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Abstract

Although up to 90% of lung cancer cases are linked to smoking, also exposure to other environmental and dietary factors and chronic inflammation are regarded as cancer risk factors. Increased cancer incidence was found for butchers and meat workers; conversely, decreased incidence was reported for people with lactose intolerance or regularly taking non-steroidal anti-inflammatory drugs. Global epidemiology points towards an association between lung cancer incidence and consumption of bovine products. A search for infectious risk factors led to the isolation of plasmid-like DNA molecules termed Bovine Meat and Milk Factors (BMMF), which have been described as cancer risk factors acting via inflammation-linked indirect carcinogenesis upon experimental detection of increased expression levels of a conserved BMMF protein (Rep) in tissues of colorectal cancer patients when compared to healthy controls by immunohistochemistry (IHC). Although vesicular BMMF structures were identified in lung cancer tissues by immune electron microscopy supported by IHC, the presence of BMMF was not systematically tested in lung cancer. In this study, Rep expression was quantified in paired tumour and peritumour tissues from lung cancer patients as well as cancer-free individuals (control) by IHC, co-immunofluorescence (IF) microscopy and immunoblot based on monoclonal anti-Rep antibodies and mass spectroscopy. In addition, publicly available scRNA-seq dataset was assessed to monitor BMMF+ cells. We observed specific and reproducible IHC staining with 5 out of 8 antibodies tested. We obtained positive staining in all peritumor tissues tested (n=42) while tumor and control tissues showed less staining (12 out of 37 tumor tissues tested positive). In peritumour tissues, a congruent staining of Rep+CD68+ cells was observed in IHC and IF in alveolar macrophages. IF triple staining with Rep, CD68 and CD163 (marker of M2-like macrophages) revealed congruent staining of Rep in CD68+CD163+ alveolar macrophages. Cell-based quantification indicated a significantly increased number of Rep+CD68+CD163+ cells in peritumour when compared to tumor and control tissues. BMMF staining was not associated with the intensity of smoking, but showed trends of an inverse correlation. The presence of Rep in lung peritumour tissues was supported by detection of bands by immunoblotting/-precipitation of tissue lysates and BMMF (Rep-specific) peptides validated by mass spectroscopy. Screening for BMMF reads in scRNA-seq data from lung adenocarcinoma patients revealed BMMF+ cells in 83% of patients (total number of patients n=30), mostly in macrophages and T-cells. In conclusion, BMMF antibody staining revealed Rep expression in CD68+(CD163+) macrophages of lung peritumour tissues and was enriched in case vs. control suggesting use as a cancer biomarker and a possible causal contribution. The identification of Rep+ macrophages in scRNA-seq, with the addition of further scRNA data, might enable to study expression changes induced by BMMF. This is of particular interest as alveolar macrophages have been described as drivers of early tumorigenesis and target for therapeutic intervention.