Research Article

The Tumor Microenvironment RegulatesSensitivity of Murine Lung Tumors toPD-1/PD-L1 Antibody BlockadeHoward Y. Li1,2, Maria McSharry2, Bonnie Bullock2, Teresa T. Nguyen2, Jeff Kwak2,Joanna M. Poczobutt2, Trisha R. Sippel2, Lynn E. Heasley3, Mary C.Weiser-Evans2,Eric T. Clambey4, and Raphael A. Nemenoff2

Abstract

Immune checkpoint inhibitors targeting the interactionbetween programmed cell death-1 (PD-1) and its ligand PD-L1induce tumor regression in a subset of non–small cell lungcancer patients. However, clinical response rates are less than25%. Evaluation of combinations of immunotherapy withexisting therapies requires appropriate preclinical animal mod-els. In this study, murine lung cancer cells (CMT167 and LLC)were implanted either orthotopically in the lung or subcuta-neously in syngeneic mice, and response to anti–PD-1/PD-L1therapy was determined. Anti–PD-1/PD-L1 therapy inhibitedCMT167 orthotopic lung tumors by 95%. The same treatmentsinhibited CMT167 subcutaneous tumors by only 30% and LLCorthotopic lung tumors by 35%. CMT167 subcutaneous tumorshad more Foxp3þ CD4þ T cells and fewer PD-1þ CD4þ T cellscompared with CMT167 orthotopic tumors. Flow cytometric

analysis also demonstrated increased abundance of PD-L1high

cells in the tumor microenvironment in CMT167 tumor–bearing lungs compared with CMT167 subcutaneous tumorsor LLC tumor–bearing lungs. Silencing PD-L1 expression inCMT167 cells resulted in smaller orthotopic tumors thatremained sensitive to anti–PD-L1 therapy, whereas implanta-tion of CMT167 cells into PD-L1� mice blocked orthotopictumor growth, indicating a role for PD-L1 in both the cancercell and the microenvironment. These findings indicate thatthe response of cancer cells to immunotherapy will be deter-mined by both intrinsic properties of the cancer cells andspecific interactions with the microenvironment. Experimentalmodels that accurately recapitulate the lung tumor microenvi-ronment are useful for evaluation of immunotherapeuticagents. Cancer Immunol Res; 5(9); 767–77. �2017 AACR.

IntroductionA breakthrough in cancer therapy is the development of

immune checkpoint inhibitors. Interaction of programmed celldeath-1 (PD-1) with its ligands, PD-L1 or PD-L2, regulatesperipheral tolerance and protects tissues from autoimmuneattack (1). However, induction of PD-L1 expression on tumorcells can inhibit cytotoxic T cells, which allows tumors toprogress (2). Evidence that PD-L1 is upregulated in non–smallcell lung cancer (NSCLC) has led to the development ofimmune-based therapies for NSCLC. However, response ratesin NSCLC patients across clinical trials of PD-1 and PD-L1antibodies have ranged from 14.5% to 20% (3–7). The com-

bination of immunotherapy with existing therapies such asradiation therapy, chemotherapy, or other immunotherapeuticagents may improve response rates. Testing of such combina-tions and development of patient selection criteria for immu-notherapy require preclinical models that mimic the humandisease.

In vivo growth of lung cancer cells has been assessed by implant-ing human cells into immune compromised mice (xenograftmodels). However, these mice lack T lymphocytes, rendering thismodel unsuitable for examining immunotherapy. To study therole of adaptive immunity in tumor progression, implantation ofmurine cancer cells into syngeneic mice is required. There are fewestablished murine lung cancer cell lines derived from C57BL/6mice. Many studies use a subcutaneous model whereby tumorsdevelop in the flank, which fails to reflect the lung tumor micro-environment (TME). To examine the role of the TME in lungcancer, our laboratory utilizes an orthotopic model in whichmurine lung cancer cells are implanted into the lungs of syngeneicC57BL/6mice (8–11). In this study, we examined the response oftumors grown in the lung or subcutaneously to PD-1/PD-L1inhibition. We found that sensitivity to PD-1/PD-L1 antibodieswas dependent on both the site of tumor growth and the cancercell line, and was associated with upregulation of PD-L1 in bothcancer cells and stromal cells. This study suggests that the responseof lung cancer to PD-1/PD-L1 inhibition will be determined byinteractions between cancer cells and noncancer cells specific tothe lung.

1Department of Medicine, Veterans Affairs Medical Center, Denver, Colorado.2Department of Medicine, University of Colorado Anschutz Medical Campus,Aurora, Colorado. 3Department of Dental Medicine, University of ColoradoAnschutz Medical Campus, Aurora, Colorado. 4Department of Anesthesiology,University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

CorrespondingAuthor:Howard Y. Li, Denver VAMedical Center, 1055 ClermontStreet, Denver CO 80220. Phone: 303-724-4881; Fax: 303-724-6042; E-mailHoward.Li@ucdenver.edu

doi: 10.1158/2326-6066.CIR-16-0365

�2017 American Association for Cancer Research.

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Materials and MethodsCell lines

CMT167 cells (12) were stably transfectedwith firefly luciferaseas previously described (11). Lewis lung carcinoma cells (LL/2)were purchased from ATCC and luciferase-expressing Lewis lungcarcinoma cells (LLC) were purchased from Caliper Life Sciences(LL/2-luc-M38). All cell lines were periodically tested for myco-plasma infection andwere last retested in February 2017. To avoidcross-contamination and phenotypic changes, cells were main-tained as frozen stocks and cultured for only 2 to 4 weeks beforeuse in experiments. Authentication of cell lines based on mor-phology, growth curve analysis, and metastatic phenotype wasperformed regularly, and no phenotypic changes were observedthrough the duration of the study.

