The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution

More than 90% of lung cancers are caused by cigarette smoke and air pollution, with polycyclic aromatic hydrocarbons (PAHs) as key carcinogens. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China, attributed to smoky coal combustion-generated PAH pollution. Here, we screened for abnormal inflammatory factors in non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used, and found that a chemokine CXCL13 was overexpressed in 63/70 (90%) of Xuanwei NSCLCs and 44/71 (62%) of smoker and 27/60 (45%) of non-smoker CR patients. CXCL13 overexpression was associated with the region Xuanwei and cigarette smoke. The key carcinogen benzo(a)pyrene (BaP) induced CXCL13 production in lung epithelial cells and in mice prior to development of detectable lung cancer. Deficiency in Cxcl13 or its receptor, Cxcr5, significantly attenuated BaP-induced lung cancer in mice, demonstrating CXCL13’s critical role in PAH-induced lung carcinogenesis. DOI: http://dx.doi.org/10.7554/eLife.09419.001


Introduction
Air pollution is a diverse mixture of pollutants that originated from anthropogenic and natural sources, is comprised of particulate matter (PM), gases (e.g., sulfur oxides, carbon monoxide, ozone), organic compounds (e.g., polycyclic aromatic hydrocarbons, PAHs), metals (e.g., lead, vanadium, and nickel), and others, such as microbes (Akimoto, 2003;Huang et al., 2014). Air pollution is a global environmental health risk that affects the populations in developed and developing countries alike, and satellite observations suggest that 80% of the global population resides in locations where the ambient pollutant concentrations exceed the World Health Organization (WHO) Air Quality Guideline (van Donkelaar et al., 2010). Outdoor air pollution in cities and rural areas was estimated to cause 3.7 million premature deaths annually worldwide in 2012, including 220,000 deaths due to lung cancer (WHO, 2014). Recently, outdoor (Loomis et al., 2013) and indoor (WHO, 2010) air pollution has been classified as a Group 1 carcinogen in humans by the International Agency for Research on Cancer (IARC) of WHO. Indeed, the risk of lung cancer rises by 18% for every increase of 5 mg/m 3 of PM smaller than 2.5 mm in diameter (PM 2.5 ) in the environment; the risk increases by 22% for every increase of 10 mg/m 3 in PM smaller than 10 mm (PM 10 ) (Raaschou-Nielsen et al., 2013). There have been efforts to investigate how air pollution causes human cancers by exposing cells and animals to PM or related chemicals (Straif et al., 2012), but the carcinogenic mechanism remains elusive, at least in part, due to the difficulty of identifying human lung cancers that are causally associated with air pollution.
Xuanwei City of the Yunnan Province in China provides a significant association between air pollution and lung cancer (Lan et al., 2002;Sinton et al., 1995;Mumford et al., 1987). Until the 1970s, residents of this region used smoky coal in unvented indoor fire pits for domestic cooking and heating, which are processes that release high concentrations of PM 2.5 /PM 10 that contain high concentration PAHs (Mumford et al., 1987). Indoor air pollution in Xuanwei is associated with a significant increase in the absolute lifetime risk of developing lung cancer (Xiao et al., 2012), and the lung cancer incidence in this region is among the highest in China (Mumford, et al., 1987;Xiao et al., 2012;Li et al., 2011). In particular, nearly all women in this region are non-smokers and cook food on the household stove, and the lung cancer incidence in women is high. In Xuanwei, the male-tofemale ratio of lung cancer incidences is 1.09:1, while that of China's national average reaches 2.08:1 (Xiao et al., 2012). Tobacco smoke was weakly and non-significantly associated with lung cancer risk in this region . In the 1990s, a reduction in the lung cancer incidence was noted after stove improvement, supporting the association between indoor air pollution and lung cancer (Lan et al., 2002). These findings had been cited by the IARC monograph to classify indoor emissions from household coal combustion as 'carcinogenic to humans (Group 1) (WHO, 2010). However, lung cancer incidence in Xuanwei is still increasing (Li et al., 2011), eLife digest Lung cancer causes the most cancer deaths worldwide. For decades, people have known that lung cancer is associated with environmental factors, and both cigarette smoke and air pollution are known to cause cancers in humans. Smoke and air pollution both contain chemicals called polycyclic aromatic hydrocarbons (or PAHs). These chemicals cause chronic inflammation of the lung, which in turn is a major risk factor for developing lung cancer.
However, it is unclear exactly how PAHs trigger inflammation and cancer. Xuanwei City in China is suited to the study of this question because until the 1970s its inhabitants used 'smoky coal' for cooking in unventilated indoor spaces; this produced high levels of small particles that contain high concentrations of PAHs. Women from this region, who traditionally do most of the cooking, have rates of lung cancer comparable to those of men. In other parts of China a woman's chance of getting lung cancer is approximately half that of a man's. Therefore, Xuanwei City provides a setting in which air pollution is a main contributor to lung cancer risk. Wang, Cheng et al. have now compared the levels of certain proteins (which are linked to inflammation) in lung cancer patients from Xuanwei City with those in control regions of China. The level of one such protein marker, called CXCL13, was particularly high in almost all patients from Xuanwei City, but only highly expressed in half of the patients from the control regions. Moreover, there was also a clear link between cigarette smoke and CXCL13 expression because, in control regions, smokers were much more likely to have high levels of CXCL13 than non-smokers. To test whether PAHs cause CXCL13 expression, Wang, Cheng et al. first exposed normal lung epithelial cells, cancer cells and then mice to a PAH. These experiments showed that CXCL13 levels did indeed increase and the mice developed lung tumours. However, when the genes for CXCL13 or its binding partner were deleted, the mice no longer got cancer when exposed to the PAH. This shows that CXCL13 signalling is an important mechanism by which PAHs cause lung cancer. Lastly, further experiments showed that CXCL13's binding partner is highly expressed on some immune cells that can promote lung cancer.
Importantly, the over-expression of CXCL13 occurred before the lung tumours developed. This might provide a new treatment strategy in which CXCL13 signalling could be inhibited after the exposure to PAHs. Future studies may now focus on discovering new drugs, or modifying existing drugs, to achieve this goal. possibly due to pollutants generated by coal-burning industrial plants that have moved into the area (Cao and Gao, 2012). Therefore, the population in this highly polluted region (HPR) provides a unique opportunity to dissect air pollution-related lung carcinogenesis. Furthermore, the key carcinogens of smoky coal combustion are PAHs (Lv et al., 2009), which are also the key carcinogens of tobacco smoke that causes more than 85% of global lung cancer deaths (Hecht, 2012). Xuanwei lung cancer is, therefore, applicable to elucidate tobacco smoke-induced lung carcinogenesis.
To systematically investigate air pollution-induced lung carcinogenesis, we used Xuanwei lung cancers to analyze the abnormalities in the cancer genomes (Yu et al., 2015), genome-wide DNA methylation, non-coding RNAs (miRNAs  and lncRNAs), and inflammation factors. The abnormalities found in the HPR lung cancers were tested in patients from control regions (CRs) where smoky coal was not used, to compare the difference between HPR and control region (CR) lung cancers. In this study, we explored the abnormal inflammatory factors in HPR non-small cell lung cancers (NSCLCs).

