number was too small to make inferences, although it did provide directions for further research.

This study shows that flow cytometry can be used as a reliable technology to study the tumour microenvironment in pituitary adenomas. Furthermore, it suggests that a novel population of activated, cytotoxic T cells might be infiltrating such tumours. Future studies shall focus on increasing sample number and validating the findings using other technologies available.

The author has declared no conflict of interest.