Kras sequence analysisTotal RNA was extracted from CMT167, LLC, and LL/2 cells

using an RNeasy Mini Plus Kit (QIAGEN). Reverse transcriptionwas performed using an iScript cDNA Synthesis Kit (Bio-Rad).Kras was amplified by PCR with the following primers: For, 50-GCCTGCTGAAAATGACTGAG-30; Rev, 50-TGCTGAGGTCTCAAT-GAACG-30. PCR products were purified using a QIAquick PCRPurification Kit (Qiagen) and sequenced using the reverse primer50-TCCAAGAGACAGGTTTCTCCA-30.

Animals and tumor modelsWild-type C57BL/6 mice and green fluorescent protein (GFP)-

expressingmice [C57BL/6-Tg(UBC-GFP)30Scha/J] were obtainedfrom The Jackson Laboratory. PD-L1 knockout (KO) mice on aC57BL/6 backgroundwere provided byDr.HaidongDong (MayoClinic, Rochester, MN). Animals were bred andmaintained in theCenter for Comparative Medicine at the University of ColoradoAnschutz Medical Campus. Experiments were performed on 8- to12-week-old male mice. All procedures were performed underprotocols approved by the Institutional Animal Care and UseCommittees at theUniversity ofColorado andDenver VAMedicalCenter.

Surgeries were performed under inhaled isoflurane anesthesia,and all efforts were made to minimize suffering. For orthotopictumors, a transverse skin incision was made along the left lateralaxillary line at the level of the xyphoid process, and subcutaneousfat was dissected away as previously described (9). CMT167 orLLC cells (105) were suspended in Hank's Buffered Salt Solution(HBSS) containing 1.35 mg/mL Matrigel (Corning) and injectedthrough the chest wall into the left lung, and the incision wasclosed using veterinary skin adhesive. For subcutaneous tumors,the inoculation site was sterilizedwith ethanol, andCMT167 cellssuspended in 100 mL HBSS were injected subcutaneously into thelower flank. Four weeks after CMT167 tumor implantation or 3weeks after LLC tumor implantation, mice were injected intra-peritoneally with D-Luciferin (PerkinElmer, 300 mg/kg) imme-diately before euthanasia. Primary tumor sizewasmeasured usingdigital calipers. Metastasis to the lungs,mediastinal lymph nodes,and liver were quantified by ex vivo bioluminescence using an IVIS50 Imaging System (Xenogen) and Living Image software(PerkinElmer).

PD-1 and PD-L1 pharmacologic antibody blockadeRatmonoclonal antibody tomouse PD-1 (clone RMP1-14), rat

monoclonal antibody to mouse PD-L1 (clone 10F.9G2), and

isotype control antibodies (rat IgG2a clone 2A3 for anti–PD-1,rat IgG2b clone LTF-2 for anti–PD-L1) were purchased fromBioXCell. Tumor-bearing mice were injected intraperitoneally[200mg in PBS per dose (8–10mg/kg)] three timesweekly starting1 week after tumor implantation.

ImmunostainingSections (5 mm thick) were cut from formalin-fixed, paraffin-

embedded (FFPE) tissue blocks. Antigens were revealed byheating slides in Antigen Unmasking Solution (Vector Labs).Slides were blocked with 3% normal goat serum, then incu-bated overnight with anti-CD3e antibody (Abcam). After wash-ing with TBST, slides were incubated with AF488 conjugatedIgG secondary antibody (Life Technologies). Slides were cover-slipped with VECTASHIELD Mounting Medium with DAPI(Vector Labs). Tumor-infiltrating lymphocytes (TIL) weredefined as lymphocytes within tumor cell nests or in directcontact with tumor cells. For comparison of CMT167 and LLCtumors, three sections from each animal were stained, and threerandom fields (40�) per section were quantified independentlyby two blinded observers (H.Y. Li and M. McSharry); the meanwas used for analysis.

Flow cytometryFor orthotopic tumors, the pulmonary circulationwas perfused

with heparinized PBS, and tumor-bearing left lungs were isolated.Flank tumors were dissected away from the subcutaneous tissueand were mechanically dissociated and digested with a mixturecontaining collagenase type 4 (8480 U/mL), elastase (7.5mg/mL), and soybean trypsin inhibitor (2 mg/mL; all fromWorthington Industries) in HBSS. Red blood cell lysis was per-formed using RBC lysis buffer [0.15 mol/L NH4Cl, 0.1 mmol/LKHCO3, 0.1 mmol/L Na2EDTA (pH 7.2)]. Antibody panels aresummarized inSupplementary TableS1. Sampleswere acquiredatthe University of Colorado Cancer Center Flow Cytometry SharedResource using a Gallios flow cytometer (Beckman Coulter) anddata were analyzed using Kaluza Software (Beckman Coulter). Todetermine the abundance of PD-L1high cells in the TME, tumorswere implanted in GFP-expressing transgenic mice, and the ratioof GFPþPD-L1high to GFPþPD-L1low cells was determined.

Recovery of lung cancer cells from tumor-bearingGFP-expressing transgenic mice

Three weeks after CMT167 tumor implantation or 2 weeksafter LLC tumor implantation, tumor-bearing left lungs wereisolated and single-cell suspensions were prepared. Cell sortingwas performed at the University of Colorado Cancer CenterFlow Cytometry Shared Resource using an XDP-100 cell sorter(Beckman Coulter). Lung cancer cells were recovered fromtumor-bearing GFP-expressing transgenic mice by sorting forGFP-negative cells, and total RNA was isolated using an RNeasyPlus kit (QIAGEN).

