Interplay between the lung microbiome and lung cancer

Accepted Manuscript Interplay between the lung microbiome and lung cancer Qixing Mao, Feng Jiang, Rong Yin, Jie Wang, Wenjie Xia, Gaochao Dong, Weidong Ma, Yao Yang, Lin Xu, Jianzhong Hu PII:

S0304-3835(17)30760-7

DOI:

10.1016/j.canlet.2017.11.036

Reference:

CAN 13626

To appear in:

Cancer Letters

Received Date: 21 September 2017 Revised Date:

23 November 2017

Accepted Date: 27 November 2017

Please cite this article as: Q. Mao, F. Jiang, R. Yin, J. Wang, W. Xia, G. Dong, W. Ma, Y. Yang, L. Xu, J. Hu, Interplay between the lung microbiome and lung cancer, Cancer Letters (2017), doi: 10.1016/ j.canlet.2017.11.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Interplay between the lung microbiome and lung cancer

2 Author list:

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Qixing Mao1,2,3,4*, Feng Jiang1,3*, Rong Yin1,3*, Jie Wang1,3, Wenjie Xia1,3,4,

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Gaochao Dong1,3, Weidong Ma1,3,4, Yao Yang2, Lin Xu1,3#, Jianzhong Hu2#

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1.

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Cancer Research, Nanjing Medical University Affiliated Cancer Hospital, Nanjing,

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210009, P.R. China

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2.

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Department of Thoracic Surgery, Jiangsu Cancer Hospital, Jiangsu Institute of

Department of Genetics and Genomic Sciences, Icahn School of Medicine at

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Mount Sinai, New York, NY10029

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3.

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Nanjing Medical University Affiliated Cancer Hospital, Nanjing, 210009, P.R. China

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4.

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Nanjing Medical University, Nanjing, 210000, P.R. China.

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Jiangsu Key Laboratory of Molecular and Translational Cancer Research,

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The Fourth Clinical College of Nanjing Medical University, Graduated College of

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# Corresponding authors:

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Jianzhong Hu

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Address: 1425 Madison Ave, New York, NY, U.S. 10029

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TEL: 212-659-6881

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FAX:212-849-2508

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Lin Xu

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Address: Baiziting 42, Nanjing, China, 210009 TEL: 86-25-83284700

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FAX: 86-25-83641062

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E-mail: [email protected]

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* These authors contribute equally to this manuscript.

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ACCEPTED MANUSCRIPT Abstract

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The human microbiome confers benefits or disease susceptibility to the human body

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through multiple pathways. Disruption of the symbiotic balance of the human

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microbiome is commonly found in systematic diseases such as diabetes, obesity, and

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chronic gastric diseases. Emerging evidence has suggested that dysbiosis of the

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microbiota may also play vital roles in carcinogenesis at multiple levels, e.g., by

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affecting metabolic, inflammatory, or immune pathways. Although the impact of the

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gut microbiome on the digestive cancer has been widely explored, few studies have

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investigated the interplay between the microbiome and lung cancer. Some recent

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studies have shown that certain microbes and microbiota dysbiosis are correlated with

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development of lung cancer. In this mini-review, we briefly summarize current

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research findings describing the relationship between the lung microbiome and lung

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cancer. We further discuss the potential mechanisms through which the lung

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microbiome may play a role in lung carcinogenesis and impact lung cancer treatment.

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A better knowledge of the interplay between the lung microbiome and lung cancer

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may promote the development of innovative strategies for early prevention and

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personalized treatment in lung cancer.

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ACCEPTED MANUSCRIPT 1

Highlights:

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The Lungs are not sterile, and the lung microbiome is associated with lung health.




The lung microbiome is linked to lung cancer.

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Microbial dysbiosis may modulate the risk of malignancy at multiple levels.

