Introduction

Lung cancer is the most prevalent cancer and the leading cause of cancer-related death, responsible for more than 2 million new diagnosis and 1.7 million deaths worldwide each year1. Approximately 85% of the diagnosed lung cancers are non-small cell lung cancer (NSCLC), with more than 50% of adenocarcinoma and 30% of squamous carcinoma. Because of the atypical symptoms, about two thirds of NSCLC patients present with advanced-stage disease at the time of diagnosis. A number of somatic mutations, oncogenic rearrangements or copy number variations in NSCLC tumors have been identified, including EGFR exon 19 deletions, L858R or T790M mutations, MET exon 14 skip** mutations, ALK or ROS1 rearrangements, MET, EGFR or HER2 copy number increases2. Various small molecular inhibitors and monoclonal antibodies have been developed to target these genetic alterations and significantly improve the prognosis of NSCLC patients3,4,5,6,7,8,9. Despite these advances, there are so far no specific therapeutic strategies for the NSCLC patients bearing mutations (G12C, G12V, or G12D) in KRAS which is the most common oncogenic driver found in 10–20% NSCLC incidences10. In addition, common co-mutational partners have been identified in KRAS-mutated NSCLC, including TP53, LKB1, and CDKN2A11. These co-mutational factors render different gene expression profiles and might determine distinct therapeutic strategies for KRAS-mutated NSCLCs. Mouse models which express the mutated KrasG12D and simultaneously inactivate Tp53 (KrasLSL−G12D/+Tp53fl/fl; KP) or Lkb1 (KrasLSL−G12D/+Lkb1fl/fl; KL) have been developed and provide powerful tools for the screen and evaluation of effective therapies for KRAS-mutated NSCLCs12,13,14.

Recently, checkpoint immunotherapies with the immune checkpoint blockers such as anti-programmed death 1 (anti-PD-1) and anti-programmed death-ligand 1 (anti-PD-L1) antibodies have significantly improved the progression-free survival (PFS) and overall survival (OS) compared with the platinum- or docetaxel-based chemotherapies for NSCLC patients15,16. In addition, a more favorable prognosis of anti-PD-1/PD-L1 checkpoint immunotherapies is observed in patients with higher expression of PD-L1 in the tumor microenvironment (TME) and without EGFR and ALK mutations17,18, suggesting that PD-L1 expression in the TME is a critical predictive marker for checkpoint immunotherapies of NSCLC. Consistently with this notion, LKB1 alterations are significantly associated with PD-L1 negativity and render PD-1 inhibitor resistance in KRAS-mutated NSCLCs19. However, even among the NSCLC patients with high levels of PD-L1 positivity, the objective response rate (ORR) is about 45%17,20, which might be due to defects in the entry or proliferation of tumor-infiltrating leukocytes or due to suppression by other molecular pathways in the TME. Therefore, administration of molecules to promote the entry, activation and expansion of tumor-infiltrating lymphocytes (TILs) would enhance the efficacy of checkpoint immunotherapies for NSCLCs.

Chemokine (C-C motif) ligand 7 (CCL7, also known as MCP-3) is a chemotactic factor and potent attractant of monocytes firstly characterized from the culture supernatants of MG-63 osteosarcoma cells21. CCL7 is expressed at low levels in endothelial cells, fibroblasts and mononuclear cells and upregulated by various stimuli including viruses, type I or type II interferons (IFNs)22. CCL7 receptors including CCR1, CCR2, and CCR3 are mainly expressed on the surface of antigen-presenting cells (APCs) such as monocytes, macrophages and dendritic cells (DCs)23. CCL7 deficient mice fail to efficiently eliminate various infected pathogens and exhibit impaired monocyte and neutrophil infiltration in infected tissues or organs24,25, indicating essential roles of CCL7 in anti-infectious immunity by recruiting immune cells to the infected microenvironment. Various studies have shown that tumor cells and stromal cells also produce high levels of CCL7, while the specific response element and signaling pathways involved are not entirely clear6. Eight-week-old male and female KP, KP7 or KL mice were intranasally injected with Ad-Cre followed by various experiments. B6.SJL (#002014) mice (8-week old) were from the Jackson Laboratory and kindly provide by Dr. Haojian Zhang (Wuhan University). CD11c-DTR mice (#004509) (8- week old) were from the Jackson Laboratory and kindly provided by Drs. **n-Yuan Zhou and Ying Wan (Third Military Medical University). Zbtb46-DTR mice (#019506) (8-week old) were from the Jackson Laboratory and kindly provided by Drs. Cliff Yang (Sun Yat-sen University) and **ao Shen (Zhejiang University). C57B/6 mice (8-week old) were purchased from GemPharmatech Co., Ltd (Nan**g, Jiangsu Province). All experimental groups contained male and female mice and all the mice housed in the specific pathogen-free animal facility (12 h/12 h light and dark cycle, 22 oC ± 2 oC) at Wuhan University. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Wuhan University.