RNA sequencing (RNA-seq)CMT167 cells were recovered from three pools of mice con-

sisting of five GFP-expressing mice each, whereas LLC cells wererecovered from five pools of mice consisting of four GFP-expres-singmice each. Total RNAwas also isolated from cells in culture atthe time of injection. RNA-seq library preparation and sequencingwere conducted at the University of Colorado Cancer CenterGenomics and Microarray Shared Resource. RNA libraries were

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constructed using an Illumina TruSEQ stranded mRNA SamplePrep Kit. Sequencing was performed on an Illumina HiSEQ 2500High-Throughput Flow Cell. RNA-seq reads were processed andaligned to the UCSC Mus musculus reference genome (buildmm10) using TopHat v2 (13). The aligned read files were pro-cessed by Cufflinks v2.0.2 (14), which uses the normalized RNA-seq fragment counts to measure the relative abundance of tran-scripts. The unit of measurement is Fragments Per Kilobase ofexon per Million fragments mapped (FPKM).

Quantitative real-time PCR (qRT-PCR)CMT167 cells were recovered from three pools of mice con-

sisting of three tumor-bearing GFP-expressingmice each, and LLCcells were recovered from four pools of mice consisting of twotumor-bearing GFP-expressing mice each, and total RNA wasisolated using an RNeasy Plus Kit (Qiagen). Total RNA was alsoisolated from cancer cells grown in culture, either in the presenceor in the absence of IFNg (100 ng/mL). Reverse transcription wasperformed using an iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR analysis was conducted in triplicate in an iCycler (Bio-Rad). Sequences of primers used were: PD-L1 (For: 50-TGCTGCA-TAATCAGCTACGG-30, Rev: 50-GCTGGTCACATTGAGAAGCA-30), b-actin (For: 50-GGCTGTATTCCCCTCCATCG-30, Rev: 50-CCAGTTGGTAACAATGCCATGT-30).

shRNA knockdown of PD-L1Three shRNAs (sh67998, sh68000, and sh68001) to mouse

PD-L1 and a nontargeting control shRNAwere obtained from theFunctional Genomics Facility at the University of Colorado Can-cer Center. Lentiviral particles were generated by transfectingshRNA vectors into 293T cells using Turbofect (Thermo Scientif-ic). CMT167 cells were transduced with lentiviral particles andplaced under puromycin selection. Cells were analyzed for PD-L1mRNA expression by qRT-PCR and protein expression by flowcytometry as described above. The pool of cells with the highestdegree of knockdown (CMT sh68001) was designated CMTshPD-L1 and used in orthotopic mouse experiments. CMT167cells transduced with nontargeting control shRNA were designat-ed CMT shCON.

CD8þ T-cell immunodepletionRat monoclonal anti-mouse CD8 (clone 53-6.72) and rat

IgG2a (clone 2A3) were purchased from BioXCell. Mice wereinjected intraperitoneally [200 mg in PBS per dose (8–10 mg/kg)]twice weekly starting 1 day prior to tumor implantation andthroughout the course of the tumor progression experiment.

Isolation of bone marrow–derived macrophages (BMDM) andtreatment with cancer cell–conditioned media (CM)

Bone marrow cells isolated from C57BL/6 mice were maturedinto macrophages using M-CSF (eBioscience, 50 ng/mL) as pre-viously described (10). Two days after isolation, 25% controlmedia, 25% LLC CM, or 25% CMT167 CM was added to bonemarrow cells (CM were normalized to total protein). Culturemedia were replaced every three days. As a positive control,macrophages matured in M-CSF only were stimulated withrecombinant mouse interferon-g (Sigma, 100 ng/mL). For neu-tralization experiments, monoclonal antibodies against IL7(BioXCell clone M25) and IL15 (eBioscience clone AIO.3) wereadded along with CM (10 mg/mL). After 10 days, total RNA wasisolated from BMDMs.

Statistical analysisData are presented asmean� SEM.Differences between groups

were identified using Student unpaired t test or one-way ANOVA.Under all circumstances, P < 0.05 was considered significant.

ResultsCMT167 cells, luciferase-expressing LLC cells, and LL/2 cellsexpress oncogenic K-Ras

Although there are hundreds of human lung cancer cell lines,there are few murine lung cancer cell lines. In this study, we usedtwo murine lung cancer cell lines derived from C57BL/6 mice,CMT167, and LLC cells. CMT167 cells have previously beenreported to harbor an activating KrasG12V mutation (15). DirectDNA sequencing confirmed that CMT167 cells harbor a KrasG12V

mutation (Supplementary Fig. S1A). The DNA sequencing chro-matogram of Kras in luciferase-expressing LLC cells purchasedfrom Caliper revealed a double peak in codon 12 that corre-sponded with a heterozygous activating KrasG12C mutation (Sup-plementary Fig. S1B). DNA sequencing of Kras in LL/2 cellspurchased from ATCC revealed an identical heterozygousKrasG12C mutation (Supplementary Fig. S1C). Thus, these celllines represent K-Ras–mutant lung cancers.