6 Keywords:

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microbiome, lung disease, lung cancer, dysbiosis

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Abbreviations:

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NGS: next-generation sequencing

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BAL: bronchoalveolar lavage

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COPD: chronic obstructive pulmonary disease

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CF: cystic fibrosis

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IPF: idiopathic pulmonary fibrosis

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PRRs: pattern recognition receptors

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TLR: toll-like receptor

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NOD-like receptors: nucleotide-binding oligomerization domain-like receptors

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SCC: squamous cell carcinoma

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AC: adenocarcinoma

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ROS: reactive oxygen species

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ACCEPTED MANUSCRIPT DCA: deoxycholic acid

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SCFAs: short-chain fatty acids

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ACCEPTED MANUSCRIPT Introduction

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Human microbiota communities have co-evolved with the host and play essential

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roles in various biological functions of human body[1]. Microbes that live on and

4

inside the human body (microbiota) consist of about 40 trillion microbial cells and

5

outnumber the quantity of human cells[2]. The identity and relative abundance of

6

members of the human microbiota are associated with different disease statuses.

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Among these various microbes, many have been identified as pathogens linked to

8

human carcinogenesis. For instance, the presence of Helicobacter pylori, a common

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Proteobacteria found in the upper gastrointestinal tract, is mainly responsible for

10

gastritis and stomach ulcers and significantly increases the risk of gastric cancer[3].

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Furthermore, Citrobacter rodentium infection has been shown to promote colon tumor

12

development in a murine model[4]. Recently, with the development of

13

high-throughput next generation sequencing (NGS), the entire spectrum of the human

14

microbiome has been surveyed, and data have suggested that specific pathogens and

15

global shifts in microbiota communities may contribute to carcinogenesis and affect

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the cancer therapies through various biological pathways, including inflammation,

17

metabolism and cell signaling.

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Although the lungs of a healthy individual are thought to be sterile, this dogma has

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been challenged by several recent studies, showing the diverse lung microbiome and

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its association with lung diseases and lung cancer[5, 6]. In this mini-review, we

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briefly summarize current research advances in the lung microbiome, the role of the

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ACCEPTED MANUSCRIPT lung microbiome in various lung diseases and lung cancer, and the mechanisms

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through which the microbiome promotes carcinogenesis.

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The environment and the lung microbiome

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The lung microbiome varies depending on the region of the lung sampled[7]. Many

5

perspective reviews have indicated that the differences in microenvironment within

6

lung, such as PH levels, oxygen tension, and immune conditions determined the

7

distinct microorganisms parasitized in lung[8, 9]. Thus, diversities in topography,

8

physiology, and immunology shape the composition of the lung microbiota.

9

Global environmental and geographical alterations play vital roles in the gut

10

microbiota. However, these effects were controversial in lung microbiome. One recent

11

study indicated that the atmospheric concentration (µg/m3) of particulate matter of 10

12

micrometers in diameter (PM10) might affect the lung microbiota, implying that

13

considerable geographic variation existed[10]. In addition, PM2.5 also played a role

14

in composition of the lung microbiome[11]. The effects of geography and

15

environment alter the microbiome, which in turn affect human health. For instance,

16

Hosgood et al found a potential role of the lung microbiota in lung cancer attributed to

17

household coal burning exposures[12]. However, the geographic variation in lung

18

microbiota was not observed in studies enrolling healthy subjects from eight US

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cities[13] as well as in community members detecting in British volunteers[14]. But

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these studies of the lower respiratory tracts of healthy volunteers have been restricted

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to Western Europe and North America[15]. More relevant studies are needed to

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ACCEPTED MANUSCRIPT confirm the relationship between the lung microbiome and the prevalence of lung

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cancer adjusting for geographical variables.

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Microbiome and lung diseases

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Several recent studies have demonstrated that a low-density, diversified microbial

5

ecosystem is present in bronchoalveolar lavage (BAL) fluid, sputum, and lung tissues.

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The lung microbiota also showed a dynamic balance of microbial immigration and

7

elimination[16-18]. The lung microbiota of healthy adults is similar to those of the

8

oropharynx,

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Proteobacteria and Bacteroidetes[13, 17, 19]. Emerging evidence has illustrated the

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complex interactions between the lung microbiome and lung health. Many microbial

11

features (summarized in Table 1) are strongly correlated with specific lung disease

12

phenotypes.

13

Chronic obstructive pulmonary disease

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Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease

15

resulting from colonization of potentially pathogenic microorganisms in the

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respiratory tract[20, 21]. A recent microbiota survey using 16s rRNA sequencing

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approach found that global changes of microbiome were associated with COPD

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status[7, 22-29]. The most differentially abundant genera between COPD patients and

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healthy controls include Pseudomonas, Streptococcus, Prevotella and Haemophilus

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when testing samples from lung tissues, BAL fluid, and sputum. The relative

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abundances of Streptococcus, Pseudomonas and Haemophilus are aggravated with

the

predominant

bacterial

phyla

include

Firmicutes,

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ACCEPTED MANUSCRIPT exacerbation of COPD. In addition, commensal fungal and viral communities have

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been identified as potential cofactors in COPD by promoting outgrowth of pathogenic

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bacteria[30, 31].

