Background
Lung cancer is the leading cause of cancer death worldwide accounting for 29% of all male and 26% of all female cancer deaths in 2012 [
1]. Cigarette smoking (CS) is the principal cause of lung cancer, and CS induced lung cancer is characterized by a deregulated inflammatory microenvironment [
2]. In addition, the association between chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, and lung cancer has been demonstrated in population-based studies [
3,
4]. Smokers with COPD have an increased risk of lung cancer compared to smokers with comparable cigarette exposure but without COPD [
5,
6]. Importantly, among former smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate as does the increased risk of lung cancer [
7]. These facts suggest a strong link between airway inflammation and lung cancer promotion.
Tumor cells produce various cytokines and chemokines that attract leukocytes including neutrophils, dendritic cells, macrophages, lymphocytes and mast cells [
6]. It is becoming increasingly clear that tumor-associated neutrophils (TANs) play a major role in cancer promotion [
8]. During immune responses, neutrophils are among the first cells to arrive at sites of inflammation. The increased number of TANs is linked to poorer outcomes in patients with bronchioloalveolar carcinoma [
9], and many patients with advanced cancer show high levels of blood neutrophils [
10]. Furthermore, in histopathologic specimens of distal lung and in bronchoalveolar lavage fluid (BALF) from COPD patients, neutrophils are prominent [
11,
12], and shown to cause COPD progression [
13]. We have previously established a COPD-like mouse model of airway inflammation induced by repetitive exposure to an aerosolized lysate of non-typeable
Haemophilus influenzae (NTHi) [
11], which is the most common bacterial colonizer of airways in COPD patients [
14]. Then we showed that this type of airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR) [
15]. This was associated with severe neutrophilic influx due to an increased level of neutrophil chemoattractant, KC, which was partially inhibited by using a natural non-specific anti-inflammatory agent, curcumin, and resulted in significant tumor suppression [
16]. Therefore, we further dissected the role of neutrophils in lung tumorigenesis by selectively targeting neutrophils, its chemokine receptor (CXCR2) and its specific enzyme (neutrophil elastase). Neutrophil depletion, CXCR2 inhibition, and lack of neutrophil elastase (NE) all resulted in significant tumor reduction in our K-ras mutant mouse model of lung cancer.
Discussion
Tumor-induced inflammation is now a cancer hallmark, and it is considered an enabling characteristic due to its contributions to the acquisition of core hallmark capabilities [
28,
29]. Tumors are complex tissues composed of multiple distinct cell types that interact with each other. The recruited stromal and inflammatory cells, which form the tumor microenvironment, are active participants in tumorigenesis and significantly influence most of the cancer hallmarks in the malignant cancer cells. In summary, the presence of these stromal and inflammatory cells within the tumor microenvironment is associated with enhanced tumor progression, cancer cell growth and spread, angiogenesis, invasion and tumor immunosuppression [
30].
Many environmental causes and risk factors for cancer are associated with chronic inflammation. According to epidemiological studies, up to 20% of cancers are linked to chronic infections and inflammation [
31]. Cigarette smoking generates an inflammatory microenvironment which can promote lung cancer [
2]. On the other hand, COPD, which is a disorder characterized by an abnormal local and systemic inflammatory response, is also strongly associated with lung cancer [
32]. In histopathologic specimens of distal lung and in BALF from COPD patients, neutrophils and macrophages are prominent [
33]. Neutrophils make up a significant portion of the inflammatory cell infiltrate found in a wide variety of murine models and human cancers [
34,
35]. They are among the first cells to arrive at sites of inflammation and, as mentioned before, the increased number of TANs is linked to poorer outcomes in patients with lung cancer. Neutrophils are present within the alveolar airspaces and within the tumor parenchyma during neoplastic development in a urethane-induced mouse model of lung cancer [
36]. We have previously shown that bacterial (NTHi) lysate induced COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR) [
15]. This was associated with severe neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. Furthermore, we and others found that K-ras mutation per se induces an inflammatory tumor microenvironment predominated by neutrophils and macrophages [
15,
17]. In this study, we provided more evidence to support the essential role for neutrophils in lung cancer promotion.