Sensitivity of CMT167 orthotopic tumors to PD-1/PD-L1inhibition

We have previously shown that murine lung cancer cellsinjected into the lungs of syngeneic C57BL/6mice form a primarytumor, which subsequently metastasizes to the other lung lobes,mediastinal lymph nodes, and liver (10, 11). Because this modelrecapitulates many features of human lung cancer, we sought todetermine whether orthotopic tumors are sensitive to anti-PD-1/PD-L1. Wild-type C57BL/6 mice were treated with anti–PD-1,anti–PD-L1, or control IgG starting 1 week after CMT167 ortho-topic tumor implantation. Treatment with either checkpointinhibitor reduced primary tumor size by approximately 95%;40%ofmice receiving anti–PD-1 and 60%ofmice receiving anti–PD-L1 had no detectable tumors (Fig. 1A). PD-1 and PD-L1inhibition reduced the metastatic burden in the other lung lobesby 81% and 87%, respectively (Fig. 1B). Metastases were detectedin some animals that had undetectable primary tumors, suggest-ing that primary tumors were initially present and regressed withcheckpoint inhibitor treatment. Immunofluorescence staining oftumor-bearing lungs for CD3e (Fig. 1C) revealed dense nests oftumor-infiltrating T lymphocytes inmice treated with anti—PD-1[Fig. 1C(c) and (d)] and anti–PD-L1 [Fig. 1C(g) and (h)].Although lymphocytes were identified in tumors from controlanimals [Fig. 1C(a), (b), (e), (f)], large nests of lymphocytes werenot observed. These data suggest that in our orthotopic model,treatment with anti–PD-1/PD-L1 leads to accumulation of TILsthat mediate antitumor responses.

PD-L1 expression in CMT167 orthotopic tumorsNext, we examined in vivo expression of PD-L1 in CMT167

orthotopic tumors. To distinguish cancer cells from normalmouse lung epithelial cells, CMT167 cells were implanted intoGFP-expressing transgenic mice. Two weeks after tumor implan-tation, tumor-bearing lungs were analyzed by flow cytometry.Uninjected mice were used for controls (Fig. 2A). In mice withCMT167 orthotopic tumors, a subset of both cancer cells (GFP–)and noncancer cells (GFPþ) expressed PD-L1 (Fig. 2B). PD-L1high

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cells were more abundant in the microenvironment of CMT167tumor–bearing mice than in control mice. Although cancer cellsexpressing high levels of PD-L1 (GFP–;PD-L1high) were detected,this population appeared smaller and expressed less PD-L1 thanhost-derived PD-L1high cells in the TME. To confirm PD-L1expression on cancer cells, CMT167 cells were recovered fromtumors using flow cytometry (Supplementary Fig. S2) and werecomparedwith cancer cells grown in culture using RNA-seq. Fromthis analysis, we determined that PD-L1 mRNA expression wasinduced in CMT167 cells in vivo (Fig. 2C). We confirmed thesedata in separate isolations of CMT167 cells recovered from GFPmice using qRT-PCR (Fig. 2D). As a positive control, CMT167 cellswere treated in vitro with IFNg to stimulate PD-L1 expression.

We have previously characterized subpopulations of macro-phages and other immune cells in the TME using flow cytometry(9). Using a similar strategy (Supplementary Fig. S3), we analyzeddistinct populations of PD-L1high cells in the TME (Table 1).Although PD-L1 was expressed by multiple cell types, the dom-inant population of PD-L1high cells inCMT167orthotopic tumorswas alveolar macrophages. The nonmacrophage PD-L1high cellswere comprised of dendritic cells, endothelial cells, and CD45�

CD31� cells that likely include fibroblasts.

Both CMT167 cells and immune cells must express PD-L1 forsensitivity to PD-L1 inhibition

To assess the specific contribution of PD-L1 expression bycancer cells, we depleted PD-L1 in CMT167 cells using shRNA(Supplementary Fig. S4A and S4B) and assessed response toPD-L1 antibody blockade in vivo. As shown in Fig. 2E, depletingPD-L1 partially reduced primary tumor growth compared withcontrol cells in mice receiving isotype antibody (25.5� 8.3 mm3

shCONvs. 9.2�2.3mm3 shPD-L1,P¼0.06).However, anti–PD-L1 antibody further inhibited growth of CMT shPD-L1 tumors(9.2� 2.3 mm3 isotype vs. 1.1� 0.6 mm3 anti–PD-L1, P < 0.01).

To evaluate the role of PD-L1 in the TME, we implantedCMT167 cells into wild type C57BL/6 and PD-L1 KO mice.CMT167 tumor growth was inhibited in PD-L1 KO mice com-pared with WT mice (Fig. 2H: WT 16.85 � 2.894 mm3 vs. PD-L1KO 1.753� 1.252mm3, P¼ 0.003). Due to the strong inhibition(�90%) of tumor growth, we were unable to determine whetherCMT167 orthotopic tumors grown in PD-L1 KOmice respond toPD-1/PD-L1 immunotherapy. Based on these data, we proposethat targeting PD-L1 on both cancer cells and cells of the TME iscritical for the antitumor effects of PD-L1 inhibition.

Sensitivity of CMT167 subcutaneous tumors to PD-1/PD-L1inhibition

To determine the importance of the lung TME inmediating theresponse to PD-1/PD-L1 inhibition, CMT167 cells wereimplanted subcutaneously in wild-type C57BL/6 mice. Tumor-bearing mice were treated with anti–PD-1 monoclonal antibody,anti–PD-L1, or control IgG starting 1 week after tumor implan-tation. In contrast to our orthotopicmodel, PD-1/PD-L1 antibodyblockade had a modest effect only on CMT167 subcutaneoustumor growth compared with tumor-bearing mice treated withcontrol IgG (Fig. 3A: IgG2a 1202 � 203.5 mm3 vs. anti–PD-1605.7 � 179.4 mm3, P ¼ 0.0412; IgG2b 1,592 � 285.6 mm3 vs.anti–PD-L1 1,125 � 251.5 mm3, P ¼ 0.2547).

We sought to determine whether the differential response ofCMT167 orthotopic tumors and subcutaneous tumors to PD-1/PD-L1 antibody blockadewas associatedwith differences in T-cellresponses. Flow cytometric analysis (Supplementary Fig. S5)

Figure 1.