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Cystic fibrosis

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Cystic fibrosis (CF) is a progressive genetic disease caused by genetic mutations in

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the CF transmembrane conductance regulator (CFTR) protein[32]. Microorganisms,

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including pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus,

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have been found in CF lungs. Several studies suggested that the diversified

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microbiome in CF might lead to variations in CF phenotypes[33-37]. A longitudinal

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study with infants and children showed reduced microbial diversity and increased

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lower airway inflammation in children with CF[35]. Furthermore, changes in the

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composition of the microbiota may be associated with partial CF exacerbations and

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could be useful in the prediction and management of CF pulmonary exacerbations[33,

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38].

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Asthma

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Risk of childhood asthma has been associated with the abundance of Moraxella

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catarrhalis,

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hypopharynx[39-41]. In human adults, studies have reported that the level of

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Proteobacteria in the lower airway was correlated with asthma[42]. Further studies

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revealed that abundances of Streptococcus II, Gemella, Rothia and Porphyromonas

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were

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Haemophilus

significantly

lower

influenza,

in

or

neutrophilic

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Streptococcus

compared

to

pneumoniae

eosinophilic

in

and

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and asthma phenotypes[43, 44]. Additionally, differences in the patterns of fungi have

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also been found between asthma patients and controls[45, 46].

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Idiopathic pulmonary fibrosis

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Previous studies suggested that infectious agents might play a role in acute

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exacerbations but not in the pathogenesis of idiopathic pulmonary fibrosis (IPF).

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However, recent studies have demonstrated the role for bacteria in the pathogenesis of

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IPF[47, 48]. An analysis of the Correlating Outcomes with biochemical Markers to

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Estimate Time-progression (COMET) study has revealed that an over-representation

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of special microbes such as Streptococcus, Prevotella, and Staphylococcus, in IPF

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patients compared with healthy individuals and showed that relatively high

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abundances of Streptococcus and Staphylococcus were associated with IPF disease

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progression[49]. However, the mechanisms mediating the roles of the microbiome in

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IPF are still unclear.

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In general, as illustrated in Figure 1, homeostasis of the lung microbiome is associated

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with the balance between immune sensing and tolerance of the commensal

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microbiota[50]. Host lung has established three major pathways to sense and defend

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against the invasion of the pathogens. Firstly, alveolar surfactant, covering on the

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surface of lung alveolar epithelial cells, participates in lung innate immunity[51].

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Secondly, the epithelial cell layer impedes the translocation of pathogens. Finally, the

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pattern recognition receptors (PRRs), including toll-like receptor (TLR) and

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ACCEPTED MANUSCRIPT nucleotide-binding oligomerization domain-like receptors (NOD-like receptors),

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which are distributed on surface of epithelial cells, dendritic cells, and macrophages,

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function to prevent the overload of pathogens or metabolites[52]. Downstream

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inflammatory signal pathways are then activated to eliminate pathogens[53].

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Conversely, tolerance of commensals is mediated by anti-inflammatory macrophages

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in alveoli by suppressing inflammatory pathways and inhibiting adaptive immune

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responses[54-56].

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The microbiome and lung cancer

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Previous studies have demonstrated that the microbiota was linked to many

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malignancies, including colorectal, gastric, hepatocellular, and pancreatic cancers[57,

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58]. The relationship between the microbiota and lung cancer, the leading cause of

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cancer-related morbidity and mortality worldwide[59], has been investigated by many

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epidemiological studies, and a significant relationship has been found between

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Mycobacterium tuberculosis (TB) and lung cancer[60]. The epidemiological links of

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the two diseases were mechanically interpreted by chronic inflammation-associated

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carcinogenesis according to previous studies[61]. One possible reason is that

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persistent infection by TB organisms induces the production of TNF and lead to

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pulmonary inflammation. In addition, pulmonary fibrosis causes by TB led to

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synthesize extra-cellular matrix (ECM), which is involved in the development of lung

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cancer. In turn, lung cancer patients are immunocompromised after chemotherapy,

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increasing the risk of TB infection. In addition, regional tumor peptides, antigens, and

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ACCEPTED MANUSCRIPT even radiotherapy may lead to granulomas microenvironment deregulation, allowing

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TB mycobacteria to proliferate[62]. Two published meta-analyses also demonstrated

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that TB was a risk factor for lung cancer[61, 63].

