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. 2016 Jun 1;76(11):3189-99.
doi: 10.1158/0008-5472.CAN-15-2840. Epub 2016 Apr 1.

IL6 Blockade Reprograms the Lung Tumor Microenvironment to Limit the Development and Progression of K-ras-Mutant Lung Cancer

Mauricio S Caetano  1 , Huiyuan Zhang  2 , Amber M Cumpian  1 , Lei Gong  1 , Nese Unver  3 , Edwin J Ostrin  4 , Soudabeh Daliri  1 , Seon Hee Chang  2 , Cesar E Ochoa  1 , Samir Hanash  3 , Carmen Behrens  5 , Ignacio I Wistuba  6 , Cinthya Sternberg  7 , Humam Kadara  6 , Carlos Gil Ferreira  7 , Stephanie S Watowich  8 , Seyed Javad Moghaddam  9
Affiliations

IL6 Blockade Reprograms the Lung Tumor Microenvironment to Limit the Development and Progression of K-ras-Mutant Lung Cancer

Mauricio S Caetano et al. Cancer Res. .

Abstract

Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras-mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL6, and activation of its responsive transcription factor STAT3 in K-ras-mutant lung tumors, which was further amplified by the tumor-enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras-mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL6 using a monoclonal anti-IL6 antibody in a K-ras-mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL6 blockade significantly inhibited lung cancer promotion, tumor cell-intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL6 inhibition reduced expression of protumor type 2 molecules (arginase 1, Fizz 1, Mgl, and IDO), number of M2-type macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses. This was accompanied by increased expression of antitumor type 1 molecule (Nos2), and antitumor Th1/CD8 T-cell responses. Our study demonstrates that IL6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also reeducates the lung microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces IL6 as a potential druggable target for prevention and treatment of K-ras-mutant lung tumors. Cancer Res; 76(11); 3189-99. ©2016 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest: No potential conflicts of interest.