PD-1 and PD-L1 antibody blockade inhibitgrowth of CMT167 orthotopic lungtumors. CMT167 orthotopic tumor-bearing mice were injected with anti–PD-1, anti–PD-L1, or isotype controlantibody (n ¼ 5 for each group). Effectsof anti–PD-1/anti–PD-L1 therapy on (A)CMT167 primary tumor volume and (B)metastatic burden in the other lunglobes. Each point represents anindividual mouse. C, Representative CD3immunostaining (FITC, green) ofsections from mice treated with controlIgG2a (a and b), anti–PD-1 (c and d),control IgG2b (e and f), and anti–PD-L1(g and h). Nuclei were stained with DAPI(blue); tumor borders are indicated byarrowheads. (a, c, e, g) Low power image(10�); scale bar, 200 mm; (b, d, f, h) highpower detail of boxed regions from lowpower images (40�); scale bar, 100 mm.

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demonstrated that CD8þ T cells were more abundant in CMT167subcutaneous tumors comparedwith CMT167 orthotopic tumor-bearing lungs (Fig. 3B), but there was no significant difference inCD4þ T cells (Fig. 3C), leading to a decreased CD4:CD8 ratio inCMT167 subcutaneous tumors (Fig. 3D). Although there was ahigher frequency of PD-1þCD8þ T cells inCMT167 subcutaneoustumors (Fig. 3E), there were fewer PD-1þ CD4þ T cells comparedwith CMT167 orthotopic tumor-bearing lungs (Fig. 3F). InCMT167 subcutaneous tumors, 69.34%� 2.48% of CD4þ T cellsexpressed Foxp3 (Fig. 3G), compared with 11.38 � 0.43% ofCD4þ T cells in CMT167 orthotopic tumors, indicating regulatoryT cells (Treg) are more abundant in subcutaneous tumors. Thus,tumors generated by the identical lung cancer cell line implantedin different anatomical sites led to different T-cell responses.

Next,we examinedPD-L1 expression inCMT167 subcutaneoustumors. CMT167 cells were implanted into GFP-expressing trans-genic C57BL/6 mice, and after 2 weeks, subcutaneous tumorswere analyzed by flow cytometry. As with CMT167 orthotopictumors, a subset of both cancer cells (GFP�) and stromal cells(GFPþ) expressed PD-L1 (Fig. 3H). Compared with CMT167orthotopic tumors, GFP� PD-L1high cancer cells were more abun-dant in CMT167 subcutaneous tumors, but GFPþ PD-L1high TMEcells were less abundant (Fig. 3H). Similar to CMT167 orthotopictumors, the dominant population of PD-L1high cells in CMT167flank tumors consisted of macrophages (Table 1). However, allthe PD-L1high macrophages within subcutaneous tumors arerecruited from the bonemarrow and circulation, and as expected,PD-L1high alveolar macrophages were not detected. There was a

Figure 2.

PD-L1 expression is upregulated on bothcancer cells and in the TME in CMT167orthotopic lung tumors. Representativeflow cytometric plot from uninjectedmice (A) and CMT167 tumor–bearingmice (B). Similar results were obtainedin 5 independent experiments, with 2mice in each pool. C, PD-L1 mRNAexpression from cancer cells recoveredfrom tumor-bearing mice was analyzedby RNA-seq and compared with cancercells in culture. Each RNA isolationrepresents a pool of 4 to 5mice.D,PD-L1mRNA expression from cancer cellsrecovered from tumor-bearing micewas determined by qRT-PCR andcompared with cells cultured in vitro,either in thepresenceor absence of IFNg(3 separate RNA isolations).E, Effects ofdepleting PD-L1 in CMT167 cells onprimary tumor size. CMT shCON isotypen ¼ 7, CMT shCON PD-L1 mAb n ¼ 8,CMT shPD-L1 isotype n ¼ 10, CMTshPD-L1 PD-L1 mAb n ¼ 10. F, CMT167cells were implanted in the left lungs ofwild-type C57BL/6 mice and PD-L1 KOmice (n¼ 4 for each group). Statisticallysignificant differences are indicated asdetermined by one-way ANOVA (C–E)or Student unpaired t test (F);�� , P < 0.01; ��� , P < 0.001.

Table 1. Subsets of PD-L1high cells in CMT167 and LLC tumors

Cells (% of GFPþPD-L1high

by flow cytometry)CMT167 orthotopic(n ¼ 4 pools of 2 mice)

CMT167 subcutaneous(n ¼ 7 tumors)

LLC orthotopic(n ¼ 4 pools of 2 mice)

Alveolar macrophages 59.4 � 4.98 N.D. 46.3 � 4.40Interstitial and monocyte-derived recruited macrophages 24.2 � 2.03 67.66 � 2.29 45.6 � 4.33Dendritic cells 4.0 � 1.46 1.8 � 1.60 1.5 � 1.43CD11bþLy6Gþ myeloid cells N.D. 28.66 � 1.60 2.5 � 1.37Endothelial cells 4.1 � 1.44 1.8 � 1.29 0.7 � 0.66Total PD-L1high (% of GFPþ cells) 16.88 � 0.84 8.05 � 1.90 9.53 � 0.95

NOTE: Statistically significant differences by ANOVA compared with CMT167 orthotopic tumors are shown in bold. N.D., not detected.

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higher proportion of CD11bþLy6GþPD-L1high cells in CMT167subcutaneous tumors, which could represent either neutrophilsor myeloid-derived suppressor cells (MDSC).