Quantitative real-time PCR

These experiments were performed as previously described54,55. Total RNA was extracted from tumor or normal tissues or cells using TRIzol reagent (Invitrogen), and the first-strand cDNA was reversed-transcribed with All-in-One cDNA Synthesis SuperMix (Biotool). Gene expression was examined with a Bio-Rad CFX Connect system (Bio-Rad CFX Manager 3.1) by a fast two-step amplification program with 2 × SYBR Green Fast qPCR Master Mix (Biotool). The value obtained for each gene was normalized to that of the gene encoding GAPDH or β-actin. Gene-specific primers are listed in Supplementary Table 6.

Tissue microarray preparation

Tissue microarray was prepared as previously described56. In brief, tumor and normal tissues from NSCLC patients with wedge resection, pulmonary lobectomy or CT-guided puncture were fixed in 4% paraformaldehyde and embedded into paraffin blocks. The paraffin blocks were punched out of the selected regions based on a hematoxylin-eosin (HE) staining analysis. The punched samples were 1.5 mm in diameter and 4–6 mm in length and assembled into a new paraffin block. The tissue microarrays were sectioned (4 μm) and stained with H&E to confirm the histological results. Tumor-burdened lungs from KP or KP7 mice were fixed in 4% paraformaldehyde and embedded into paraffin blocks which were sectioned (5 μm) for subsequent analysis.

IHC assays

The sections were deparaffinized with xylene, rehydrated in 100, 85 and 70% ethanol for 10 min, quenched for endogenous peroxidase activity in 3% hydrogen peroxide, and processed for antigen retrieval in 0.5 mM EDTA (pH8.0) buffer by heating in a microwave oven for 20 min. The sections were cooled down naturally to room temperature and stained with various antibodies diluted in PBS containing 1% BSA and incubated at room temperature for over 6 h. Immunostaining was performed using the Maixin_Bio Detection Kit peroxidase/diaminobenzidine (DAB) rabbit/mouse (Kit-9710, DAB-0031; Maixi_Bio, Fuzhou), which resulted in a brown-colored precipitate at the antigen site. Subsequently, sections were counterstained with hematoxylin (Zymed Laboratories) for 5 min and coverslipped. The information and dilution of antibodies have been listed in Table S5. Images were acquired with the Leica Aperio VERSA 8 (Aperio imagescope (v12.3.2.8013) multifunctional scanner. The intensities of DAB staining were measured and quantified with IOD or cell intensity by Image Pro Plus 6 (Media Cybernetics).

Induction of tumorigenesis in KP or KL mouse model

Eight-to-ten-week-old KP or KP7 mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (w/v = 1:7), followed by intranasal injection of Ad-Cre viruses (Obio Technology, Shanghai) (1~2 × 106 pfu in 60 μl PBS per mouse) or Lentiviruses expressing empty vector, CCL7, Cre or Cre and CCL7 (2 × 106 pfu in 60 μl PBS per mouse). The survival of mice was recorded until the end of the study. Alternatively, at the indicated time points after infection, mice were euthanized and the BALF, lungs or dLNs were removed for subsequent analysis.

Preparation, concentration, and titration of lentiviruses

The phage-6tag vector was modified to generate Lenti-Vec, Lenti-Cre, Lenti-CCL7, or Lenti-Cre-CCL7 constructs. In brief, the gene encoding Puromycin downstream the PGK promoter was removed and the DNA encoding Cre recombinase or GFP was inserted by Pst I and Sph I. Such vectors were designated as Lenti-Cre or Lenti-Vec. The DNA encoding mouse CCL7 was inserted into the multiple clone site of the Lenti-Cre or Lenti-Vec vector with Not I and Xho I and the resulted constructs were named Lenti-Cre-CCL7 or Lenti-CCL7. The Lenti vectors were cotransfected with the package plasmids pSPAX2 and pMD2G into HEK293T cells. The medium was changed with fresh full medium (10% FBS, 1% streptomycin-penicillin and 10 μM β-mercaptoethanol) after 8 h. Forty hours later, the supernatants were harvested and filtered with a 0.45 μm filter and mixed with a Virus Precipitation Solution (5×) at 4 °C for 12 h (Cat# EMB810A-1, Excell Bio). The viruses were harvested by centrifugation at 3000 × g for 30 min. The supernatants were discarded and the precipitants containing Lentiviruses were re-suspended with PBS and stored at −80 °C. The resulted Lenti-Cre or Lenti-Cre-CCL7 and Lenti-Vec or Lenti-CCL7 or their serial dilutions were used to infect 3T3loxp-RFP-stop-loxp-GFP cells or 3T3 cells (kindly provided by Dr. Hong-Bin Ji, Institute for Biochemistry and Cell Biology, Chinese Academy of Science, Shanghai) for forty-eight hours, respectively, and the titers was determined by flow cytometry analysis57.