Abnormal inflammatory factors in HPR NSCLCs
Using a microarray analysis of 84 cytokines/chemokines in tumor samples and their adjacent normal lung tissues of eight HPR NSCLCs, we found that the expression of four cytokines (IL-1F5, IL-1F9, MIF, and SPP1) and seven chemokines (CXCL13, CCL7, CCL20, CCL26, CXCL6, CXCL9, and CXCL14) was increased in tumors compared with their counterpart normal lung tissues ( Figure 1A). Among them, CXCL13 was the most significantly up-regulated gene, with an average of 63-fold (10.48-173.65) higher expression in the tumor samples than in the normal controls. The expression of three cytokines (IL-1a, IL-5, and TNF-a) and nine chemokines (CCL4, CCL17, CCL18, CCL21, CCL23, CXCL1, CXCL2, CXCL3, and CXCL5) was lower in the tumor samples than in the normal controls ( Figure 1A).

Overexpression of CXCL13 in NSCLCs
We expanded these observations, and found that the expression of CXCL13 was elevated in the tumor tissues from 63/70 (90%) HPR patients ( Figure 1B) and 71/131 (54.2%) CR patients (Table 1) compared with their normal controls. CXCL13 expression was much higher in the HPR patients than the CR cases (p<0.002; Figure 1C). Using immunohistochemistry (IHC) and immunoreactivity scoring, we showed that the expression of the CXCL13 protein was significantly higher in the tumor samples than their adjacent normal controls ( Figure 1D, E). CXCL13 expression was higher in the HPR NSCLCs than the CR patients, and the CR smoker NSCLCs had higher CXCL13 levels than the CR non-smoker patients ( Figure 1B, D, E, and Table 1). Using Western blot assays, we found that CXCL13 was much higher in the tumor samples than their adjacent normal lung tissues ( Figure 1F). ELISA showed that the CXCL13 serum concentration was higher in the HPR patients compared with the CR patients, while the latter was higher than the healthy donors ( Figure 1G).