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However, increasing evidence revealed that globe changes of microbiome played a

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central role in the development of lung cancer. In general, alpha diversity (the number

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[richness] and distribution [evenness] of taxa expected within a sample) is

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significantly higher in non-malignant lung tissues than in tumor lung tissues. Beta

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diversity (diversity in the microbial community between different samples) is not

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significantly different between non-malignant and tumor tissues[10, 64]. However,

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several taxa have been shown to be enriched in cancer cases compared with control

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cases. Laroumagne et al. identified gram-negative bacteria such as Haemophilus

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influenzae, Enterobacter spp., and Escherichia coli, as colonizing in lung cancer by

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analyzing 216 bronchoscopic samples[65]. Hosgood et al. studied oral and sputum

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samples from women in China and found that the Granulicatella, Abiotrophia, and

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Streptococcus genera were enriched in lung cancer patients compared with healthy

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controls[12]. Moreover, higher alpha diversity of the lung microbiota was observed in

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cases who used smoky coal for cooking and heating compared with those using

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smokeless coal in sputum samples. In contrast, no significant difference was found in

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oral samples[12]. Yan’s study showed that Capnocytophaga, Selenomonas, Veillonella,

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and Neisseria were significantly altered in squamous cell carcinoma and

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adenocarcinoma patients compared to controls in salivary samples. Additionally, the

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ACCEPTED MANUSCRIPT combination of two bacterial biomarkers, i.e., Capnocytophaga and Veillonella,

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showed good performance in the prediction of squamous cell carcinoma (SCC) and

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adenocarcinoma (AC), which might attribute to lung cancer screening[66]. Moreover,

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a pilot study of 10 cases, found that Streptococcus viridans and 16 other species were

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only significantly abundant in lung cancer samples, whereas seven bacterial species

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were only found in controls[67]. In addition, according to a study by Lee et al., two

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phyla (Firmicutes and TM7) and two genera (Veillonella and Megasphaera) were

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relatively more abundant in BAL fluid from lung cancer patients. In lung cancer

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patients, a significantly higher ratio of Firmicutes to Bacteroidetes was observed in

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smokers than in non-smokers. Notably, an increase in the phylum TM7 was observed

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in both COPD and lung cancer cases, indicating that TM7 might play a potential role

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in the transformation of COPD to lung cancer. A combination of Megasphaera and

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Veillonella, which showed significantly high AUC value in predicting lung cancer,

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could serve as a biomarker for lung cancer[68].

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Because lung biopsy is not ethical in healthy human subjects, analysis of saliva,

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sputum, bronchoscopic samples, and BAL fluid is typically used in the research field

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as the alternative approaches to resemble the alternations of microbiome in lung.

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However, because samples from these alternative locations may contain possible

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contamination from the upper respiratory tract[9], analysis of lung tissue may provide

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a more accurate assessment of the microbiome in lung cancer. Yu et al. found that the

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lung microbiota was unique and different from the digestive tract microbiota in

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ACCEPTED MANUSCRIPT healthy subjects. Moreover, a lower alpha diversity was observed in lung tumor

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tissues compared with that in normal tissues, which has also been observed in other

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respiratory diseases. Additionally, higher phylogenetic diversity with increased

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relative abundance of Thermus and decreased relative abundance of Ralstonia was

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observed in adenocarcinoma compared with squamous cell carcinoma, implying that

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the microbiota might be correlated with cancer histology. Further analysis revealed

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that Legionella was highly abundant in metastasis cases, suggesting that Legionella

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might play a role in tumor progression through multiple pathways[10]. Another

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important study reported that significant decreases in microbial diversity were

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observed in patients with lung cancer in comparison with controls as well as that

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alpha diversity steadily declined from healthy site to noncancerous to cancerous site

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using 24 lung cancer patients with unilateral lobar masses and 18 healthy controls

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undergoing bronchoscopies. At genus level, Streptococcus was significantly more

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abundant in cancer cases than in controls and exhibited moderate classification

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potential, whereas Staphylococcus was more abundant in the controls, indicating that

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changes of the microenvironment were correlated with the development of lung

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cancer[69]. However, to date, most studies were cross-sectional and only conducted

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with modest sample sizes. Further large-scaled studies are needed to validate

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microbial biomarkers or microbial therapies for lung cancer patients.