Orthotopic lung tumors differ in sensitivity to anti–PD-1/PD-L1 therapy

We examined the response of a second K-Ras–mutant murinecell line, LLC, in our orthotopic model. In contrast with CMT167tumors, LLC orthotopic tumor growth was not inhibited by

PD-1/PD-L1 antibody blockade (Fig. 4A and B). Immunofluo-rescent staining of LLC tumors for CD3e (Fig. 4C) failed to showthe dense nests of TILs found in CMT167 tumors, even in micetreated with PD-1/PD-L1-neutralizing antibodies. In cancer cellsrecovered from GFP-expressing transgenic mice, PD-L1 mRNAexpression was lower in LLC cells (Fig. 2C and D), indicating thatLLC cells express less PD-L1 thanCMT167 cells in vivo. Increases inPD-L1 expression on cancer cells and in PD-L1high cells in the TMEwere less in LLC tumors than in CMT167 tumors (Fig. 4D and E).

Figure 3.

CMT167 subcutaneous tumors induce aT-cell response that is skewed towardCD8þ T cells and are less sensitivethan CMT167 orthotopic tumors toanti–PD-1/PD-L1 therapy. A, CMT167subcutaneous tumor-bearing micewere injected with anti–PD-1,anti–PD-L1, or isotype control antibody(n¼ 10 for IgG2a and anti–PD-1 groups,n¼ 5 for IgG2b and anti–PD-L1 groups).CMT167 orthotopic tumor-bearinglungs (n¼9) andCMT167 subcutaneoustumors (n ¼ 7) were analyzed by flowcytometry for (B) CD8þ T cells, (C)CD4þ T cells, (D) CD4:CD8 ratio,(E) PD-1 expression on CD8þ T cells,(F) PD-1 expressiononCD4þTcells, and(G) Foxp3 expression in CD4þ T cells.Statistically significant differences areindicated as determined by Studentunpaired t test; �� , P < 0.01;��� , P < 0.001. H, PD-L1 expression wasanalyzed by flow cytometry in CMT167subcutaneous tumors. Similar resultswere obtained in five independentmice.

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CMT167 and LLCorthotopic tumors activate T lymphocytes to asimilar extent

To determinewhether the differential response of CMT167 andLLC orthotopic lung tumors to PD-1/PD-L1 inhibitionwas due todifferences in T-cell activation, FFPE lung sections from tumor-bearing animals were stained for CD3þ cells and TILs werecounted (Fig. 5A and B). Although there were more tumor-infiltrating CD3þ T lymphocytes per high-power field in CMT167tumors (Fig. 5C), abundant CD3þ T lymphocytes were alsodetected within LLC tumors. Immunodepletion of CD8þ T cellsincreased LLC tumor growth to a similar degree as CMT167tumors (Fig. 5D), indicating that CD8þ T cells constrain CMT167and LLC tumors to a similar extent. Flow cytometric analysis oforthotopic tumor-bearing lungs demonstrated a decrease inCD8þ T cells (as a percentage of CD45þ cells) in LLC orthotopictumors compared with uninjected lungs (Fig. 5E), but no differ-

ence in CD4þ T cells (Fig. 5F). CD44 and CD69 expression onCD8þ T cells was detected in both CMT167 and LLC orthotopiclung tumors (Supplementary Fig. S6B and S6C), suggesting T cellsare activated by both tumors. Moreover, both CMT167 and LLCorthotopic lung tumors induced PD-1 expression on CD8þ andCD4þ T cells (Fig. 5G and H). Thus, in contrast with CMT167subcutaneous tumors, the insensitivity of LLC tumors to anti–PD-1/PD-L1 was not associated with differences in PD-1 expressionon T lymphocytes.

Induction of PD-L1 expression on macrophages in vitro ismediated by IL7 and IL15

We hypothesized that differences in PD-L1 expression onnontumor cells in the lung were mediated by differences betweenthe secretomes of CMT167 and LLC cells. To identify factors thatupregulate PD-L1 expression on macrophages, BMDMs were

Figure 4.

LLC orthotopic tumors are less sensitivethan CMT167 orthotopic tumors to anti–PD-1/PD-L1 therapy. LLC orthotopictumor-bearing mice were injected withanti–PD-1, anti–PD-L1, or isotype controlantibody (n ¼ 4 for each group). Effectsof anti–PD-1/anti–PD-L1 therapy on (A)LLC primary tumor volume and (B)number of liver metastases. C,Representative CD3 immunostaining oftissue sections from mice treated withcontrol IgG2a (a and b), anti–PD-1 (c andd), control IgG2b (e and f), andanti–PD-L1 (g andh). Nucleiwere stainedwith DAPI (blue); tumor borders areindicated by arrowheads. (a, c, e, g) Lowpower image (10�); scale bar, 200 mm;(b, d, f, h) high-power detail of boxedregions from low power images (40�);scale bar, 100 mm. D, Representativeflow cytometric plot from LLC tumor–bearing mice. Similar results wereobtained in 5 independent experimentsconsisting of 2 mice each. E, Thepercentage of GFPþPD-L1high cells inmice implanted with CMT167 tumors orLLC tumors was compared withuninjected mice. Each point representsan individual animal (n ¼ 5 for eachgroup). �� , P < 0.01; ��� , P < 0.001.

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stimulated with CM from each cell line. Addition of CM fromeither cell line increased PD-L1 expression at both the transcrip-tional and protein levels (Fig. 6A and B). However, CMT167 CMincreased PD-L1 expressionmore than LLCCM(Fig. 6A). Analysisof genes that code for secreted proteins expressed �5-fold higherin CMT167 compared with LLC cells in vivo revealed increases inIL7 and IL15 (Supplementary Fig. S7; Fig. 6C), which have beenshown to induce PD-L1 expression onmacrophages (16). In vitro,neutralization of both IL7 and IL15 with monoclonal antibodiesblocked upregulation of PD-L1 on BMDMs induced by CMT167

CM, whereas neutralization of either IL7 or IL15 alone had noeffect (Fig. 6D).