Analysis of association between CXCL13 expression and clinical characteristics
CXCL13 expression was not significantly different in smoker and non-smoker HPR patients (p=0.17; Table 1), suggesting that severe air pollution had a carcinogenic effect on humans. In NSCLCs from CRs, however, the expression of CXCL13 was significantly higher in smokers (44/71, 62%) than in  Table 1), suggesting a potential association between tobacco smoke and CXCL13 expression. The multivariate logistic analyses showed that among the 201 NSCLCs, CXCL13-high was associated with HPR (p=4.6Â10 -6 ) and tobacco smoke (p=0.032; Table 2).
To investigate CXCL13 expression in NSCLCs of other cohorts, a cancer microarray database Oncomine (Rhodes et al., 2004) (www.oncomine.org) was applied. We found that in several works of this database (Okayama et al., 2012;Bhattacharjee et al., 2001;Hou et al., 2010;Landi et al., 2008;Selamat et al., 2012;Talbot et al., 2005;Su et al., 2007;Stearman et al., 2005), CXCL13 in tumor samples was elevated compared with their paired normal lung tissues or other normal controls ( Figure 1H). CXCL13 was also higher in smoker NSCLCs than non-smoker patients in some studies (Okayama et al., 2012;Landi et al., 2008;Selamat et al., 2012) ( Figure 1I). In microarray data sets GSE6135 (Ji et al., 2007), GSE21581 (Carretero et al., 2010), and GSE54353 (Xu et al., 2014) deposited in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) from genetically engineered mouse models of lung cancer, Cxcl13 was increased in Kras G12D Stk11 -/mice  Table 3 for their baseline demographic characteristics). The median follow-up was 1087 days (range, 187-1845 days). DOI: 10.7554/eLife.09419.003 The following source data and figure supplement are available for figure 1: (especially in metastatic tumors), as compared with Kras G12D Stk11 wt mice ( Figure 1J). These results suggest that CXCL13 overexpression was not specific to the Chinese cohorts, and Cxcl13 may play a role in Stk11-related lung tumorigenesis. We showed patients of both HPR and CR regions with relatively earlier disease (stages I and II) had lower CXCL13, while those with advanced disease (stages III and IV) had higher CXCL13 ( Figure 1K), and multivariate logistic analyses showed that CXCL13-high was associated with TNM stage (Table 2, p=0.003). In 54 CR patients whose survival information was available (Table 3), the median survival time of CXCL13-high patients (965 days) was much shorter than the CXCL13-low cases (1193 days, p=0.03; Figure 1L). Kaplan-Meier estimates of survival of patients with NSCLC according to age (

BaP induces CXCL13 production in vitro and in vivo
PAHs were reported to be the major carcinogens in the PM 2.5 /PM 10 in HPR, as well as in the PM at urban locations in Beijing, Shanghai, Guangzhou and Xi'an in China during January 2013 (Huang et al., 2014). Clinically, a long latency is required for individuals to develop lung cancer since they were first exposed to smoking or air pollution. To test the effects of PAHs on cytokine/ chemokine production, the normal human lung epithelial 16HBE cells (Cozens et al., 1994) were exposed to a representative PAH compound benzo(a)pyrene (BaP) at 1 mM for a long period of time (30 days). We found that CXCL13 was the most significantly up-regulated gene among the 84 cytokines/chemokines ( Figure 2A). We confirmed that BaP up-regulated CXCL13 at both the mRNA and protein levels in a dose-and time-dependent manner in 16HBE and A549 lung cancer cells ( Figure 2B, C). To test the in vitro observations in vivo, A/J mice were treated with vehicle control (corn oil) or 50-100 mg/kg BaP (Figure 2-figure supplement 1A, modified from previous studies (Wattenberg and Estensen, 1996). All BaP-treated animals developed lung cancer within 6 months, as detected by a microCT scan (Figure 2-figure supplement 1B,C) and histopathology ( Figure 2D). We found that Cxcl13 was significantly up-regulated in the tumor samples compared with their normal counterparts at both the mRNA ( Figure 2E) and protein ( Figure 2D) levels, which paralleled the increase in Cxcl13 serum concentrations ( Figure 2F). Notably, BaP increased Cxcl13 concentrations in peripheral blood of the mice at 1 month after the beginning of the treatment ( Figure  To determine the source of Cxcl13, we performed IHC and immunofluorescence assays in lung cancer tissues of the mice. To do this, thyroid transcription factor-1 (Ttf-1) which is expressed in the lung epithelial cells (Lazzaro et al., 1991), was used to mark lung cancer cells, and Cd68 stain was employed to mark macrophages which can produce Cxcl13 in inflammatory lesions with lymphoid neogenesis (Carlsen et al., 2004). By IHC assay, we found that both the Ttf1-positive lung cancer cells and Cd68-positive macrophages were stained positive for Cxcl13, but Ttf1-positive cells constituted more than 95% of the cellular component of the lung tumor tissues of the mice ( Figure 2G). This observation was confirmed by immunofluorescence assay ( Figure 2H) using antibodies against Cxcl13 (green), Cd68 (red), and Ttf1 (white). These results indicate that Ttf1 positive lung cancer cells were the main source of Cxcl13 in mice exposed to BaP.
We further showed that the anti-inflammatory drug dexamethasone (DEX) (Wattenberg and Estensen, 1996) down-regulated Cxcl13 mRNA and protein, and reduced the Cxcl13 serum concentration ( Figure 2D-F). DEX reduced the tumor burden (Figure 2-figure supplement 1B,C, Figure 2D) and prolonged the survival ( Figure 2I) of the mice. Cxcl12, which was shown to be associated with lung cancer (Teicher and Fricker, 2010), was not significantly changed in the BaPtreated mice (Figure 2-figure supplement 1E).  BaP induces CXCL13 production by facilitating AhR translocation to the nucleus We screened for transcription factors that could regulate CXCL13, and found potential aryl hydrocarbon receptor (AhR) ( Figure 3A) and V-Rel Avian Reticuloendotheliosis Viral Oncogene Homolog A (RelA) (data not shown) binding sites in its promoter. AhR is a ligand-activated transcription factor that binds the xenobiotic-responsive element (XRE) or aryl hydrocarbon response element (AHRE) to regulate genes in response to the planar aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Fujisawa-Sehara et al., 1987;Lo and Matthews, 2012;Shimizu et al., 2000). The potential XRE-like sequence, 5'-GCCCAGGCTGGAGTGCAG-3', is located at 1.7 kb downstream from the transcription start site (TSS; Figure 3A). The potential interaction between AhR and CXCL13 was analyzed by the chromatin immunoprecipitation (ChIP) assay, and the results showed that AhR could not bind CXCL13 in A549 cells without BaP treatment. Interestingly, AhR-CXCL13 interaction was detected and CXCL13 was significantly up-regulated in the presence of BaP, revealed by quantitative PCR (qPCR) ( Figure 3B). However, significant interaction between AhR and CXCL12, CXCL14, CXCL11, or CXCL2 was not detected ( Figure 3B). BaP significantly induced luciferase activity driven by the CXCL13 promoter containing XRE-like sequence; deletion of the XRE-like element, co-incubation with the AhR antagonist a-NF (Wilhelmsson et al., 1994) or transfection of an AhR-specific siRNA significantly reduced luciferase activity ( Figure 3C-E). TCDD can activate AhR by inducing its translocation from the cytoplasm to nucleus (Pollenz et al., 1994). We reported that a-NF and siAhR drastically inhibited BaP caused translocation of AhR to nucleus in 16HBE cells ( Figure 3F-I). These results might explain why a-NF and siAhR significantly decreased CXCL13 expression ( Figure 3J) and concentration in the supernatant ( Figure 3K) in BaP-treated 16HBE cells.