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Possible mechanisms linking microbiome with carcinogenesis

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Microbiome dysbiosis

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ACCEPTED MANUSCRIPT Symbiotic relationship between host and the microbiome is based on multi-level

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barriers and the immune sensing system[70, 71]. Once the barrier defects or immune

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defects disappear, perturbation of the composition of the microbiome and bacterial

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translocation occur, resulting into pathological interactions between the microbiome

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and epithelial cells or the immune system[72, 73]. This may boost dysbiosis and

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consequent chain-reactions, leading to carcinogenesis. Other factors such as activation

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of inflammatory signaling, dietary changes, infections, and NOD2-deficiency, can

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also lead to dysbiosis[74-76]. Dysbiosis of the microbiome causes commensal

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microbes to decrease and inflammation-inducing bacteria to increase, which can

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induce carcinogenesis by multi-levels.

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The underlying mechanisms mediating the microbiome and carcinogenesis have been

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proposed and examined by many studies; the results suggested that dysbiosis of the

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microbiota modulated the susceptibility of malignancies in multi-levels, including

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increased genotoxic and virulence effects, altered metabolism, immune response, and

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pro-inflammation (illustrated in Figure 2).

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Genotoxicity and virulence effect

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Reactive oxygen species (ROS) has been identified to mediate DNA damage

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responses by previous reports. Recent studies implicated that dysbiosis of microbiota

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would alter ROS level to induce the DNA damage response and carcinogenesis.

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Bacterial toxins, such as Cytolethal distending toxin(CDT), cytotoxic necrotizing

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factor 1, and Bacteroides fragilis toxin, were identified as mediums triggering

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ACCEPTED MANUSCRIPT double-stranded DNA damage responses[74, 77-81]. In addition, bacterial-driven

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hydrogen sulfide and superoxide radicals were found to be responsible for genomic

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instability[82]. Furthermore, Fad A, secreted by Fusobacterium nucleatum, regulates

4

the catenin signaling pathway by interacting with E-cadherin[83]. With regard to

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virulence effect, Burns et al. found an enrichment of virulence-associated bacterial

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genes in the microenvironment of colorectal cancer, which might be dependent on the

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genomes of Fusobacterium and Providencia[84].

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Metabolism

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The microbiome has been shown to participate in regulating host metabolism, which

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is relevant for detoxification, hormone and bile acid production, and nutrient and

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vitamin levels[85]. Previous studies have reported that the bacterial microbiota

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contributed to generate acetaldehyde, which is a crucial carcinogen[86, 87]. In

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addition, deoxycholic acid (DCA), an obesity-induced gut microbial metabolite,

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contributes to obesity-associated development of hepatocellular carcinoma[88, 89].

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Furthermore, recent studies found that dietary fiber would facilitate the fermentation

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of short-chain fatty acids (SCFAs) by the gut microbiome[90, 91]. SCFAs exert

17

anti-inflammatory effects and decrease the incidence of colon and mammary

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cancer[92, 93].

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Inflammation

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Activation of inflammation pathways, such as microbe-associated molecular pattern

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(MAMP) or PRR signaling, not only senses the status of the microbiota but also

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ACCEPTED MANUSCRIPT triggers the proliferation and survival of epithelial cells under certain circumstances,

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thereby promoting the development of cancer. Accumulating evidence indicated that

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activation of TLRs played a central role in mediating carcinogenesis in colon, gastric,

4

liver, and pancreatic cancers[94-96]. Knocking out of TLR4 in mice suppresses

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carcinogenesis. The carcinogenic effects of TLRs are mediated by activating the

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nuclear factor-κB (NF-κΒ) pathway and the transducer signal transducer and activator

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of transcription 3 (STAT3) to promote the survival of malignant cells [97].