Figures

Figure 1
Figure 1. IL-6/STAT3 pathway as a potential druggable target for K-ras mutant lung tumors
(A) All adenocarcinomas (N=152) and (B) KRAS-mutant adenocarcinomas (N=39) from PROSPECT Cohort were stratified based on STAT3 median mRNA expression (high, black; low, grey). Patient subgroups were then analyzed for differences in disease-free survival using the Kaplan-Meier method for estimation of survival probability and the log-rank test. (C–D) The cell lines were incubated with tocilizumab or siltuximab at different concentrations alone or in combination with the respective IC50 of cisplatin (Combo) for 48 hours. Data represent means ± SEM. (n=4 independent experiments) (*p<0.05, **p<0.005, ***p<0.001, ****p<0.0001).
Figure 2
Figure 2. Anti-IL-6 treatment reduces lung tumor burden, tumor cell proliferation, tumor angiogenesis, and STAT3 activation
(A) Histopathologic appearance of lung and lung surface tumor number in CC-LR mice at age of 14 weeks after IgG1 (n=8) or anti-IL-6 treatment (n=12) (4×; Scale bar for, 2mm). (B–C) Representative photomicrographs and quantitative analysis of positive tumor cells for Ki-67, and P-STAT3 in lungs of CC-LR mice at the age of 14 weeks after IgG1 (n=4) or IL-6 (n=5) blockade (20×; Scale bar for, 100μm) (data represent means ± SEM **** p<0,001). (D) Representative photomicrographs of lung tumors stained for CD31, VEGF, and MMP-9 in CC-LR mice at the age of 14 weeks after IgG1 (n=4) or IL-6 (n=5) blockade (40×; Scale bar for, 100μm).
Figure 3
Figure 3. Anti-IL-6 treatment changes the lung microenvironment in K-ras induced lung tumors
(A) Total inflammatory cell and lineage-specific leukocyte numbers from BALF of CC-LR mice at the age of 14 weeks after IgG1 (n=5) or IL-6 blockade (n=8). (B) Representative flow cytometry analysis of (live/CD45+) alveolar macrophage population (CD11c+F4/80+) in BALF of CC-LR mice after IgG1 (n=4) or IL-6 blockade (n=6) at the age of 14 weeks. (C) Total (CD11c+F4/80+) cell number in BALF of CC-LR mice after IgG1 (n=4) or IL-6 blockade (n=5) at the age of 14 weeks. (D) Relative expression of Ccl2 mRNA in whole lungs from CC-LR mice after IgG1 (n=4) or IL-6 blockade (n=5) at the age of 14 weeks. (E) Relative expression of type 2 macrophage mRNA signature in BALF cells from CC-LR mice after IgG1 (n=3) or IL-6 blockade (n=3) at the age of 14 weeks. (F) ELISA analysis on BALF of CC-LR mice after IgG1 (n=3) or anti-IL-6 treatment (n=3) at the age of 14 weeks. (G) Relative expression of IL17, Tgfb, Foxp3, Infg, Gzmb and Tbx21 mRNA in whole lungs of CC-LR mice at the age of 14 weeks after IgG1 (n=4) or IL-6 blockade (n=5), normalized by Cd45 expression (data represent means ± SEM *p<0,05, **p<0.005, ****p<0,001).
Figure 4
Figure 4. Anti-IL-6 treatment reduces lung tumor cell proliferation, tumor angiogenesis, and STAT3 activation in COPD setting
(A) Histopathologic appearance of lung and lung surface tumor number in CC-LR mice at the age of 14 weeks after NTHi exposure and IgG1 (n=9) or anti-IL-6 treatment (n=12). (B–C) Representative photomicrographs and quantitative analysis of positive tumor cells for Ki-67, and P-STAT3 in lungs of CC-LR mice at the age of 14 weeks after IgG1 (n=4) or IL-6 (n=5) blockade (data represent means ± SEM **** p<0,001). (D) Representative photomicrographs of lung tumors stained for CD31, VEGF, and MMP-9 in CC-LR mice at the age of 14 weeks after IgG1 (control) (n=4) or IL-6 (n=5) blockade. (40×; Scale bar for, 100μm).
Figure 5
Figure 5. Anti-IL-6 treatment changes the lung microenvironment in K-ras induced lung tumors in COPD setting
(A) Total inflammatory cell and lineage-specific leukocyte numbers from BALFs of CC-LR mice at the age of 14 weeks after NTHi exposure and IgG1 (n=6) or IL-6 blockade (n=8). (B) Relative expression of Cd45 and Ccl2 mRNA in whole lungs from CC-LR mice after NTHi exposure and IgG1 (n=4) or anti-IL6 treatment (n=5) at the age of 14 weeks. (C) Representative flow cytometry analysis and total number of (live/CD45+) macrophage population (F4/80+) in BALF of CC-LR mice after NTHi exposure and IgG1 (n=3) or IL-6 blockade (n=4) at the age of 14 weeks. (D) Representative flow cytometry data and percentage of (live/CD45+) activated M1 macrophage population (Ly6C−MHCII+) in BALF of CC-LR mice after NTHi exposure and IgG1 (n=3) or IL-6 blockade (n=4) at the age of 14 weeks. (E) Representative flow cytometry data and total number of (live/CD45+) G-MDSC population (Ly6G+CD11b+) in BALF of CC-LR mice after NTHi exposure and IgG1 (n=3) or IL-6 blockade (n=4) at the age of 14 weeks. (F) M-MDSC (CD45+CD11b+Ly6G−) and G-MDSC (CD45+CD11b+Ly6G+) populations in BALF of CC-LR mice after NTHi exposure and IgG1 or IL-6 blockade at the age of 14 weeks were isolated by fluorescence-activated cell sorting, pooled, and relative expression of Arg1 and Ido mRNA were measured and compared in anti-IL-6 (n=3) or IgG1(n=3) treated groups. (G) ELISA analysis of BALFs from CC-LR mice after NTHi exposure and IgG1 (n=3) or IL-6 blockade (n=3) at the age of 14 weeks. (H) Relative expression of Tgfb, Ccl2, Foxp3, Nos2, Gzmb and Tbx21 mRNA in whole lungs of CC-LR mice after NTHi exposure and IgG1 (n=4) or IL-6 blockade (n=5) at the age of 14 weeks normalized by Cd45 expression of anti-IL-6 treated group compared with controls treated with IgG1 (data represent means ± SEM *P < 0.05. **P < 0.05.)
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