Knockdown of Cxcl13 attenuates BaP-induced lung cancer
To uncover the role of CXCL13 in BaP-induced lung cancer, Cxcl13 knockout mice (Cyster et al., 2000) were treated with 100 mg/kg BaP twice a week for 8 weeks (Figure 4-figure supplement  1A,B), and the tumor burden of the mice was evaluated. First, tumor nodules in histologic sections of mice upon BaP treatment were analyzed as described (Tan et al., 2013), and the results showed that at treatment time points of 120 days, 180 days, and 240 days, Cxcl13 -/mice had much fewer lesions than Cxcl13 +/and Cxcl13 +/+ mice ( Figure 4A). Then, microCT was used to detect visible tumors in mice 240 days after BaP treatment. We found that Cxcl13 -/mice harbored much fewer lung tumors than Cxcl13 +/and Cxcl13 +/+ mice ( Figure 4B). Consistent with this observation, the tumor volume of Cxcl13 -/mice was significantly smaller than Cxcl13 +/and Cxcl13 +/+ mice ( Figure 4C). In Cxcl13 +/+ and Cxcl13 -/+ mice, the Cxcl13 serum concentrations were elevated 3 months after BaP treatment, at which time point no tumors were detected ( Figure 4D). Moreover, the life span of BaP-treated Cxcl13 -/mice was prolonged compared with the Cxcl13 +/+ and Cxcl13 -/+ mice ( Figure 4E). These results indicate that CXCL13 is required for BaP-induced lung cancer.
CXCL13-CXCR5 signaling is critical for BaP-induced lung cancer CXCL13 primarily functions by binding to the G protein coupled receptor CXCR5, and CXCL13 is currently the only known ligand of CXCR5 (Fö rster et al., 1996;Lazennec and Richmond, 2010). We tested the expression of CXCR5 in 24 NSCLCs, and found that it was expressed by the tumor samples but was not significantly higher than in paired normal lung tissues (p=0.089; Figure 4F). In A/J mice treated with BaP, Cxcr5 was slightly up-regulated (p=0.07; Figure 4G). To further show the role CXCR5 plays in lung carcinogenesis, Cxcr5 -/mice (Fö rster et al., 1996) were treated with BaP at 100 mg/kg BaP twice a week for 8 weeks (Figure 4-figure supplement 1B,C). We showed that BaP treatment induced lung cancer in Cxcr5 +/+ mice ( Figure 4H,I). However, BaP-treated Cxcr5 +/mice developed fewer tumors, and BaP-treated Cxcr5 -/mice had much ewer lung tumors ( Figure 4H,I). Consistently, the tumor volume of Cxcr5 -/mice was significantly lower than Cxcr5 +/and Cxcr5 +/+ mice ( Figure 4J). Furthermore, Cxcr5 -/mice had a prolonged life span compared with the wild-type and Cxcr5 +/mice ( Figure 4K). These results demonstrate that CXCR5 is also critical for BaP-induced lung cancer.
CXCR5-expressing macrophages promote lung cancer progression CXCR5 is expressed by B cells, T cells and macrophages (Fö rster et al., 1996;Nigrovic and Lee, 2005). We investigated which cells were involved in BaP-induced lung cancer by analyzing cell surface markers in the tumor microenvironment using antibodies against CD45, CD19, CD4, and CD68. Flow cytometry analysis showed a significant increase in Cd68 + macrophages in the lung tumor samples from BaP-treated A/J mice ( Figure 5A,B), which was confirmed by IHC ( Figure 5C). We found that 94.7% of these macrophages strongly expressed Cxcr5 ( Figure 5D). However, B cells and T cells were not enriched in the tumor microenvironment ( Figure 5-figure supplement 1). Moreover, CD68 + macrophages were enriched in human lung tumor tissues revealed by IHC ( Figure 5C) and immunofluorescence assays ( Figure 5E). The Tamm-Horsfall protein (THP-1) macrophage cell line also expressed high levels of CXCR5 and CD68 ( Figure 5E).
We investigated the potential role for these macrophages in promoting lung cancer. In a transwell migration assay, THP-1 cells induced the migration of A549-Luciferase (Luc) cells and H1975 cells from the upper chamber to the lower chamber ( Figure 5F). Stable transfection of CXCL13 into A549-Luc cells (designated A549-Luc-CXCL13 cells) significantly enhanced the migration activity, which was antagonized by an anti-CXCR5 antibody ( Figure 5F). Addition of CXCL13 to THP-1 cells enhanced the migration of H1975 cells, while an anti-CXCR5 antibody inhibited this effect ( Figure 5F).
A549-Luc-CXCL13 cells were injected into the right lung of the non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (whose macrophages were functionally immature), and bioluminescence was recorded 30 days later. We showed that compared with the A549-Luc cells, the A549-Luc-CXCL13 cells significantly increased the tumor burden in the recipient mice ( Figure 5G,H). Furthermore, tail vein-injection of THP-1 cells increased the volume of tumors formed by the A549-Luc-CXCL13 cells ( Figure 5G,H) and caused obvious metastases to the left lung of the mice ( Figure 5I).