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Interestingly, cancer-associated modulation of TLRs increases susceptibility of

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particular infections in order to promote carcinogenic process by increasing

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expression of certain TLRs[98]. In addition, the microbiota induces MYD88 in

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myeloid cells, triggering IL-23 signaling to promote tumor progression and the

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development of a tumoral IL-17 response[99, 100]. One recent study showed that

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deficiency of IL-17C would promote the growth and metastasis in lung cancer

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model[101]. NLRs are another subfamily of PRRs localized to the cell membrane.

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NLRs launch a series of defensive mechanisms against the invasive bacteria. NOD1

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exerts protective effects, acting as a barrier to prevent the transition from

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inflammation to carcinogenesis[102]. NOD2 plays a vital role in modulation of

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microbiota and decreases the susceptibility of CRC[103, 104]. Knockout of NOD2 in

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mice results into overload of bacteria and inflammation. In addition, NLRP6

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deficiency in mice decreases IL-18 production and increases the susceptibility of

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colorectal cancer[105]. Similar results have been found in NLRP12 deficiency mice,

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ACCEPTED MANUSCRIPT 1

which

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carcinogenesis[106].

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Immune response

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The microbiome plays a significant role in shaping the adaptive immunity throughout

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life time. Therefore, the central role of how microbiome modulates the immune

6

responses in cancers must call for attention. Enterotoxigenic Bacteroides fragilis

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(ETBF) triggers the activation of STAT3 by a selective T helper type 17 (Th17)

8

response in mice, indicating that human commensal bacteria can induce cancer via a

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Th17-dependent pathway[107]. In addition, activation of the tumor-associated

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microbiota and TLR signaling stimulates the expression of calcineurin and nuclear

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factor of activated T cells (NFAT) factors, which sustains the survival and

12

proliferation of cancer stem cells[108]. Another investigation demonstrated that

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microbial-derived butyrate could expand the pool of regulator T cells by activating the

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forkhead box P3 (FOXP3) and G protein-coupled receptors[109-111]. Furthermore,

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the pathological microbiota promotes the epithelial IL-17C expression in COPD

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patients, thereby enhancing tumor growth by neutrophilic inflammation in the tumor

17

microenvironment[101].

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Studies of the mechanisms linking the lung microbiome and lung cancer are still

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preliminary. Chronic lung inflammation, such as COPD, is defined as a risk factor for

20

lung cancer[112]. Recent studies have revealed the role of variations in the lung

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microbiome in mediating the development and progression of lung cancer. Jungnickel

an

important

role

in

dysbiosis

of

the

microbiome

and

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ACCEPTED MANUSCRIPT et al found that the epithelial cytokine IL-17C, induced by aberrant bacteria such as

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nontypeable Haemophilus influenzae (NTHi), in COPD patients, mediates the

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tumor-promoting effects of bacteria by increasing neutrophilic inflammation. Thus,

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IL-17C promotes tumor-associated inflammation and tumor proliferation[101].

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Another study demonstrated that exposure to the combination of smoking and NTHi

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promoted metastatic growth and proliferation of lung cancer as a result of

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smoking-induced translocation of bacterial factors[113]. In addition, IL-6 has been

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identified to play essential role in lung cancer by promoting COPD-like

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inflammation[114]. Furthermore, microbiota-induced Th17 cells could promote lung

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cancer cell proliferation and angiogenesis[115]. Commensal bacteria are crucial to

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maintaining immune homeostasis in host. Cheng et al. demonstrated that the

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commensal microbiota contributed to the γδT17 cell response against lung cancer in a

13

mouse model[116].

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Prospective

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A few preliminary studies provided us an initial overview of the interplay between the

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lung microbiome and lung cancer. However, many questions still remain to be

17

answered, and the current knowledge on lung cancer is far less than that on other

18

cancers, such as gastric and colon cancers. Among those major challenges in

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researches of lung microbiome and lung cancers, several critical challenges include:

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First of all, it is known that many risk factors including tobacco smoking, exposure to

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various carcinogens, air pollution, family history and etc. are contributors to the lung

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ACCEPTED MANUSCRIPT cancer susceptibility. However, there is barely any current lung microbiome study

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containing a large sample size, which is statistically sufficient to adjust for many

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important cancer confounding risk factors in the multivariable analysis. For the future

4

studies, a national /international study consortium is essential to establish a

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standardized protocol for sample collection and processing, quantification of bacterial

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loading, and the NGS sequencing analysis pipeline in order to carry out comparative

7

and meta-analysis on interplays between the lung microbiome and lung cancer.

