STK11/LKB1 deficiency promotes neutrophil recruitment and proinflammatory cytokine production to suppress T cell activity in the lung tumor microenvironment

Shohei Koyama1,2*, Esra A. Akbay2,3*, Yvonne Y. Li2,3*, Amir R. Aref2,3, FerdinandosSkoulidis4, Grit S. Herter-Sprie2,3, Kevin A. Buczkowski3 , Yan Liu2,3, Mark M. Awad2,3, Warren L. Denning4,Lixia Diao4, Jing Wang4, Edwin R. Parra4, Ignacio I. Wistuba4, Margaret Soucheray5, Tran C. Thai3, Hajime Asahina2,3,Shunsuke Kitajima3,Abigail Altabef3, Jillian D. Cavanaugh3, Kevin Rhee3, Peng Gao3, Haikuo Zhang2,3, Peter E. Fecci6, Takeshi Shimamura5, Matthew D. Hellmann7, John V. Heymach4, F. Stephen Hodi2,3,Gordon J. Freeman1,2, David A. Barbie2,3, Glenn Dranoff1,2,**, Peter S. Hammerman2,3,** and Kwok-Kin Wong2,3,8,**

1Department of Medical Oncology and Cancer Vaccine Center, Dana Farber Cancer Institute, Boston, MA

2Depatment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston MA

3Department of Medical Oncology, Dana Farber Cancer Institute, Boston MA

4Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas

5Department of Molecular Pharmacology and Therapeutics, Oncology Research Institute, Loyola University Chicago, Illinois

6Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina

7Department of Medicine, Memorial Sloan Kettering Cancer Center,New York, NY

8Belfer Institute for Applied Cancer Science, Dana Farber Cancer Institute, Boston, MA

* These authors equally contributed to this work

** Corresponding author

SUPPLEMENTAL MATERIAL

Supplemental Methods

Antibody list

Antigen / Clone / Antigen / Clone
CD45 / 30-F11 / PD-1 / 29F.1A12
CD3ε / 145-2C11 / TIM-3 / RMT3-23
CD4 / RM4-5 / LAG-3 / C9B7W
CD8a / 53-6.7 / PD-L1 / 10F.9G2
CD19 / 6D5 / CCR3 / J073E5
DX5 / DX5 / CXCR2 / TG11/CXCR2
NKp46 / 29A1.4 / EpCAM / G8.8
CD11c / N418 / CD16/32 / 2.4G2
CD11b / M1/70 / FOXP3 / FJK-16s
Ly-6G / 1A8 / CTLA-4 / UC10-4B9
Ly-6C / HK1.4 / IFNg / XMG1.2
Siglec F / E50-2440 / Ki-67 / 16A8
CD103 / 2E7 / LGALS9 / RG9-35
PD-L1(human) / 29E.2A3

Gating method for FACS analysis and sorting

Gating method was performed as described with some modifications (1,2)

After gating live, single CD45+ cells from total lung cells, cells were differentiated into 3 populations: 1) lymphocyte population (CD11c-CD11b-) which is mainly composedof CD4 T cells, CD8 T cells, B cells and NK cells, 2) CD11c+CD11b-whichincludes CD103- alveolar macrophages (AM) and CD103+CD11c+ dendritic cells,3) CD11b+ which include Ly-6G+ neutrophils (CXCR2+) and Ly-6G- population. CD11b+Ly-6G- population is composed of sub-populations, such asSiglecF+CCR3+ eosinophils and SiglecF-Ly-6Chimonocytes.

Mouse RNA sequence data analysis

RNA-seq reads were aligned to the mm9 Ensembl transcript annotation (release 65) using the PRADA pipeline (10.1093/bioinformatics/btu169), and FPKM expression values were determined using Cufflinks (3) with mm9 RefSeq gene annotations. FPKM values were log2-transformed and then used to calculate fold-change, where a fold change over 1.5 denoted overexpressed and less than -1.5 denoted underexpressed. For heatmaps, the log2-transformed FPKM values were colored on a blue-red scale, where blue/red represents lower/higher than the mean expression for that row/gene, respectively.

Human gene expressionand proteomic data analysis (CCLE, TCGA and PROSPECT)

We downloaded RNA-seq data for 230 lung adenocarcinoma primary samples from TCGA (4) as well as KRAS and STK11/LKB1 mutation data for these samples from cBioPortal ( We downloaded RNA-seq data for 173 lung tumor cell-lines from the CCLE (5) as well as KRAS and STK11 mutation data for 172 of these samples from the CCLE Hybrid capture sequencing dataset and the COSMIC cell line project ().

For each dataset, we selected KRAS-mutated samples with G12 activating mutations and LKB1-mutated samples with homozygous (where annotated) nonsense, splice site, or frameshift mutations. We segregated the samples into those with KRAS and STK11/LKB1 mutations (KL) versus those with KRAS but no STK11 mutations of any kind (also including missense mutations) (K).

Details regarding sample collection, storage, selection and nucleic acid extraction for

PROSPECT tumors have been previously published(6). Gene expression data for the PROSPECT cohort have been previously deposited in the GEO repository (GSE42127). RPPA analysis was performed as previously reported with samples assayed using an Aushon Biosystems 2470 arrayer (Burlington, MA) (4,7).

Measurement of soluble factor concentrations in BALFs from mice and culture supernatants from murine and human cell lines

BALFs collection was performed as described previously (1). For analysis of cell culture supernatants, 5x105 or 8x105 cells were spread on 6-well plates and supernatants were collected at 12hr, 24hr or 48hr. Collected BALFs and supernatants were kept at -80º before performing the ELISA. Cytokine and chemokines were measured with ELISA kits according to the manufacturer`s protocol; mouse and human IL-6 (BD biosciences), mouse G-CSF, MFG-E8, CXCL7 (R&D Systems), mouse IL-1α, IL-10 (eBioscience) and human G-CSF, CXCL7 (R&D Systems).

SUPPLEMENTARY REFERENCES

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