Background: Several studies have confirmed the important role of the gut microbiota in the regulation of immune functions and its correlation with different diseases, including cancer. While brain-gut and liver-gut axes have already been demonstrated, the existence of a lung-gut axis has been suggested more recently, with the idea that changes in the gut microbiota could affect the lung microbiota, and vice versa. Likewise, the close connection between gut microbiota and cancer of proximal sites (intestines, kidneys, liver, etc.) is already well established. However, little is known whether there is a similar relation when looking at world's number one cause of death from cancer-lung cancer. Objective: Firstly, this study aims to characterise the gut, lung, and upper airways (UAs) microbiota in patients with non-small cell lung cancer (NSCLC) treated with surgery or neoadjuvant chemotherapy plus surgery. Secondly, it aims to evaluate a chemotherapy effect on site-specific microbiota and its influence on immune profile. To our knowledge, this is the 1st study that will analyse multi-site microbiota in NSCLC patients along with site-specific immune response. Methods: The study is a case-controlled observational trial. Forty NSCLC patients will be divided into 2 groups depending on their anamnesis: Pchir, patients eligible for surgery, or Pct-chir, patients eligible for neoadjuvant chemotherapy plus surgery. Composition of the UAs (saliva), gut (faeces), and lung microbiota (from broncho-alveolar lavage fluid (BALF) and 3 lung pieces: "healthy" tissue distal to tumour, peritumoural tissue and tumour itself) will be analysed in both groups. Immune properties will be evaluated on the local (evaluation of the tumour immune cell infiltrate, tumour classification and properties, immune cell phenotyping in BALF; human neutrophil protein (HNP) 1-3, b-defensin 2, and calprotectin in faeces) and systemic level (blood cytokine and immune cell profile). Short-chain fatty acids (SCFAs) (major products of bacterial fermentation with an effect on immune system) will be dosed in faecal samples. Other factors such as nutrition and smoking status will be recorded for each patient. We hypothesise that smoking status and tumour type/grade will be major factors influencing both microbiota and immune/inflammatory profile of all sampling sites. Furthermore, due to non-selectivity, the same effect is expected from chemotherapy. Abbreviations: ANSM = The French National Agency for Medicines and Health Products Safety (Agence nationale de sécurité du médicament et des produits de santé), BAL = broncho-alveolar lavage, BALF = broncho-alveolar lavage fluid, BMI = body mass index, CIFRE = Industrial Research Training Agreements grant (Convention industrielle de formation par la recherché), CRP = C-reactive protein, CT = chemotherapy, ELISA = enzyme-linked immunosorbent assay, FDR = false discovery rate, GF = germ-free, GI = gastrointestinal, HBSS = Hank's balanced salt solution, HNP = human neutrophil peptide, ICI = immune checkpoint inhibitor, IL = interleukin, MCLB = mammalian cell lysis buffer, NSCLC = non-small cell lung cancer, OTU = outer taxonomic unit, Pchir = patient surgery, fr. «patient chirurgie», Pct-chir = patients chemotherapy plus surgery, fr. «patient chimiothérapie-chirurgie», qPCR = quantitative polymerase chain reaction, SCFAs = short-chain fatty acids, UAs = upper airways.