CXCL13 induces SPP1 production by macrophages
The above results suggested that CXCL13 may induce macrophages to produce critical factors that promote lung cancer progression/metastasis. To identify these factors, a whole genome gene expression assay was performed in the eight patients ( Figure 1A), and 436 genes were found to be associated with CXCL13 (a coefficient >0.5 or <À0.5; Figure 6-source data 1). These genes were enriched in cytokine-cytokine receptor interactions, the Wnt signaling pathway, the calcium signaling pathway, and others ( Figure 6A). Among the secretion-related genes, MMP12, secreted phosphoprotein 1 (SPP1), which encodes a macrophage-secreted cytokine SPP1 or osteopontin (Kohri et al., 1992), and MMP7 had the highest coefficient values for interacting with CXCL13 ( Figure 6B). We tested 11 secretion-related genes in THP-1 cells, and found that the expression of SPP1 was the highest ( Figure 6C). Therefore, it was chosen for further investigation. In THP-1 cells, CXCL13 treatment significantly increased SPP1 concentrations in the supernatant ( Figure 6D). In trans-well assays, the addition of CXCL13 to THP-1 cells in the lower chamber enhanced the migration of H1975 cells, while SPP1 silencing by siSPP1 in THP-1 cells attenuated this effect ( Figure 6E, left). Transfection of CXCL13 into A549 cells increased their migration activity, while SPP1 silencing in THP-1 cells inhibited A549-CXCL13 cell migration ( Figure 6E, right panel). In BaP-treated A/J mice, the Spp1 serum concentration was significantly increased ( Figure 6F), and the tumor samples showed significantly increased Spp1 IHC staining compared with the control mice ( Figure 6G). By IHC assay, we found that Cd68 positive macrophages were also strongly stained by Spp1, whereas Ttf1-positive lung cancer cells were only weakly stained positive for Spp1 ( Figure 6H). This observation was confirmed by immunofluorescence assay ( Figure 6I) using antibodies against Spp1 (green), Cd68 (red), and Ttf (white). These results indicate that Cd68-positive macrophages were the main source of Spp1 in mice exposed to BaP.

CXCL13-CXCR5-SPP1 signaling induces an EMT phenotype
At least two tumors were found in left and/or right lungs of each BaP-treated mouse (Figure 2-figure supplement 1B), and CXCL13 induced cancer cell migration and metastasis ( Figure 5F-I, Figure 6E). We tested the epithelial mesenchymal transition (EMT) phenotypes that are associated with cancer progression and metastasis (Kalluri and Weinberg, 2009) in tumor samples from the BaP-treated A/J mice, and found that the expression of E-Cadherin was down-regulated, while N-Cadherin, Vimentin, Slug and Snail were up-regulated ( Figure 6-figure supplement 1A). At protein level, E-Cadherin was down-regulated, while N-Cadherin and Vimentin were up-regulated in the tumors (Figure 6-figure supplement 1B). b-catenin transport to the nucleus is critical for cells to enter into an EMT and acquire an invasive phenotype (Kim, 2002). We showed that the nuclear bcatenin levels were increased in the tumor samples from A/J mice ( Figure 6-  figure supplement 1D,E) and BaP-treated Cxcr5 +/+ mice ( Figure 6-figure supplement 1F,G), the expression of E-Cadherin was low, while the N-Cadherin and nuclear b-catenin were high. SPP1 was shown to activate b-catenin (Robertson and Chellaiah, 2010). To study the role of SPP1 in promoting the progression of lung cancer, A549 cells were transfected with pcDNA3.1-flag-SPP1. We found that SPP1 overexpression decreased the cytoplasmic expression but increased the nuclear expression of b-catenin ( Figure 6-figure supplement 1H,I). SPP1 overexpression in cancer cells led to the down-regulation of E-Cadherin and up-regulation of N-Cadherin and Slug at both the mRNA and protein levels ( Figure 6-figure supplement 1J,K), and up-regulation of Vimentin and Snail at the mRNA level ( Figure 6-figure supplement 1J). These findings were also observed in A549 cells supplemented with supernatants from THP-1 cells that were co-incubated with CXCL13 ( Figure 6-figure supplement 1H-K). These results suggest that SPP1 may transactivate b-catenin to induce an EMT in BaP-treated mice.