DiscussionNSCLCcells and immune cells in theTMEcan inhibit antitumor

T cells throughmultiple pathways, including through coinhibitoryreceptorssuchasPD-1.PharmacologicantibodiestargetingPD-1orits ligandPD-L1canrelieveinhibitionofantitumorTcells, resultingin immune attack on tumors (2). These agents have shownefficacy

Figure 5.

Both CMT167 orthotopic lung tumorsand LLC orthotopic lung tumorsgenerate an adaptive immuneresponse and induce PD-1 expressionon T cells. Tissue sections from CMT167(representative image shown in A) andLLC (representative image shown in B)tumor-bearing mice were stained forCD3 (FITC, green) and nuclei werestained with DAPI (blue).Magnification, �20. C, Quantificationof tumor-infiltrating lymphocytes inCMT167 tumors (n¼ 9) and LLC tumors(n ¼ 6). D, Effects of CD8þ T cellimmunodepletion on growth ofCMT167 (n ¼ 7 each group) and LLCorthotopic tumors (n ¼ 6 each group).CMT167 (n¼9, samemice as in Fig. 3B–F) or LLC tumor–bearing lungs (n ¼ 9)were analyzed by flow cytometry andcompared with lungs from naive mice(n¼ 8) for (E) CD8þ T cells, (F) CD4þ Tcells, (G) PD-1 expression on CD8þ Tcells, and (H) PD-1 expression on CD4þ

T cells. Statistically significantdifferences are indicated asdetermined by Student unpaired t test(C–D) or by one-way ANOVA (E–H);� , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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in clinical trials for several typesof cancer (17,18), resulting inFDAapproval for use of these agents for treatment of NSCLC andmelanoma. However, response rates in NSCLC patients haveranged from 14.5% to 20% (3–7). Developing patient-selectioncriteria for these agents and rational combinations with othertherapies requires a better understanding of factors that mediateresponse to immune checkpoint inhibitors.One critical issue is thecontribution of the TME to anti–PD-1/PD-L1 therapy.

In this study, we have compared responses to anti–PD-1/PD-L1therapy in several immunocompetent models of lung cancer. Ourdata support a system in which intrinsic properties of the cancercells, aswell as interactions between cancer cells and immune cellsin the TME, determine the response to PD-1/PD-L1 inhibitors.Whereas anti–PD-1/PD-L1 therapy inhibited CMT167 orthotopiclung tumors by 95%, CMT167 subcutaneous tumors were onlyinhibited by 30%. Silencing PD-L1 expression in CMT167 cellsresulted in smaller orthotopic tumors that remained sensitive toanti–PD-L1 therapy, suggesting PD-L1 expression in the TMEplays a role in immune escape of tumors. Moreover, CMT167orthotopic tumors in PD-L1 KO mice are approximately 1/10ththe size of CMT167 orthotopic tumors in WT mice, which indi-cates that PD-L1 on nontumor cells can limit antitumor immu-nity. Because the immunogenicity of CMT167 cells is identical inboth orthotopic and subcutaneous models, we propose thatspecific features of the lung TME are critical for mediating theresponse to PD-1/PD-L1 inhibitors.

TILs have been investigated as a biomarker for response to anti–PD-1/PD-L1 therapy. The presence of TILs was associated withlocal PD-L1 expression in both primary andmetastaticmelanomatumors (19). The presence of tumor CD8þ T cells and PD-1expression in patients may represent positive predictive biomar-kers for anti–PD-1/PD-L1 therapy (20). A threshold level ofintratumor CD8þ PD-1 expression dictates therapeutic responseto anti–PD-1 therapy (21). In our studies, PD-1 expression onCD8þ T cells did not predict response to anti–PD-1/PD-L1 ther-apy, because the frequency of PD-1þ CD8þ T cells was higher inCMT167 subcutaneous tumors than CMT167 orthotopic tumors,and comparable between CMT167 orthotopic tumors and LLCorthotopic tumors. However, PD-1 can be induced in multiplecontexts, including recently activated T cells and subsets ofexhausted cells (22). Given this complexity and the evolvingunderstanding of which cells confer the therapeutic benefit ofPD-1/PD-L1 blockade (23–25), we have avoided describing thePD-1þ T cells found in our studies as exhausted. However, weobserved more Tregs in CMT167 subcutaneous tumors than inCMT167 orthotopic tumors, which may explain why subcutane-ous tumors are less responsive to anti–PD-1/PD-L1 therapy. Inour studies, there were also fewer PD-1þ CD4þ T cells in CMT167subcutaneous tumors than inCMT167orthotopic tumors. Severalroles for CD4þ T cells in antitumor immunity have been reported,including secretion of effector cytokines such as IFNg (26, 27) andIL4 (28, 29), recruitment of effector cells including eosinophils

Figure 6.

CM from CMT167 cells induce higherlevels of PD-L1 in bonemarrow–derivedmacrophages than CM from LLC cells.A,Bonemarrow–derivedmacrophageswere treated with CM from CMT167cells or LLC cells. Data represent themean of 3 experiments using 3separate isolations of CM. B, Arepresentative overlay of PD-L1expression is shown from untreatedBMDMs (blue), BMDMs treated withLLC CM (green), and BMDMs treatedwith CMT167 CM (red). Similar resultswere obtained in 3 independentexperiments using 3 separate isolationsof CM. C, IL7 and IL15mRNA expressionwas determined by RNA-seq fromCMT167 cells and LLC cells, both in vitroand in vivo. D, Effects of IL7 and IL15neutralization on upregulation of PD-L1on BMDMs induced by CMT167 CM.Data represent themean and SEM from3 independent experiments.Statistically significant differences areindicated as determined by one-wayANOVA; � , P < 0.05; �� , P < 0.01;��� , P < 0.001.