Isolation of mouse lung epithelial cells

Mouse primary lung epithelial cells were isolated as described previously58. Lungs from C57B/6 mice were perfused through cardiac lavage with PBS. Dispase solution (2 ml at 3.6 unit/ml; 17105–41; Gibco) was instilled into the lungs through a tracheal catheter. Lungs were removed from mice and incubated in the dispase solution for 1 h at room temperature. The lungs were microdissected and cell suspensions were filtered through nylon monofilament. The recovered cells were centrifuged at 1500 × g for 5 min and resusbended in PBS containing 1.5% FBS. The cells were incubated with anti-CD45 microbeads for 30 min at 4 °C and the CD45+ cells were depleted by flow-through a magnet column (Miltenyi Biotec). The resulted cells were resuspended in DMEM containing 10% FBS, 1% streptomycin-penicillin and 10 μM β-mercaptoethanol and were seeded into 48-well plates at a density of 1 × 105 cells per well for overnight culture, followed by various treatments.

Chromatin immunoprecipitation (ChIP) assays

These experiments were performed as previously described59,60. Cells with various stimuli were fixed with 1% formaldehyde for 15 min and washed with PBS for three times. The cells were lyzed in ChIP lysis buffer (50 mM Tris·HCl pH 8.0, 1% SDS, 5 mM EDTA) followed by sonication to generate DNA fragments of 300–500 bp. The lysates were centrifuged at 4 °C for 15 min and ChIP dilution buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) was added to the supernatant (4:1 volume). The resulting lysates were then incubated with protein G beads and anti-pSTAT1 (Cat9167S, CST) or control IgG at 4 °C for 4 h. DNA was eluted by ChIP elution buffer (0.1 M NaHCO3, 1% SDS, 30 μg/mL proteinase K) followed by incubation at 65 °C for overnight. The DNA was purified with a DNA purification kit (TIANGEN) and was assayed by quantitative PCR using the SFX connect system with the 2 × SYBR Green fast qPCR master mix kit (Biotool). The qPCR primer sequences of CCL7 or Ccl7 promoter were listed in Supplementary Table 6.

JAK1 inhibitor treatment

KP mice intranasally injected with Ad-Cre (2 × 106 pfu in 60 μl PBS per mouse) for six weeks were prepared for JAK1 inhibitor treatment. Ruxolitinib phosphate (T3043, TargetMol) was dissolved in DMSO (100 mg/ml) and diluted with PBS containing 5% (v/v) PEG300/dextrose (PEG:dex, 1:3, v/w) buffer until use. The mice were administered orally twice daily in an application dosage of 60 mg/kg bodyweight. After 2 weeks treatment of ruxolitnib, the lungs of the KP mice were separated and analyzed by qRT-RCR assay.

Preparation of single-cell suspensions from tumor-burdened lungs

Tumor-burdened lungs from KP or KP7 mice were perfused through alveolar lavage and cardiac lavage with PBS. The lungs from one mouse were cut into small pieces (2~4 mm in diameter) and transferred into a gentleMACS C Tube with the enzyme mix containing 2.35 ml of DMEM, 100 μl of Enzyme D, 50 μl of Enzyme R, and 12.5 μl of Enzyme A from a Tumor Dissociation Kit (Miltenyi Biotech). The C Tube was tightly closed and attached onto the sleeve of the gentleMACSTM Octo Dissociator (Miltenyi Biotech) with the tumor isolation program. After termination of the program, C tube was detached from the Dissociator and incubated at 37 °C for 40 min. Then repeat the tumor isolation program twice and perform a short spin up to 1500 × g to collect the sample at the bottom of the tube. After dissociation, the sample re-suspended was applied to MACS SmartStrainers (70 μm) to prepare single-cell suspension.

