title: Role of neutrophils in metastatic non-small cell lung cancer
title: The role of neutrophils in metastatic non-small cell lung cancer
titleType: AlternativeTitle
Creators: name: Bruce, Caroline Margaret
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: lung cancer
subject: metastasis
subject: non-small cell lung cancer
subject: NSCLC
subject: neutrophils
subject: T cells
subject: pneumonia pleural fluid

description: Lung cancer is a leading cause of cancer deaths globally. Metastasis, the process by which cancer spreads from its site of origin to a more distant site, accounts for approximately 90% of cancer deaths. Lung cancer is broadly split into non-small cell lung cancer (NSCLC), which represents around 85% of lung cancer cases, and small cell lung cancer (SCLC). When cancer cells have spread from the lung to the pleural fluid around the lung (malignant pleural effusion), this is considered metastatic disease. Neutrophils are a heterogenous population and have been shown to be polarised to anti-tumour or pro-tumour roles depending on their tumour microenvironment. T cells are known to be important effector cells in cancer. Neutrophils and T cells are both found in malignant pleural effusions, but little is known about their interaction in this context. The objective of this project was to gain a better understanding of how neutrophils interact with T cells in the NSCLC pleural fluid metastatic niche. Using pleural fluid from NSCLC patients to model the metastatic niche, I have shown when healthy donor T cells are cultured in the presence of NSCLC pleural fluid, there is an uplift in the percentage of CD4+ and CD8+ T cells that divide. However, when healthy donor neutrophils are added to this co-culture model, this pleural fluid uplift in the percentage of CD4+ and CD8+ T cells that divide is significantly suppressed. Further to this I have observed that the neutrophil induced suppression of T cell proliferation is a result of reduced entry into proliferation as opposed to inhibiting the ability of T cells to continue to divide once they have started to divide. This suppression is contact dependent. I have also identified that the neutrophil induced T cell suppression also occurs in the presence of pleural fluid from pneumonia patients, suggesting a common mechanism induced by both the infective and metastatic niche. Additionally, at 72 hours post stimulation, neutrophils cause a suppression in T cell activation as measured by reduced CD25 and CD71 and increased CD62L T cell expression when T cells are cultured with neutrophils and NSCLC or pneumonia pleural fluid compared to when cultured with pleural fluid alone. Taken together, this data suggests neutrophils are reducing T cell activation and causing a block to T cell entry into proliferation, suggestive of the neutrophils having an effect early in the T cell activation process. This is supported by the failure of T cells to induce expression of the early activation markers CD69 and phosphorylated ribosomal protein S6 (pRPS6) in the presence of neutrophils and NSCLC pleural fluid compared to pleural fluid alone 4 hours following stimulation. I have established that the suppressive neutrophil induced effect in the presence of NSCLC pleural fluid is dependent on active signalling, as the neutrophils were unable to cause suppression when they were fixed prior to co-culture, and is likely to involve a protein factor, as the suppressive effect was lost when the NSCLC pleural fluid was heat treated. Proteomic analysis revealed the majority of the 358 proteins identified were expressed to similar degrees in both NSCLC and pneumonia pleural fluid. Metabolomic analysis of pleural fluid showed both methionine and tryptophan essential amino acids were present in both types of pleural fluid although at higher levels in NSCLC compared to pneumonia. NanoString transcript data of CD8+ T cells cultured with NSCLC pleural fluid alone or with NSCLC pleural fluid and neutrophils then sorted into CD69+ or CD69- T cells showed some of the genes differentially expressed between CD69+ (“activated”) and CD69- (“non-activated”) T cells were perturbed by the addition of neutrophils in the co-culture. Interestingly SMAD3 was the only gene that was upregulated in both CD69+ and CD69- T cells in the presence of neutrophils compared to without neutrophils. Despite this, neither SMAD3 nor transforming growth factor beta (TGFβ) inhibitors reversed the neutrophil induced T cell suppression shown in the context of NSCLC pleural fluid. In summary, I have established a robust phenotype of neutrophils causing suppression of T cell activation and entry into proliferation in the context of NSCLC pleural fluid. I have shown this to be the case for both CD4+ and CD8+ T cells and across multiple NSCLC pleural fluids and pneumonia pleural fluids. I have used proteomic, metabolomic and transcriptomic data to try to understand how the suppressive effect may be manifesting. Further work is required to identify a target and functionally prove reversal of the suppressive effect. Establishing mechanisms by which neutrophils cause a suppressive effect on T cells may help provide therapeutic targets to optimise the overall anti-tumour response in NSCLC treatments.