mLy-6G Ab is used to deplete neutrophils in murine disease models. It binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes [
19]. In our study, depletion of neutrophils by mLy-6G Ab suppressed lung tumor promotion which was associated with decreased tumor cell proliferation and angiogenesis. Similar to tumor associated macrophages (TAMs), TANs also can be divided into an antitumorigenic (N1) phenotype and a protumorigenic (N2) phenotype [
20]. Analysis of cytokines and chemokines in BALF of CC-LR mice showed suppression of proinflammtory cytokines (IL-6, IL-17, TGF-β) by mLy-6G, with further inhibition of N2 type cytokines and molecules (CCL5, arginase 1). Surprisingly, mLy-6G Ab was not able to suppress the number of neutrophils in BALF of NTHi-exposed mice which can imply that there exists a pool of neutrophil that can be mobilized. However, mLy-6G Ab reduced the numbers of macrophages in the BALF and the number of neutrophils infiltrated in the lung parenchyma. Interestingly, many of these infiltrating neutrophils were found to be stained by Ki-67 in the tumor site, showing a proliferating state for these cells. These cells mostly express pro-tumor N2 phenotype markers and can be viewed as myeloid-derived suppressor cells (MDSCs), which represent a heterogeneous group of immature myeloid cells comprising macrophages, monocytes, neutrophils, and DCs [
37] or myeloid progenitor cells at different stages of development that have been reported to be recruited along with PMNs to sites of inflammation and proliferate in tissue [
38].
Interleukin-8 (IL-8), which is known as keratinocyte-derived chemokine (KC) in the murine, is a proinflammatory CXC chemokine associated with the neutrophil chemotaxis and degranulation [
24]. It is also reported to be a transcriptional target of Ras Signaling, but its role in Ras-induced tumorigenesis has not been fully defined [
39]. Expression and secretion of IL-8 by tumor cells enhance proliferation and neutrophil infiltration into the tumor [
40‐
42]. According to our previous study, the expression of KC in the BALF of the CC-LR mice was higher compared to the WT mice [
15]. The application of curcumin, which is a naturally occurring polyphenolic phytochemical isolated from the rhizome of the medical plant Curcuma longa, with non-specific anti-inflammatory effect, could suppress lung tumor promotion partly by suppressing KC production and subsequent inhibition of neutrophil recruitment to the lung [
16]. In the current study, KC level was also suppressed by mLy-6G Ab in the non-NTHi-exposed group.
The protein encoded by the CXCR2 gene is a member of the G-protein couple receptor family. It is the receptor for IL-8. This receptor mediates neutrophil migration to sites of inflammation and is essential for neutrophil recruitment [
43,
44]. There is a remarkable up-regulation of CXCR2 in airway epithelial cells in COPD, and this correlates with the increased number of neutrophils in the airways [
45]. Neutrophil recruitment into tumors also appears to be dependent on chemokines that bind to CXCR2 expressed by neutrophils [
40]. Furthermore, CXC chemokines are potent promoters of angiogenesis, and mediate their angiogenic activity via signal-coupling of CXCR2 on endothelium [
41,
46,
47]. It has recently been shown that CXCR2 expression in tumor cells is a poor prognostic factor and promotes invasion and metastasis in lung adenocarcinoma [
48]. We found that inhibiting CXCR2 by a selective antagonist could efficiently suppress neutrophilic inflammation and lung tumor progression. This was associated with significant reduction in tumor cell proliferation and angiogenesis. This is similar to the findings from other groups showing that CXCR2 inhibition using a neutralizing antibody inhibits the progression of premalignant alveolar lesions by reducing vascular density in another mouse model of lung cancer [
49]. Analysis of the cytokines and chemokines in the BALF of CC-LR mice showed that the secretion of pro-inflammatory cytokines (IL-6, IL-17, TGF-β) decreased after treatment with CXCR2 inhibitor with further reduction in levels of N2 type markers, CCL-5. This was associated with decreased expression of arginase 1, while the expression of iNOS increased. These results indicate that CXCR2 inhibition not only suppresses the recruitment of neutrophils but also may shift their differentiation from a N2 phenotype to a N1 phenotype. There was a much larger reduction in angiogenesis with CXCR2 inhibition as opposed to neutrophil depletion with mLy-6G Ab treatment. This could be due to inhibition of additive direct effect of CXCR2 axis activation on tumor endothelial cells that leads to less tumor angiogenesis.
