Lipopolysaccharide-Mediated Chronic Inflammation Promotes Tobacco Carcinogen-Induced Lung Cancer and Determines the Efficacy of Immunotherapy

Abstract

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti-PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti-PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti-PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. SIGNIFICANCE: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen-induced lung cancer receiving immune checkpoint blockade therapy.

Figure. 1.. Recurrent exposure to lipopolysaccharide (LPS) promotes tobacco smoke carcinogen-induced lung tumorigenesis.

(A) FVB/NJ (7 weeks, female) mice were exposed with PBS, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 3 mg i.p. biweekly for 4 weeks), LPS (5μg weekly i.n. instillation for 16 weeks), or NNK and LPS (NNK/LPS) combined. (B) H&E staining of the tumor-bearing lungs. Arrows indicate lung tumors. (C) Quantification of the number of tumors in mice exposed to PBS (n=5), NNK (n=15), LPS (n=9) or NNK/LPS (n=11), Results are mean ± S.D. of one representative dataset of four independent experiments. ****p < 0.0001 using one-way ANOVA with post-hoc Bonferroni correction. (D) Tumor incidence rate (percent) in various exposure groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using Chi-squared test. (E) The number of alveolar hyperplasia foci, adenomas, and adenocarcinomas in the lungs of NNK compared to NNK/LPS exposure mice. Values are means ± S.D., ** p < 0.01, **** p < 0.0001 using two-way ANOVA with post-hoc Bonferroni correction. (F) Tumor area (percent) was calculated in NNK (n=5) and NNK/LPS exposure (n=5). Values are means ± S.D., *** p < 0.001 using Student’s t-test. (G) H&E staining of tumors derived from NNK- and NNK/LPS-treated mice. Bottom, high-magnification images. Arrow indicates tumor-infiltrating leukocytes. Scale bars = 100 μm. Abbreviations: AH = alveolar hyperplasia; i.n. = intranasal; i.p. = intraperitoneal; PBS = phosphate-buffered saline.

Figure. 2.. Lipopolysaccharide (LPS) alters tobacco smoke carcinogen-induced inflammatory profiles and increased the accumulation of immunosuppressive cells in the lungs.

(A) FVB/NJ (7 weeks, female) mice were exposed with PBS, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 3 mg i.p. biweekly for 4 weeks), LPS (5μg weekly i.n. instillation for 16 weeks), or NNK and LPS (NNK/LPS) combined. Quantification of total cell number from bronchoalveolar lavage (BAL) of exposure groups (n=5 for each group). (B) Differential cell counts of inflammatory cells in BAL fluid. (C-G) Cytokines/Chemokines in BAL and protein extracts (n=5 for each group) of exposure groups were analyzed by Luminex assay and enzyme-linked immunosorbent assay. Flow cytometry analysis of immune cell population of mouse lungs in different exposure groups harvested at week 17 after initiation of NNK and LPS treatment as indicated in Fig. 1A. The cellular markers included (H) Th1, (I) Th17, (J) Tregs, (K) Granulocytic MDSCs, (L) Monocytic MDSCs, and (M) Tc1. Values are means ± S.D. and representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001 using one-way ANOVA with post-hoc Bonferroni correction. Abbreviations: CTL = control; Th1 = T-helper cell type 1; Th17 = T-helper cell type 17; Treg = regulatory T cell; MDSC = myeloid-derived suppressor cell; Tc1 = CD8⁺ cytotoxic T lymphocyte.

Figure. 3.. Lipopolysaccharide (LPS)-mediated chronic inflammation increased cell-related immunity and induces T-cell exhaustion.

FVB/NJ (7 weeks, female) mice were exposed with PBS, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 3 mg i.p. biweekly for 4 weeks), LPS (5μg weekly i.n. instillation for 16 weeks), or NNK and LPS (NNK/LPS) combined. Mouse lung mRNA was analyzed by microarray (n=3 for each group) was analyzed. (A) Unsupervised hierarchical clustering heatmap based on the differentially expressed genes (n=1179, absolute fold change ≥ 2, FDR < 0.05) between NNK/LPS and NNK. Arrows indicate lymphocyte recruitment CXCL9, CXCL10, CXCL13, and CCL20, and inhibitory checkpoint receptor PDCD1 transcripts. (B) Pathways identified by Ingenuity Pathway Analysis based on 1179 differentially expressed genes. (C) Levels of inhibitory checkpoint receptors, PDPD1, CTLA-4, LAG3 and TIM-3 in each exposure group were quantified by quantitative real-time polymerase chain reaction (n = 6/group). Values are means ± S.D. ** p < 0.01, *** p < 0.001, **** p < 0.0001 using one-way ANOVA with post-hoc Bonferroni correction. Abbreviations: CXCL9: chemokine (C-X-C motif) ligand 9, CXCL10: chemokine (C-X-C motif) ligand 10, CXCL13: chemokine (C-X-C motif) ligand 13, CCL20: chemokine (C-C motif) ligand 20, PDCD1: programmed cell death 1, CTLA-4: cytotoxic T-lymphocyte associated protein 4, LAG3: lymphocyte-activation gene 3, TIM-3: T-cell immunoglobulin mucin-3.

Fig. 4.. Combined NNK and LPS exposure increases tumor-infiltrating lymphocytes (TILs) with co-localized PD-1 and increases tumor PD-L1 expression.

