Fernando Guimaraes

Which tumour immunosuppressive pathways prevent natural killer cell activation?

Background: Despite advances in treatment and earlier detection, cancer is still a main cause of cancer death worldwide. Natural killer (NK) cells are circulating innate lymphocytes that naturally protect against tumor spread (metastasis), and recently showed by our group as dysfunctional in the tumour microenvironment (TME) established by cancers at distant organs for future metastatic spread. Yet, despite knowing that NK cells do control cancer metastasis, our knowledge of how cancer cells evade NK cell control is still very poor. This project aims to examine several immune suppressive pathways that cancers likely manipulate to avoid NK cells and spread. These include factors the transforming growth factor (TGF)-β superfamily that are elevated in the tumor environment. These molecules have great potential to suppress the normally high killing and anti-metastatic activity mediated by NK cells, but to date we still need to elucidate how relatively important each pathway might be.

Proposed research program: The intrinsic NK cell function under suppressive factors stimulation will be assessed with NK cells purified from mouse spleen (wild type) by cell sorter, and in vitro challenge with activating cytokines and suppressive factors. Aim-1: Which suppressive factor is a major inhibitor of NK cell killing activity? This aim will be screened by killing activity of NK cells versus target tumour cells in co-culture systems. Aim-2: Which suppressive factor is a major inhibitor of NK cell cytokine secretion? This aim will assess NK cell cytokine production by intracellular cytokine (e.g. IFN-gamma) staining (flow cytometry) and secreted IFN-gamma, among others, from culture supernatants (ELISA); Aim-3: What is the cellular signalling status under suppressive conditions? The identification of altered cellular signalling will be screened by intracellular staining of phosphorylated signalling molecules (phosphor(p)-AKT, p-ERK1/2, p-p38, p-phospholipase C-gamma2, p-phosphotyrosine, p-SMAD2,3, p-STAT4, p-STAT5 and p-ZAP70 (PhosphoFlow).

These experimental tools will determine which is the most important suppressive pathway in inhibiting NK cell functions. Information we obtain from this work will allow us to design rationale approaches to increase NK cell function in personalised immunotherapy approaches.