Similar to mLy-6G Ab treatment, neutrophils in BALF of NTHi-exposed mice were not affected by the CXCR2 inhibitor, but it reduced the number of infiltrating neutrophils with MDSC/progenitor cell characteristics in the lung parenchyma and tumor sites. Failure of the anti-neutrophil Ab and CXCR2 inhibitor in inhibiting BALF neutrophilic influx in response to repetitive exposure to NTHi indicates involvement of other chemokines and inflammatory signaling pathways besides the IL-8/CXCR2 pathway in this phenomenon.
Products secreted from neutrophils, such as, reactive oxygen species and proteinases, have defined and specific roles in regulating tumor cell proliferation, angiogenesis, and metastasis [
34]. Among them, NE is generally considered the major effector of neutrophil function, making up approximately 2% of the dry weight of a neutrophil [
50]. It contributes to cigarette smoke-induced emphysema in mice [
51]. NE KO mice are significantly protected from the development of CS-induced emphysema and recruitment of both neutrophils and macrophages. We have also found that lack of NE reduces lung macrophages and inhibits lung cancer promotion in our K-ras mutant mouse model indicating an essential promoting role for this enzyme in lung tumorigenesis. This is consistent with the study from Houghton et al., where they found the similar promoting effect for NE through degradation of IRS-1 in another mouse model of lung cancer [
52]. NE exposure can activate PI3K/Akt pathway resulting in tumor cell proliferation and survival [
52]. Lack of NE in our K-ras mutant model was associated with decreased proliferation and increased apoptosis of tumor cells which could be secondary to lack of PI3K/Akt pathway activity in absence of NE. Tumor regression was also accompanied with decreased levels of TGF-β similar to the finding by Kang-Yun et al., showing that NE up-regulates TGF-β gene expression and release via the MyD88/IRAK/NF-κB pathway [
53]. It has been previously shown that TANs were involved in tumor angiogenesis by the production of proangiogenic factors such as VEGF and IL-8 [
8], proteases such as MMPs [
54] and elastases [
55]. We have also found decreased expression of VEGF and numbers of CD31 positive cells in our K-ras mutant mouse with lack of NE, indicating a role for NE in tumor angiogenesis. It has also been shown that NE can cleave VEGF to generate a diffusible VEGF fragments that stimulate inflammatory cell recruitment and activation via VEGF receptor 1/Akt pathway [
56] that could be considered as a tumor promoting mechanism of NE in our model. Similar to our findings Wada et al. have demonstrated that NE released from activated neutrophils stimulates the growth and progression of cancer cells by releasing the growth factors such as VEGF on the cell surface and that sivelestat, a specific NE inhibitor, blocks these processes [
57].
It is known that K-ras induces the secretion of the cytokine IL-6 in various epithelial cell types including lung epithelial cells [
58]. We have previously revealed increased levels of IL-6 and subsequent activation of the STAT3 pathway in our K-ras induced mouse model of lung cancer in absence and presence of COPD-like airway inflammation [
59]. We further demonstrated that genetic ablation of IL-6 results in significant tumor suppression in this model in vivo, indicating an essential role for IL-6 in lung cancer promotion in this model. Therefore, reduced levels of IL-6 in response to neutrophil depletion, CXCR2 inhibition, or lack of NE could be considered as another mechanism of tumor regression. However, this could simply be a reflection of the reduced production of this cytokine from K-ras mutant cells due to reduction in tumor burden.
