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BackgroundDespite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth.ObjectiveThis study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer.MethodsIn this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations.ResultsTo investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects.ConclusionOverall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

Abstract Neoplasms of the lungs are the leading cause of cancer incidence and mortality worldwide. Although immunotherapy has increased the overall survival of patients with lung cancer, there is the need to improve this treatment. At this regard, blood lipid levels are thought to be linked to cancer risk and thus a preventive intervention through regulation of the nutrition of patients with lung cancer is gaining much attention. In this study, we therefore asked about the contribution of serum lipids and cholesterol cellular metabolism in lung cancer development and progression. We measured different serum lipids and analyzed cholesterol synthesis enzymes 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) and acetyl-coenzyme A cholesterol acetyltransferase 1 (ACAT1) as well as the cholesterol cellular export protein ATP-binding cassette (ABC) A-1 mRNA by quantitative PCR (qPCR) in the control and tumoral regions of post-surgery lung tissues to analyze the accumulation of cholesterol in cancer cells in a cohort of patients with lung adenocarcinoma (LUAD). We found that triglycerides in serum directly correlated with the body mass index (BMI) in patients with LUAD. By contrast, we found that high-density lipoprotein (HDL) cholesterol inversely correlated with the BMI, C-reactive protein (CRP) and overall survival and total cholesterol inversely correlated with the tumor diameter, serum CRP and overall survival in these LUAD patients. Functionally, the role of cholesterol is indispensable for the growth and development of normal animal cells where it is tightly regulated. Excess of cellular cholesterol regulated by HMGCR is converted to cholesteryl esters by the enzyme ACAT1 and exported extracellularly by the cholesterol transporter ABCA1. Here we found HMGCR and ACAT1 upregulated and ABCA1 downregulated in the lung’s tumoral region of our LUAD cohort, indicating cholesterol dysregulated cellular export in lung tumor cells.

Although lung cancer is the leading cause of cancer deaths worldwide, the mechanisms how lung cancer cells evade the immune system remain incompletely understood. Here, we discovered IL-9-dependent signaling mechanisms that drive immune evasion in non-small cell lung cancer (NSCLC). We found increased IL-9 and IL-21 production by T cells in the tumoral region of the lung of patients with NSCLC, suggesting the presence of Th9 cells in the lung tumor microenvironment. Moreover, we noted IL-9 producing Tregs in NSCLC. IL-9 target cells in NSCLC consisted of IL-9R+ tumor cells and tumor-infiltrating lymphocytes. In two murine experimental models of NSCLC, and in vitro, IL-9 prevented cell death and controlled growth of lung adenocarcinoma cells. Targeted deletion of IL-9 resulted in successful lung tumor rejection in vivo associated with an induction of IL-21 and reduction of Treg cells. Finally, anti-IL-9 antibody immunotherapy resulted in suppression of tumor development even in established experimental NSCLC and was associated with reduced IL-10 production in the lung. In conclusion, our findings indicate that IL-9 drives immune escape of lung tumor cells via effects on tumor cell survival and tumor infiltrating T cells. Thus, strategies blocking IL-9 emerge as a new approach for clinical therapy of lung cancer.

Airway infection is a major cause of mortality worldwide. The identification of new mechanisms aiding in effective host immune response is therefore required. Here, we show that the specific depletion of the pleural immune cell compartment during bacterial pneumonia resulted in a reduced pulmonary immune response and increased mortality in mice. Bacterial airway infection provoked early pleural space (PS) inflammation characterized by innate response activator (IRA) B cell development and pleural large resident macrophage (LRM) necroptosis, the repopulation of LRMs being driven by cellular proliferation in situ. Necroptotic LRMs amplified PS inflammation by stimulating pleural Mincle-expressing macrophages whereas IRA B cells contributed partially to GM-CSF-induced PS inflammation. Upon pulmonary infection, the induction of PS inflammation resulted in reduced bacterial burden whereas the specific depletion of pleural resident macrophages led to increased mortality and bacterial burden and reduced pulmonary immunity. Moreover, mice in which B cells were unable to produce GM-CSF exhibited reduced CD103+ dendritic cells and reduced CD4+ T cell numbers in the draining lymph node. Altogether, our results describe a previously unrecognized mechanism of pleural space inflammation necessary for effective protection against bacterial airway infection.

BackgroundLung cancer is the second common cancer type in western countries and has a high mortality. During the development and progression of the tumor, the nutrients in its environment play a central role. The tumor cells depend crucially on glucose metabolism and uptake. Tumor cell metabolism is dominated by the Warburg effect, where tumor cells produce large amounts of lactate from pyruvate under aerobic conditions. We thus reasoned that, reducing carbohydrates in the diet might support anti-tumoral effects of current immunotherapy and additionally target tumor immune escape.ObjectivesThe link between reducing carbohydrates to improve current immunotherapy is not clear. We thus aimed at analyzing the effects of different glucose levels on the tumor development, progression and the anti-tumoral immune response.MethodsWe correlated the clinical parameters of our LUAD cohort with different metabolic markers. Additionally, we performed cell culture experiments with A549 tumor cell line under different glucose levels. Lastly, we investigated the effect of low and high carbohydrate diet in an experimental murine model of lung cancer on the tumor progression and different immune subsets.ResultsHere we found a positive correlation between the body mass index (BMI), blood glucose levels, reduced overall survival (OS) and the expression of Insulin-like growth factor-1 receptor (IGF1R) in the lung tumoral region of patients with lung adenocarcinoma (LUAD). Furthermore, increasing extracellular glucose induced IGF1R expression in A549 LUAD cells. Functional studies in a murine model of LUAD demonstrated that, glucose restricted diet resulted in decreased tumor load in vivo. This finding was associated with increased presence of lung infiltrating cytotoxic CD8+ T effector memory (TEM), tissue resident memory T (TRM) and natural killer cells as well as reduced IGFR mRNA expression, suggesting that glucose restriction regulates lung immunity in the tumor microenvironment.ConclusionsThese results indicate that, glucose restricted diet improves lung immune responses of the host and suppresses tumor growth in experimental lung adenocarcinoma. As glucose levels in LUAD patients were negatively correlated to postoperative survival rates, glucose-restricted diet emerges as therapeutic avenue for patients with LUAD.

Despite the opposite roles of Tbet and Foxp3 in the immune system as well as in tumour biology, recent studies have demonstrated the presence of of CD4+ T cells, expressing both, Tbet and Foxp3. Although Tbet+Foxp3+ T cells are currently a subject of intense research, less is known about their biological function especially in cancer. Here we found a considerable accumulation of Tbet+Foxp3+CD4+ T cells, mediated by the immunosuppressive cytokine TGFβ in the lungs of tumour bearing mice. This is in line with previous studies, demonstrating the important role of TGFβ for the immunopathogenesis of cancer. By gathering results both in murine model and in human disease, we demonstrate that, the conversion of IFNγ producing anti-tumoral T-bet+Th1 CD4+ T cells into immunosuppressive Tbet and Foxp3-PD1 co-expressing regulatory cells could represent an additional important mechanism of TGFβ-mediated blockade of anti-tumour immunity.