Preparation of lung LILs

The obtained single-cell suspensions were centrifuged at 1,500 g for 5 min at room temperature, and the precipitants were re-suspended with 40% Percoll (Cat17-0891-09, GE Healthcare) in PBS (v/v). The suspension was centrifuged at 1,500 g for 20 min at room temperature and the supernatant was discarded. The precipitants containing LILs were re-suspended in 10% FBS DMEM containing PMA (50 ng/ml, P8139, Sigma), Ionomycin (500 ng/ml, I0634, Sigma), Golgi-stop (1:1000, Cat# 554724, BD Biosciences) and cultured for 4 h at 37 °C, followed by staining and flow cytometry analysis.

Flow cytometry analysis

Flow cytometry protocol has previously described61,62. The single-cell suspensions of tumor-burdened lungs, bronchial dLN or the obtained LILs were re-suspended in FACS buffer (PBS, 1%BSA) and blocked with anti-mouse CD16/32 antibodies for 10 min prior to staining with the antibodies of the surface markers. For intracellular cytokine staining, cells were fixed and permealized with a fixation and permeabilization solution kit (Cat# 424401, Biolegend) followed by staining with the specific antibodies against intracellular cytokines. Antibodies used for flow cytometry analysis were listed in Table S5. Flow cytometry data were acquired on a FACSCelesta flow cytometer (BD Biosciences, BD FACSDiVa Software v8.0.1.1) and analyzed with Flowjo 10.6.2 software (TreeStar). The staining antibodies were listed in Supplementary Table 7.

Bone marrow transfer and LCMV infection

C57BL/6 mice (8-week old) were irradiated (8 Gy, 4 Gy for twice) followed by injection of mixed bone marrow cells from CD45.1+ (wild-type) mice (1 × 106) and CD45.2+ (Ccl7−/−) mice (1 × 106) through tail vein. Eight weeks later, the mice were intraperitoneally injected with LCMV (2 × 105 pfu per mouse) (Armstrong) which was kindly provided by Dr. **n-Yuan Zhou (Third Military Medical University). The mice were sacrificed one week after infection and the spleenocytes were left unstimulated or stimulated with PMA and ionomycin plus Golgi stop followed by surface and intracellular staining with GP31-41 tetramer, CD45.1, CD45.2, and IFNγ and flow cytometry analysis.

Diphtheria toxin-mediated depletion of CD11c+ or Zbtb46+ DCs

Bone marrow cells were isolated from the femur of CD11c-DTR or Zbtb46-DTR donor mice. Ten-week-old recipient KP or KP7 mice were irradiated with 8 Gy (4 Gy for twice) by small animal X-ray irradiater (RS2000Pro, Rad Source) and immediately injected the isolated CD11c-DTR or Zbtb46-DTR bone marrow cells through the tail vein (106 cells per mouse). Eight weeks later, the recipient KP or KP7 mice were intranasally infected with Ad-Cre (2 × 106 pfu per mouse). At the fifth week after tumor induction, the recipient KP or KP7 mice were injected intraperitoneally with DT (4 ng/g body weight, D0564, Sigma) or PBS every three days for 4 weeks. DC depletion efficiency in the recipient KP or KP7 mice were examined by flow cytometry and IHC analysis.

Combinational treatment of CCL7 and anti-PD-1

Eight-week-old KP mice were infected intranasally with Ad-Cre (2 × 106 pfu in 60 μl PBS per mouse). At fifth week after tumor induction, mice were intranasally injected with Lenti-GFP or Lenti-GFP-CCL7 (2 × 106 pfu in 60 μl PBS per mouse). These mice were either intraperitoneally injected with control lgG (BE0091, BioXcell) or anti-PD-1 (J43BE0033-2, BioXcell) (0.2 mg in 200 μl PBS per mouse each time) twice a week until death or for 4 weeks for histological analysis.

Hematoxylin-eosin staining analysis

Lungs from mice were fixed in 4% paraformaldehyde and embedded into paraffin blocks as previously described63. The paraffin blocks were sectioned (5 μm) for H&E staining (Beyotime Biotech) followed by coverslipped. Images were acquired using a Aperio VERSA 8 (Leica) multifunctional scanner.

Statistical analysis

Differences between experimental and control groups were tested using Student’s t-test. The number of repeats for each experiment is also indicated in the respective figure legends. N in the figure legends indicates the number of mice or replicates in the experiments. P values < 0.05 were considered statistically significant. For animal survival analysis, the Kaplan–Meier method was adopted to generate graphs, and the survival curves were analyzed with log-rank analysis. Prism 6 was used to generate graphs and perform statistical analysis.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.