Materials and methods
Animals
CCSP
Cre/LSL-K-ras
G12D mice (CC-LR) were generated as previously described [
15]. Briefly, this is a mouse generated by crossing a mouse harboring the LSL-K-ras
G12D allele with a mouse containing Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus [
15]. CC-LR mice were crossed with neutrophil elastase (NE) knock out (KO) mice [
52,
60], which were kindly provided by Dr. Steven Shapiro (University of Pittsburgh) to generate CC-LR-NEKO mice. All mice were housed in specific pathogen-free conditions and handled in accordance with the Institutional Animal Care and Use Committee of M. D. Anderson Cancer Center. Mice were monitored daily for evidence of disease or death.
NTHi lysate aerosol exposure
A lysate of NTHi strain 12 was prepared as previously described [
15], the protein concentration was adjusted to 2.5 mg/ml in phosphate buffered saline (PBS), and the lysate was frozen in 10 ml aliquots at −80°C. To deliver the lysate to mice by aerosol, a thawed aliquot was placed in an AeroMist CA-209 nebulizer (CIS-US, Bedford, MA) driven by 10 liter/min of room air supplemented with 5% CO
2 for 20 min.
Neutrophils depletion and CXCR2 inhibition
Neutrophils were depleted with the anti-neutrophils antibody (Ab), mLy-6G (clone: RB6-8C5; Bio X Cell, West Lebanon, NH, USA), 100ul at the concentration of 1 mg/ml per mouse, twice a week by intraperitoneal (i.p.) injection. CXCR2 was inhibited by using a selective inhibitor, SB332235Z, 50 mg/kg orally, twice daily by gavage (GSK, Brentford, UK).
Histochemistry
The tracheas of euthanized mice were cannulated with PE-50 tubing and sutured into place. The lungs were infused with 10% buffered formalin (Sigma, St. Louis, MO) and then removed and placed in 10% buffered formalin for 18 hours. Tissues then were transferred to 75% ethanol, embedded in paraffin blocks, and sectioned at 5-mm thickness. The sections on glass slides were dried at 60°C for 15 minutes and then were deparaffinized and stained with hematoxylin and eosin (H&E) by incubating the tissues in Harris hematoxylin (Sigma) followed by serial eosin (Sigma) and graded ethanol steps. The H&E-stained slides were examined by a pathologist blinded to genotype and treatment, and the proliferative lesions of the lungs were evaluated in accordance with the recommendations of the Mouse Models of Human Cancer Consortium [
61].
Immunohistochemistry
Previously sectioned lung samples on slides were immunohistochemically (IHC) stained and evaluated for expression of Ki-67 (1:200; ab16667; abcam, MA, USA), P40 (1:100; MCA771G; Serotec, Oxford, UK), CD31 (1:10, 550274, BD Biosciences, CA, USA), VEGF (1:500; sc-152, Santa Cruz, CA, USA), cleaved caspase-3 (1:500, ab13847, abcam), Anti-Ly6g (1:100, ab25377, abcam), Arginase 1 (1:100, Thermo scientific, PA5-22009) DAPI (Signa, D9564-10MG), Alexa Fluor 488 Dye and Alexa Fluor 594 (Invitrogen). All antibodies reacted with mouse antigens. Heat-induced antigen retrieval was performed using 10 mmol/L of citrate buffer (pH 6) in a pressure cooker for 20 min. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 30 min at room temperature. Blocking was performed with non-immune normal serum. Immunoreactivity for immunohistochemistry was detected using biotinylated IgG secondary antibodies specific for each primary antibody followed by incubation with ABC kit (Vector Laboratory, Burlingame, CA) for 30 min, and stained with diaminobenzidine chromogenic substrate for 4–10 min. Slides were counter-stained with Harris hematoxylin in for 30 sec, followed by dehydration and mounted with cytoseal 60 (ThermoFisher Scientific, Cheshire, UK). Images were obtained by an OLYMPUS BX 60 inverted microscope at ×4, ×20 or ×40 magnification with Image-Pro Plus, version 4.5.1.22. The numbers of labeled positive cells for any of these markers were quantitated as a fraction of total tumor nuclei per high power field (40×) in 10 fields from three mice of each group. Results were expressed as percentage of positive cells ± SE.