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Secondly, current lung microbiome research is lacking of the adequate controls to

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adjust for the systematic biases from sampling types and environmental contaminants.

10

Using different specimen types, such as lung tissues, sputum, bronchoscopic samples,

11

and BAL fluid, to represent the lung microbiome can be problematic and give

12

different results merely due to sampling bias. In addition, certain sample types contain

13

environmental contaminants from the oral sites, and the oral microbe could pass to

14

bronchoscope through the mouth. Lung biopsies may be better than other sample

15

types in term of avoiding oral contamination. Furthermore, for some low biomass

16

lung sample types, such as BAL fluid and lung biopsies, even low environmental

17

contamination in samples may contribute dominantly to the PCR or NGS results.

18

Therefore, it is of particular importance in the lung microbiome study. In a typical

19

sample collection process, lung samples should be collected with sterile reagents and

20

devices. However, in wet-lab practice, it is challenging to obtain absolutely

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contaminant-free DNA/RNA from those samples. To filter the signal from those

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ACCEPTED MANUSCRIPT possible contamination from reagents and equipment, blank reagents/tools should also

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be included as systematic controls in the analysis.

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Thirdly, more hypothesis-driven studies are required to explore the causative

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relationships between lung microbiome and lung cancer. For instance, the existence of

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the gut-lung axis and its potential influences on the chronic lung diseases, such as

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COPD, CF, and asthma, has been proposed by several studies[117]. However, the

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underlying mechanisms through which the gut microbiome may affect the

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development of lung cancer is still need to be investigated. Further studies with

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longitudinal and larger sample sizes are essential to investigate the mechanistic links

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between the microbiome and lung cancer.

11 Acknowledgements & Funding

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This work was supported by the National Natural Science Foundation of China (Nos.

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81372321, 81472702, 81501977, 81672294), Natural Science Foundation of Jiangsu

15

Province

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Development Project of Jiangsu Province (No. BM2015004). This work was also

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supported by International exchange and cooperation program for graduate education

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of Nanjing Medical University for oversea study.

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number:

SBK016030028),

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(grant

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Conflict of interest

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None.

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and

the

Innovation

Capability

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15 Figure legend

17

Figure 1. Maintenance of hemostasis depends on delicate equilibrium between

18

immune system and resident microorganisms. The commensal microbiota contributes

19

to immune tolerance through decreasing lung inflammation and dendritic cells

20

recruitment (left panel). Surfactants containing sIgA and epithelial cells protect the

21

host by clearing the potential pathogens. Dendritic cells are activated by microbiota

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ACCEPTED MANUSCRIPT pathogens and present antigen to T cells. Macrophages and T cells respond to

2

microbial colonization and prevent the overload of pathogens or metabolites. The

3

pattern recognition receptors (PRRs), including Toll-like receptor (TLR) and

4

nucleotide-binding oligomer-ization domain-like receptors (NOD-like receptors),

5

activate the NF-κB signaling pathway to produce series of inflammatory factors.

6

Induced B cells differentiate to secrete sIgA into the surfactant (right panel). SCFA

7

decreases the inflammatory response through interacting with G-protein couple

8

receptor. SCFA: Short-Chain Fatty Acids; LPS: lipopolysaccharide; GPR43:

9

G-protein-coupled receptor 43.

10

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Figure 2. The mechanisms of microbiota modulating carcinogenesis. The microbiota

12

produce the cytotoxicity-related components, inducing the DNA damage of host cells.

13

The microbiota and its metabolites activate the TLRs and result into downstream

14

inflammatory reactions. These inflammatory activators triggers downstream critical

15

signaling pathways, which promote the malignant behaviors of host cells. NLRs serve

16

as tumor suppressers through decreasing the inflammatory responses. The

17

translocation of microbial pathogens leads to a series of immune responses, which

18

promote the carcinogenesis of host cells. EMT: epithelial–mesenchymal transition;

19

DCA: deoxycholic acid; CDT: cytolethal distending toxin.

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Table Legend

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ACCEPTED MANUSCRIPT Table 1. Current findings of relationship between lung microbiota and lung disease.

2

Table 2. Current findings on relationship between lung microbiota and lung cancer.