(A) Representative CD4 (red), CD8 (magenta), and PD-1 (green) cell infiltration in lung tumors. Nuclei are identified by DAPI staining. Scale bars = 50 μm. Dotted lines outline tumor boundaries. (B) Quantification of CD4⁺ and CD8⁺ TILs and percentage of PD-1 expression in CD4⁺ and CD8⁺ TILs. * p < 0.05, ** p < 0.01, **** p < 0.0001 using Student’s t-test. (C) PD-L1 levels in lung lysates from NNK (n=4) and NNK/LPS-treated mice (n=4) of representative results from one experiment of three individual experiments. * p < 0.05 using Mann-Whitney test. (D) Representative PD-L1 IHC staining of NNK and LPS/NNK-exposed lung tumors, and the positive tumor cell surface staining was scored. Scale bars = 100 μm. Abbreviations: DAPI = 4’,6-diamidino-2-phenylindole, IHC = immunohistochemistry; LPS: lipopolysaccharide, NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, PD-1 = programmed cell death 1 receptor; PD-L1 = programmed death-ligand 1

Fig. 5.. LPS-mediated inflammation alters immune contexture and correlates with PD-1 blockade efficacy.

(A) The treatment paradigm of anti-PD-1. Mice were exposed to NNK or NNK combined with 10-week LPS exposure and treated with either IgG control (IgG2a) or anti-PD-1 for 6 weeks. (B) After treatments from week 10 to week 16 without additional LPS exposure, anti-PD-1 treatment decreased lung tumor number in NNK/LPS mice but did not differ in NNK mice as compared to IgG2a control mice. **p < 0.01, ***p < 0.001 using Student’s t-test. (C) The treatment paradigm of anti-PD-1 and anti-Ly6G. Mice were exposed to NNK combined with 16-week LPS treated for either IgG control (n=8), anti-PD-1 (n=8), anti-Ly6G (n=8), or combined anti-PD-1 and anti-Ly6G (n=8) for 6 weeks. (D) After 16 weeks, anti-PD-1 treatment only slightly decreased lung tumors in NNK/LPS mice and was not statistically significant as compared to IgG2a control. Anti-Ly6G antibody effectively inhibited NNK/LPS-induced lung tumors and further enhanced treatment efficacy in combination with ant-PD-1 antibody. Values are means ± S.D. and representative of two independent experiments. ***p < 0.001, ****p < 0.0001 using one-way ANOVA with post-hoc Bonferroni correction. (E) Flow cytometry analysis of granulocytic MDSCs of mouse lungs in 10-week and 16-week NNK/LPS group (n=10 for each group). Values are means ± S.D. ****p < 0.0001 using Student’s t-test. The granulocytic MDSC signature of the 10-week and 16-week NNK/LPS group in the mouse microarray dataset was analyzed. Values are means ± S.D. *p < 0.05 using Mann-Whitney test. Abbreviations: IgG: immunoglobulin G. LPS: lipopolysaccharide, Ly6G: lymphocyte antigen 6 G, NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, PD-1 = programmed cell death 1 receptor

Fig. 6.. Immune gene signatures predict responsiveness to PD-1 blockade and are associated with progression-free survival.

Tumor samples of 35 NSCLC patients before anti-PD-1 treatment were analyzed for the gene expression using the GSE93157 dataset. (A) Expression profiles of 130 differentially expressed genes from NPD and PD tumors (red = increased and blue = decreased as compared to means for each gene). Overall response (NPD; PD), drug response (CR; PR; SD; PD), and histological type (Non-SqNSCLC; SqNSCLC) are indicated at the top of the heatmap. Immune cell gene signatures for T cell, B cell, Th17 cell, and NK cell are indicated. (B) Gene signatures across patients with PD and NPD status. *p < 0.05, **p < 0.001 using Student’s t-test. (C) Kaplan–Meier survival analysis based on selected gene signatures in Non-SqNSCLC and SqNSCLC. p-value determined with log-rank test. Abbreviations: CR = complete response; NK cell = natural killer cells; Non-SqNSCLC = non-squamous cell non-small cell lung cancer; NPD = non-progressive disease, including CR, PR, and SD; PD = progressive disease; PR = partial response; SD = stable disease; SqNSCLC = squamous cell non-small cell lung cancer; Th17 = T-helper cell type 17.

Fig. 7.. Immune gene signatures demonstrate the clinical relevance of the mouse lung cancer model to NSCLC patients.

(A) The immune gene profiling of mouse lungs after treatments of PBS, NNK, LPS, and NNK/LPS for 16 weeks. Mouse orthologs (n = 127 transcripts) were identified from the human immune gene panel (n=130 transcripts) extracted from GSE93157 (Supplementary Table S2). The immune gene profiling of mouse lungs after treatments was presented by clustering heatmap. (Red = increased and blue = decreased as compared to means for each gene). Immune cell gene signatures including T cell, B cell, Th17 cell, and NK cell were labeled. (B) Immune cell gene signatures for T cell, B cell, Th17 cell, and NK cell were quantified. Values are means ± S.D. *p < 0.05, ***p < 0.001 using Mann-Whitney test. Abbreviations: LPS: lipopolysaccharide, NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, PBS: phosphate-buffered saline.