Discussion
In this study, we took the advantage of the epidemiology profile of Xuanwei lung cancer to systematically explore the abnormalities in inflammatory factors that are critical to air pollution-induced lung cancer, and reported that CXCL13 was up-regulated in 63/70 (90%) HPR NSCLCs ( Table 1). BaP induced lung epithelial cells to secret CXCL13 (Figure 2), which recruited tumor-associated macrophages (TAMs) and induced SPP1 production ( Figure 5, 6). SPP1 activated b-catenin by facilitating its translocation to the nucleus and promoted an EMT, leading to the progression of lung cancer ( Figure 6-figure supplement 1). CXCL13-CXCR5 signaling was required for PAH-induced lung cancer, because knockdown of Cxcl13 or Cxcr5 attenuated BaP-induced lung cancer in mice (Figure 4). These results suggest that the ubiquitous carcinogen BaP may induce tumor-promoting inflammation to facilitate the invasive growth and metastasis of lung cancer, shedding new insights into the carcinogenic mechanisms of air pollution.
CXCL13 may be associated with lung cancer risk in smokers (Shiels et al., 2013). We showed that in HPR, smoker and non-smoker NSCLCs had approximately equal expression levels of CXCL13, suggesting a carcinogenic effect of air pollution. In CR, however, smoker patients had higher CXCL13 expression than non-smoker cases ( Figure 1 and Table 1). Moreover, high CXCL13 expression was associated with advanced stage cancer and poor prognosis (Figure 2). These results indicate that CXCL13 is a common and critical factor in air pollution-induced and tobacco smoke-induced lung carcinogenesis. Furthermore, the serum concentration of CXCL13 was elevated in mice before the emergence of detectable lung tumor by microCT (Figure 2 and Figure 2-figure supplement 1), suggesting that this chemokine may represent a biomarker for early diagnosis, which warrants further investigation.
PAHs are key carcinogens in air pollution and tobacco smoke (Huang et al., 2014;Hecht, 2012), and cause mutations in TP53 and K-RAS, resulting in uncontrolled cell growth (Hecht, 2012). PAHs also perturb the immune system and induce production of the inflammatory factors to facilitate cancer progression in cellular and animal models (Zaccaria and McClure, 2013;N'Diaye et al., 2006;Umannová et al., 2011;Chen et al., 2012;Dreij et al., 2010). By treating 16HBE cells with a relatively low concentration (1 mM) of BaP for a long time course (30 days) to mimic BaP-induced lung cancer in humans, we showed that CXCL13 was the most significantly up-regulated gene among the 84 cytokines/chemokines ( Figure 2). Furthermore, CXCL13 was increased in 134/201 (66.7%) NSCLCs (Table 1), suggesting the clinical relevance of CXCL13 in BaP-induced lung cancer. AhR is critical for the carcinogenic action of BaP (Shimizu et al., 2000). We found that CXCL13 was a direct target of AhR (Figure 3), and deficiency in CXCL13 or its receptor, CXCR5, abrogated BaP-induced lung cancer (Figure 4), while an anti-CXCR5 antibody significantly inhibited lung cancer cell migration ( Figure 5). These results further demonstrate CXCL13's critical role in environmental pollutantinduced lung cancer, but its role in pre-malignancy and cell transformation remains to be addressed.
Cross-talk between cancer cells and cells of the neoplastic stroma is involved in the acquired ability for invasive growth and metastasis (Hanahan and Weinberg, 2011). Using a whole -genome gene expression array (Figure 6), we discovered that TAMs secreted SPP1, which activates b-catenin and is regulated by this transcription factor (Robertson and Chellaiah, 2010;El-Tanani et al., 2001). SPP1 induced the nuclear localization and activation of b-catenin in epithelial and cancer cells, resulting in an EMT phenotype (Figure 6-figure supplement 1H-K). At this stage, the detailed mechanisms of CXCL13-CXCR5-induced SPP1 production were unclear. However, our results suggested that SPP1-b-catenin could form a positive feedback loop to promote EMT and lung cancer progression. CXCL13 was shown to be able to induce an EMT in breast cancer via RANKL and Src activation (Biswas et al., 2014). We found that BaP or CXCL13 treatment or SPP1 overexpression in lung epithelial or cancer cells did not up-regulate RANKL or Src (data not shown), but did activate b-catenin ( Figure 6-figure supplement 1), suggesting that CXCL13 may induce an EMT via different mechanisms in different settings.
Chemokines can modulate tumor cell proliferation, survival, angiogenesis, senescence and metastasis . For example, CCL20 is induced by another important carcinogen in tobacco smoke, nicotine-derived nitrosaminoketone, and is overexpressed in smoker lung cancers and inversely associated with prognosis. CCL20 promotes lung cancer cell proliferation and migration . CXCL12 and CCR7 regulate the metastasis of breast cancers and NSCLCs (Phillips et al., 2003;Müller et al., 2001;Takanami, 2003), while CXCL1/2 enhance cell survival and promote chemoresistance (Acharyya et al., 2012). CXCL13 is overexpressed in breast cancer (Panse et al., 2008) and mediates prostate cancer cell proliferation (El-Haibi et al., 2011). Given their critical roles in cancer, pharmaceutical development pipelines are filled with new chemokine-targeting drugs to treat malignancies (Klarenbeek et al., 2012). DEX is a synthetic glucocorticoid used to counteract certain side effects of chemotherapies or as a direct chemotherapeutic agent in certain malignancies. It reduces lung tumor multiplicities in the tobacco smoke-exposed and non-exposed mice (Witschi et al., 2005). We showed that DEX inhibited CXCL13 production by epithelial cells and SPP1 production by TAMs (Figure 3 and  (Figure 2), revealing a new mechanism for this existing drug. Because DEX is a wide-spectrum anti-inflammatory drug that may cause severe side effects, more specific, CXCL13/ CXCR5-targeting antibodies or small molecules should be developed to combat lung cancer, the leading cause of cancer-related mortality accounting for 1.59 million deaths worldwide in 2012 (WHO, 2012).

