Download Supplementary Table 1, Supplementary Methods, and

SUPPLEMENTAL MATERIAL
Supplemental Methods:
Table 1. List of Q-RT-PCR primers
Genes
Forward
Reverse
Actb
GGCTGTATTCCCCTCCATCG
CCAGTTGGTAACAATGCCATGT
Cd45
ACCACCAGGTGAATGTCAATTT
CTTGCTTTCCCTCGGTTCTTT
Arg1
TTTTTCCAGCAGACCAGCTT
AGAGATTATCGGAGCGCCTT
Ido
TGGCGTATGTGTGGAACCG
CTCGCAGTAGGGAACAGCAA
Il17
CTCCAGAAGGCCCTCAGACTAC
GGGTCTTCATTGCGGTGG
Tgfb
CTCCCGTGGCTTCTAGTGC
GCCTTAGTTTGGACAGGATCTG
Ifng
GATGCATTCATGAGTATTGCCAAGT
GTGGACCACTCGGATGAGCTC
Ccl2
TTAAAAACCTGGATCGGAACCAA
GCATTAGCTTCAGATTTACGGGT
Foxp3
GGCCCTTCTCCAGGACAGA
GCTGATCATGGCTGGGTTGT
Tbx21
CAACAACCCCTTTGCCAAAG
TCCCCCAAGCAGTTGACA
Gzb
CCACTCTCGACCCTACATGG
GGCCCCCAAAGTGACATTTATT
Fizz1
TGCTGGGATGACTGCTACTG
AGCTGGGTTCTCCACCTCTT
Mgl2
GGATCCCAAAATTCCCAGTT
TCCCTCTTCTCCAGTGTGCT
Mrc1
ATGCCAAGTGGGAAAATCTG
TGTAGCAGTGGCCTGCATAG
Nos2
GTTCTCAGCCCAACAATACAAGA
GTGGACGGGTCGATGTCAC
Scgb1
CCTTTCAACCCTGGCTCAGA
AGGGTATCCACCAGTCTCTTCAG
Supplementary method 1. Mass Spectrometry Analysis. Mass spectrometry
performed as previously described in Taguchi A et al, 2011 Cancer Cell (see below
reference 1). Briefly, lung cancer cell lines were grown in RPMI1640/10% FBS
supplemented with 13C-lysine per standard SILAC protocol. Extracts from the nucleus,
surface, cell culture media, and a total cell extract were analyzed separately. The
tandem mass spectra were searched against a composite database of IPI human
(v3.57) and IPI bovine (v3.43), with a fixed modification of 6.020129 mass units added
to lysine residues for database searching to account for incorporation of 13C-lysine.
PeptideProphet and ProteinProphet software tools were used to estimate significance
and abundance of peptide and protein matches (Faca et al). Initial peptides identified
with PeptideProphet were filtered for those only matching the human and not the bovine
database, which were then submitted to ProteinProphet. Protein matches were filtered
to a maximum 5% error, with the total number of spectral counts for each protein group
used for semi-quantitative analyses. Each dataset was normalized to the total number
of spectral counts of each compartment.
Supplementary method 2. Western Blot Analysis. After 24 and 48 hours, protein was
collected from cell lines by lysis using RIPA buffer including protease and phosphatase
inhibitors (Roche). Protein concentration was measured using Bradford Assay (Bio-Rad
Laboratories). Western blot analysis was performed using 20 µg protein/lane according
to standard procedures using polyvinylidene fluoride (PVDF) membranes and an
enhanced chemiluminescence system (GE Healthcare). Anti-phospho STAT3 (Tyr705)
(Clone D3A7, Cell Signaling Technology) and anti- β-actin (Clone AC-15, Sigma) were
used at 1:2000 and 1:20000 dilution, respectively.
Supplemental Figure Legends:
Supplementary Figure 1. Gating method for FACS analysis and sorting. After gating
live, single CD45+ cells, each population were differentiated as:1) Alveolar Macrophage
(CD11c+F4/80+); 2) Tumor Associated Macrophages (M1) (F4/80+/ Ly6C-/MHCII+); 3)
M-MDSC (F4/80+/Ly6Chi/MHCII-) and 4) G-MDSC (CD11b+/Ly6Ghi).
Supplementary Figure 2. (A) Heatmap representing IL-6/STAT3 pathway genes
expression status. Gene (mRNA) expression status for IL6, IL6ST, IL6R and STAT3 in
39 non-small cell lung cancer cell lines. The table summarizes histology, KRAS status,
plus IL6 and STAT3 expression in four representative cell lines used. (B-C) Mass
spectrometric quantification of IL-6 and STAT3 in lung cancer cell lines. Analysis
was performed separately on cell culture media and a nuclear, surface, and total cell
extract. (B) IL-6 is predominantly found secreted into the cell culture media in four lung
cancer cell lines. (C) STAT3 is found predominantly in the nuclear and surface
compartments of these cells. (D) Siltuximab and Tocilizumab decreased STAT3
activation in NSCLC cell lines. NSCLC cell lines were incubated with siltuximab (S)
(10g/mL) or tocilizumab (T) (1g/mL) for 24h or 48h and western blot was performed
to detect p-STAT3 expression.
Supplementary Figure 3. Increased levels of IL-6 in bronchoalveolar lavage fluid
(BALF) of CC-LR mice during tumor progression. (A) BALF from LR and CC-LR
mice were collected at the age of 14 weeks (B) BALF were collected at the age of 6
weeks, and 14 weeks from CC-LR mice treated or not treated with anti-IL-6 antibody
(aIL-6). The IL-6 levels were measured by ELISA. (n=4 animals per group) (data
represent means ± SEM **p<0.005. ****p<0.0001).
Supplementary Figure 4. Lung lesion distribution in lungs of CC-LR mice. Multiple
slides from CC-LR mice at the age of 14 weeks were evaluated for distribution of
hyperplastic vs adenoma/adenocarcinoma lesions and overall percentages of lesion
compared between anti IL-6 treated and non-treated groups were compared.
Supplementary Figure 5. Relative expression of Arg1 mRNA normalized by Cd45
expression in lungs of CC-LR mice at the age of 14 weeks after IgG1 (n=4) or IL-6
blockade (n=5) (data represent means ± SEM ****P < 0.0001).
Supplementary Figure 6. Increased levels of IL-6 in bronchoalveolar lavage fluid
(BALF) of CC-LR mice after NTHi exposure. (A) BALF from LR and CC-LR mice were
collected after 8 weeks of NTHi exposure (B) BALF from CC-LR were collected after 8
weeks of NTHi exposure and after IL-6 blockade. The IL-6 levels were measured by
ELISA. (n=4 animals per group) (data represent means ± SEM **p<0.005.
****p<0.0001).
Supplementary Figure 7. Relative expression of Scgb1 (CCSP) mRNA in lungs of CCLR mice at the age of 14 weeks after IgG1 (n=4) or IL-6 blockade (n=4) in the absence
or presence of NTHi-induced COPD-type inflammation (data represent means ± SEM).
Supplemental References: 1. Taguchi A, Politi K, Pitteri SJ, Lockwood WW, Faca vM,
Kelly-Spratt K, and et al. Lung cancer signatures in plasma based on proteome profiling
of mouse tumor models. Cancer Cell. 2011 Sep 13;20(3):289-99