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Table 1. Current findings of relationship between lung microbiota and lung disease.

RI PT

Propionibacterium 

24

phyla: Firmicutes
genera: Lactobacillus


SC

Sze et al

25

Pragman et al

26

27

Garcia-Nuñez et al

Disease

Sample type

26

lung tissue

24

lung tissue broncho alveolar

phyla: Fusobacteria
genera: Leptotrichia
Fusobacterium


32 16

sputum

17

sputum

8

sputum

134

sputum

genera: Staphylococcus
Burkholderia
Streptococcus


4

sputum

genera: Pseudomonas
Burkholderia


23

sputum mouthwash

genera: Pseudomonas
Corynebacterium
Moraxella


Millares et al

Obstructive Pulmonary

size

phyla: Firmicutes
genera: Ochrobactrum
Stenotrophomonas 

23

Kim et al

Chronic

Sample

Differential taxa features*

M AN U

condition

References

phyla: Proteobacteria
Firmicutes
Actinobacteria  Bacteroidetes 

lavage

phyla: Bacteroidetes
Firmicutes
Fusobacteria
Actinobacteria
Proteobacteria 

28

Lee et al

genera: Prevotella
Porphyromonas
Veillonella
Fusobacterium
Streptococcus
stable: H influenzae


29

Garcha et al

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Medical

33

Carmody et al Fodor et al

34

Frayman et al

35

Cystic Fibrosis

EP

exacerbation: S pneumoniae
M catarrhalis


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1

genera: Staphylococcus
Streptococcus
Pseudomonas
genera: Streptococcus
Burkholderia
Prevotella
Haemophilus


36

Laguna et al

Porphyromonas, and Veillonella
37

Feigelman et al

genera: Pseudomonas
Staphylococcus
Stenotrophomonas
Achromobacter


95 12 17

broncho alveolar lavage broncho alveolar lavage sputum

ACCEPTED MANUSCRIPT

22

Hilty et al

44

phyla: Proteobacteria
genera: Streptococcus
39

genera: S. pneumoniae
M. catarrhalis
H. influenzae
genera: Haemophilus
Neisseria
Fusobacterium


41

Durack et al

Porphyromonas
48

Molyneaux et al

genera: Haemophilus


Streptococcus
Neisseria
Veillonella


49

Han et al

M AN U

Idiopathic

genera: Staphylococcus
Streptococcus


Fibrosis 47

Molyneaux et al

genera: Streptococcus
Prevotella
Veillonella
Pseudomonas


Haemophilus


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: Microbiota increases in cases compared to controls; : Microbiota decrease in cases compared to controls;

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20

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Teo et al

Bisgaard et al

Pulmonary

bronchoscopy

phyla: Proteobacteria
Firmicutes Actinobacteria

40

Asthma

genera: Haemophilus spp.


SC

Marri et al

phyla: Proteobacteria


sputum

234

nasopharyngeal

321

hypopharyngeal

42

bronchoscopy

65 55 35

broncho alveolar lavage broncho alveolar lavage bronchoscopy

ACCEPTED MANUSCRIPT

Table 2. Current findings on relationship between lung microbiota and lung cancer. Sample

Differential taxa features 65

Laroumagne et al 15

Hosgood et al

size

Genera: Haemophilus influenzae
Enterobacter sp.
Escherichia coli
Genera: Granulicatella
Abiotrophia
Streptococcus Genera: Capnocytophaga
Selenomonas
Veillonella
Neisseria

66

Yan et al

Streptococcus

Cameron et al

Streptococcus Genera: Escherichia coli Fusobacterium nucleatum

Lee et al

Genera: Thermus
Ralstonia


69

Genera: Streptococcus
Staphylococcus

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Liu et al

EP

13

Yu et al

2

Genera: Veillonella
Megasphaera


TE D

68

216

bronchoscopy

16

sputum saliver

30

saliver

10

sputum

28

broncho alveolar lavage

165

lung tissues

M AN U

Genera: Granulicatella adicens
Mycobacterium tuberculosis
67

Sample type

RI PT

References

SC

1

42

lung tissues and bronchoscopy

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ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT 1

Highlights:

2




3

The Lungs are not sterile, and the lung microbiome is associated with lung health.




The lung microbiome is linked to lung cancer.

5




Microbial dysbiosis may modulate the risk of malignancy at multiple levels.

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