Patient samples
The study was approved by the local research ethics committees of all participating sites; all lung cancer samples were collected with informed consent. A total of 201 previously untreated NSCLCs from HPRs or CRs were included ( Table 1). The HPR patients were diagnosed in the last 5 years in participant hospitals in Yunnan Province, and the diagnosis of lung cancer was confirmed by at least three pathologists. Those who fulfilled the following criteria were selected for this study: (1) Residents of Xuanwei where the smoky coal was used.
(2) Resided in their communities and never stayed in other regions for a long time (6 months or more). (3) Previously untreated primary lung cancer. (4) The tissue samples were taken at the time of surgery and quickly frozen in liquid nitrogen. The tumor samples contained a tumor cellularity of greater than 60% and the matched control samples had no tumor content. Serum samples were obtained from 80 untreated NSCLCs (40 from HPR and 40 from CR) and 40 healthy donors.

Assessment of CXCL13 expression
The expression of inflammatory factors in eight HPR patients ( Figure 1A) was determined using the Human Inflammatory Cytokines & Receptors RT 2 Profiler PCR Array, which contains 84 cytokines/ chemokines and their receptors. CXCL13 expression was detected in additional 193 HPR and CR patients, in BaP-treated cells, and in BaP-treated A/J, Cxcl13 -/-48 or Cxcr5 -/-50 mice by real-time PCR, Western blot analysis, ELISA, or IHC using anti-CXCL13 and anti-SPP1 antibodies. The immunoreactivity score (IRS) was calculated as IRS (0-12)=CP (0-4)ÂSI (0-3), where CP is the percentage of CXCL13-positive epithelial cells and SI is the staining intensity (Remmele and Stegner, 1987).

Cell culture and preparation of conditioned medium
The lung adenocarcinoma cancer cell lines A549 and NCI-H1975 were obtained from ATCC (Manassas, VA, USA). Human normal bronchial epithelial cell line 16HBE was obtained from Clonetics (Walkersville, MD) and cultured according to standard protocols. The macrophages were obtained by incubation of THP-1 cells (ATCC) with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 hr and then the culture medium was refreshed with serum-free Dulbecco's Modified Eagle Medium (DMEM) for another 24 hr (Tsuchiya et al., 1982). For preparation of conditioned medium, PMA-differentiated THP-1 cells were plated in six-well plates and co-cultured with CXCL13 for 48 hr. The medium was then centrifuged at 200Âg for 10 min at 4˚C to remove cell debris and stored at À80˚C until use. For the analysis of EMT signaling by western blot analysis, supernatants were ultrafiltered for protein enrichment using a centrifugal filter device (Millipore, Darmstadt, Germany).

RT-PCR
The total RNA was isolated using the TRIZOL reagent (Invitrogen) and the phenol-chloroform extraction method according to the manufacturer's instruction. Total RNA (2 mg) was annealed with random primers at 65˚C for 5 min. The cDNA was synthesized using a 1st-STRAND cDNA Synthesis Kit (Fermentas, Pittsburgh, PA). Quantitative real-time PCR was carried out using SYBR Premix ExTaq (Takara). The primer sequences for real-time PCR are listed in Figure 1-source data 1. Each sample was analyzed in triplicate three times.

Migration
Trans-wells with 8-mm pores were incubated at 37˚C in a CO 2 incubator for at least 1 hr. The differentiated THP-1 cells were seeded in the lower chamber, and A549-Luc-CXCL13 or H1975 cells (1Â10 4 ) with serum-free DMEM/RPMI 1640 were added to the upper chamber and allowed to migrate for 24 hr. Cells on the inserts were fixed with 90% ethanol, stained with 0.0005% Gentian Violet Solution, and washed with PBS. Non-migrated cells on the upper side of the inserts were wiped off with a cotton swab. Migrated cells were counted in five microscopic fields at 4Â magnification, and the counts were averaged (Meijer et al., 2006).