Assessment of lung tumor burden and inflammation
On the first day after the final NTHi exposure, animals were euthanized by i.p. injection of a lethal dose of avertin (Sigma). In all mice (n = 8 per group per time point), lung surface tumor numbers were counted and then in some of them (n = 8 per group per time point), the lungs were prepared for histologic analysis as described above. In other mice (n = 8 per group per time point), bronchoalveolar lavage fluid (BALF) was obtained by sequentially instilling and collecting 2 aliquots of 1 mL PBS through a tracheostomy cannula. Total leukocyte count was determined using a hemacytometer, and cell populations were determined by cytocentrifugation of 300 mL of BALF followed by Wright-Giemsa staining. The remaining BALF (1,400 mL) was centrifuged at 1,250 × g for 10 min, and supernatants were collected and stored at −80°C.
Cytokines and chemokines measurement
The levels of KC, TNF, IL-6, IL-10, IL-17A, CCL2, CCL5 and VEGF in the BALF were assessed using MCYTOMAG-70 K assay (Millipore, St Charles, MO), according to the manufacturer’s instructions). Due to reagent incompatibilities, TGF-β was assayed separately from the other cytokines using the TGFB-64 K-01 (Millipore, MO, USA) assay. Data were collected using a Luminex 100 (Luminex Corporation, TX, USA). Standard curves were generated using a 5-parameter logistic curve-fitting equation weighted by 1/y (StarStation V 2.0; Applied Cytometry Systems, CA, USA). Each sample reading was interpolated from the appropriate standard curve.
Quantitative RT-PCR analysis
Total RNA was isolated from whole lung according to the TRIzol reagent protocol (Invitrogen, NY, USA) and purified by E.Z.N.A. total RNA kit I (OMEGA, GA, USA). Reverse transcription PCR was performed using the qScript cDNA SuperMix (Quanta biosciences, MD, USA). Real-time PCR was carried out according to a standard protocol using the PerfeCTaFast Mix II (Quanta) with the TaqMan probe for arginase 1 and iNOS (ABI, NY, USA), and products measured on an ABI Viia 7 PCR system (ABI). Relative expression of each gene was calculated and graphed for comparison among the treatment groups. GAPDH RNA was measured for reference.
Western blot analysis
Total proteins were prepared from each group of mouse lungs. Lung samples were removed and immediately placed in RIPA buffer and a protease inhibitor mixture. The samples were then homogenized and centrifuged at 14000 g for 20 min at 4°C. The supernatants were collected as the total proteins. Equal amounts (40 μg) of the total proteins were boiled for 5 min in the presence of loading buffer, loaded on each lane, and separated by 10% SDS-PAGE. The gels were then transferred to nitrocellulose membranes. Equal amounts of protein loading for each lane was checked by Ponceau (Sigma Chemical Co., MO, USA) staining. The anti-arginase 1 (Thermo Scientific, clone PA5-22009, MA, USA) antibody was diluted to the concentration according to commercial recommendations (1:1000). Immunoreactive bands were detected with Pierce ECL Western Blotting Substrate (Pierce, IL, USA).
Statistical methods
Summary statistics for cell counts in BALF, IHC positive cells in lung tissue, and mRNA expression were computed within treatment groups, and analysis of variance with adjustment for multiple comparisons was conducted to examine the differences between the control group and each of the treatment groups in the presence or absence of NTHi exposure. For tumor counts, comparisons of groups were made using Student’s t test. Differences were considered significant for P < 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LG carried out the mouse in vivo study including mLy6-G and CXCR2 injection, weekly NTHi exposure, and lung tissue extraction, and participated in preparing the figures and drafting the manuscript. AMC, MSC, CEO, and DJL participated in the mouse colony maintenance, genotyping, and lung tissue extraction and western blot analysis. MMD and SGM carried out the histopathology examination and analysis of the lung tissues and participated in preparing the figures. BFD and QZ participated in the design of the study and the drafting the manuscript. SJM designed the study, assessed lung tumor burden and inflammation, performed the statistical analysis, and participated in preparing the figures and drafting the manuscript. All authors read and approved the final manuscript.