Chromatin immunoprecipitation (ChIP) assay
A549 cells (5Â10 6 ) were seeded onto 10-cm dishes and treated with or without BaP (5 mM) for 2 h, and ChIP assay was performed as described . Both input and immunoprecipitated DNA samples were analyzed by qPCR to determine the relative amounts of DNA from the CXCL13 gene promoter region present in the samples. The primer pairs used here were listed in Figure 1-source data 1.

Flow cytometry
Mouse lung cancer tissues were dissected into 2-mm fragments, followed by collagenase IV (0.2%, Sigma) digestion for 40 min at 37˚C. A single-cell suspension was generated through a 200-mmstainless steel wire mesh. The dissociated cancer cells labeled with indicated cell surface markers were sorted by MoFlo XDP Cell Sorter (Beckman Coulter, Brea, CA), and the data were analyzed on the Summit Software v5.0 (Beckman Coulter). All FACS analyses and sorting were paired with matched isotype control. Dead cells were excluded based on scatter profile.

Immunofluorescence microscopy
Cells grown on coverslip (24 mmÂ24 mm) were fixed with 4% paraformaldehyde for 15 min, washed with 150 mM glycine in PBS, and permeabilized with 0.3% Triton X-100 in PBS for 20 min at room temperature. After blocking with 5% BSA, the cell smears were incubated with the indicated primary antibodies overnight at 4˚C, washed, and FITC/PE-labeled secondary antibody in PBS was added to the cell smears. Images were taken by a laser scanning confocal microscopy (Zeiss, Oberkochen, Germany).

Immunohistochemistry analysis
IHC assay was performed using anti-CXCL13 and anti-SPP1 antibodies as previously described (Ma et al., 2011). Briefly, formalin-fixed, paraffin-embedded human or mouse lung cancer tissue specimens (5 mm) were deparaffinized through xylene and graded alcohol, and subjected to a heatinduced epitope retrieval step in citrate buffer solution. The sections were then blocked with 5% BSA for 30 min and incubated with indicated antibodies at 4˚C overnight, followed by incubation with secondary antibodies for 90 min at 37˚C. Detection was achieved with 3, 3'-diaminobenzidine (DAB, Zhongshan Golden Bridge Biotechnology, Beijing, China) and counterstained with hematoxylin, dehydrated, cleared and mounted as in routine processing. The scoring of immunoreactivity was performed as described (Remmele and Stegner, 1987 ).

ELISA
Concentration of CXCL13 in serum and cell culture supernatant was determined by ELISA using a commercially available ELISA kit (R&D). The absorbance of the plates was read at 450 nm using an automated microplate reader (Bio-Tek, Winooski, VT, USA).

Microarray experiment and analysis
Eight pairs of human lung cancer tissues and their adjacent normal lung tissues were homogenized by Biopulverizer (Biospec, Bartlesville, OK). Total RNA was extracted from homogenized samples using the RNeasy Mini Kit (Qiagen, Valencia, CA). Total RNA (1 mg) was labeled and hybridized with a One-ColorQuick Amp Labeling Kit and Gene Expression Hybridization Kit (Agilent Technologies, Santa Clara, CA). Hybridization signals were detected using a DNA microarray scanner G2565BA (Agilent Technologies), and all scanned images were analyzed using Agilent Feature Extraction Software. Quantile normalization and subsequent data processing were performed using the Gene-Spring GX v11.5.1 software package (Agilent Technologies). Quality assessment of mRNA data after filtering was carried out by Box Plot and Scatter-Plot. To identify differentially expressed mRNAs with statistical significance, Volcano Plot filtering between the two groups (fold change ! 1.5, p values 0.05) was performed. Pathway analysis was performed using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. The genes correlated with CXCL13 were analyzed by Pearson's correlation coefficient, and the results were listed in Figure 6-source data 1.

Animal studies
The animal studies were approved by the Institutional Review Board of Institute of Zoology, Chinese Academy of Sciences. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences. Female NOD/ SCID mice (5-6 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). A/J mice (5-6 weeks old, female), homozygous Cxcl13-deficient, and homozygous Cxcr5deficient mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). The exposure protocols were modified from previous studies (Wattenberg and Estensen, 1996;Estensen and Wattenberg, 1993). The A/J mice were exposed to BaP (0 mg/kg, 50 mg/kg, 100 mg/kg twice per week for 5 weeks) in corn oil via oral gavage, and treated with or without DEX (0.5 mg/kg per day for 10 weeks in diet; Figure 2-figure supplement 1A). The Cxcl13 -/and Cxcr5 -/mice were treated with BaP at 100 mg/kg twice per week for 8 weeks (Figure 4-figure supplement 1B). For microCT (PerkinElmer, Waltham, MA) and bioluminescence (using an IVIS Spectrum In Vivo Imaging System, PerkinElmer) analyses, mice were anesthetized by mixture of oxygen/isoflurane inhalation and positioned with legs fully extended, and assayed according to manufacturers' instruction. Survival of the mice was evaluated from the first day of treatment until death or became moribund, at which time points the mice were sacrificed.

Statistical analysis
All statistical analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, CA) and SPSS 12.0 software for Windows (Chicago, IL). Statistically significant differences were determined by Student's t-test, Wilcoxon rank sum test, one-way analysis of variance, the chi-squared test, or multivariate logistic analysis. P values less than 0.05 were considered statistically significant in all cases.