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andmacrophages (30, 31), and provision of help to CD8þ T cellsfor optimal priming and effector function (32, 33) and generationof CD8þ memory T cells (34). Whether subsets of PD-1þ T cellsdiffer between CMT167 orthotopic tumors, LLC orthotopictumors, and CMT167 subcutaneous tumors will be the subjectof future studies.

In CMT167 orthotopic tumors, PD-L1 was upregulated oncancer cells and alveolar macrophages. However, in CMT167subcutaneous tumors, PD-L1 was expressed mainly on cancercells, macrophages recruited from the circulation, and CD11bþ

Ly6Gþ cells thatmay representneutrophils orMDSCs. Because thepopulationsofmacrophages in subcutaneous tumors are differentfrom those in lung tumors, we hypothesize PD-L1þ alveolarmacrophages have different functions than PD-L1þ macrophagesrecruited from the circulation. However, proving that alveolarmacrophages are responsible for differences between orthotopictumors and subcutaneous tumors is challenging, because residentalveolar macrophages cannot be selectively depleted from thelungs. Conversely, alveolar macrophages injected into the flankwould not be expected to maintain their phenotype in a differentmicroenvironment. Effector cell populations responsible for kill-ing tumor cells are also likely different between CMT167 ortho-topic tumors and subcutaneous tumors, similar to what wasshown for antibody-mediated killing of melanoma cells grownin different anatomical sites (35). One challenge in comparingorthotopic tumors and subcutaneous tumors is that analysis of thelung TME includes regions of normal lung, whereas analysis of thesubcutaneous TME is limited to tumor-infiltrating cells. Thus,comparison of the relative abundance of immune cell subpopula-tions in different tumor models is difficult.

A second lung cancer cell line (LLC) implanted into the samelungmicroenvironment as CMT167 cells is less sensitive to PD-1/PD-L1 inhibition,with less inductionof PD-L1 expressionon cellsin the TME. Consistent with these data, CMT167 CM upregulatedPD-L1 expression on BMDMs in vitro more than LLC CM, indi-cating soluble factors produced by cancer cells contribute toinduction of PD-L1 in the TME. Analysis of genes that code forsecreted proteins demonstrated that CMT167 cells express higherlevels of IL7 and IL15, which have been shown to induce PD-L1expression on macrophages (16). In vitro, neutralization of bothIL7 and IL15 with monoclonal antibodies blocked upregulationof PD-L1 on BMDMs induced by CMT167 CM. Neutralization ofeither IL7 or IL15 alone had no effect, which suggests thesecytokines synergize to induce PD-L1 expression on BMDMs.Future studies will explore this potential synergy, and whetherblocking these factorsmay represent an approach to prevent T-cellexhaustion in lung cancer. The relative contribution of PD-L1 oncancer cells and cells in the TME to sensitivity to PD-1/PD-L1inhibition also remains a subject for future investigation.

Because our data demonstrate that both CMT167 and LLC celllines express oncogenic K-Ras, the differences in response toPD-L1/PD-1 inhibitors are not related to the oncogenic driver.The differential responses of CMT167 and LLC tumors to PD-L1antibody blockade are also unlikely to be due to differences in theimmunogenicity of the cancer cells, because activated T lympho-cytes were detectedwithin both tumors, and growth of orthotopictumors formed by both cell lines was increased to the same extentby CD8þ T-cell immunodepletion. Moreover, both CMT167 andLLC orthotopic tumors induce expression of CD44, CD69, andPD-1 on T cells. One difference is that CMT167 cells represent anepithelial cell line that expresses abundant E-cadherin, whereas

LLC cells are mesenchymal and express little E-cadherin (Supple-mentary Fig. S8). Mesenchymal NSCLC cell lines express higherlevels of PD-L1 and are more responsive to anti–PD-L1 blockade(36).Our study demonstrates the reverse, with themore epithelialCMT167 cells expressing higher levels of PD-L1 in vivo anddemonstrating greater response to PD-1/PD-L1 inhibition thanthe more mesenchymal LLC cells. However, CMT167 cells andLLC cells also differ in their cytokine profile, both in vitro andin vivo. Thus, the soluble factors produced by the cancer cell maybe a better predictor of response to PD-1/PD-L1 inhibition thanthe state of differentiation. Although JAKmutations are a primaryresistance mechanism to anti–PD-1 therapy in melanoma andcolon cancer (37), genetic analysis of LLC cells revealed nomutations in either JAK1 or JAK2 (Supplementary Fig. S8), andJAK mutations are therefore not the reason LLC tumors areinsensitive to anti–PD-1/PD-L1 therapy.

In summary, we conclude that understanding basic mechan-isms regulating immune checkpoints and testing rational combi-nations of checkpoint inhibitors require an orthotopic model, inwhich tumors develop in the appropriate microenvironment. Theresponse to these agents will depend on the nature of the cancercells, as well as interactions between cancer cells and noncancercells present in the lung. Defining the factors that regulate PD-L1expression on both cancer cells and cells of the TME will providetargets for the treatment of lung cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H.Y. Li, T.R. Sippel, R.A. NemenoffDevelopment of methodology: H.Y. Li, J.M. Poczobutt, T.R. Sippel,M.C. Weiser-EvansAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.Y. Li, M. McSharry, B. Bullock, T.T. NguyenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H.Y. Li, J.M. Poczobutt, M.C. Weiser-Evans,E.T. Clambey, R.A. NemenoffWriting, review, and/or revision of themanuscript:H.Y. Li, B. Bullock, J. Kwak,L.E. Heasley, M.C. Weiser-Evans, E.T. Clambey, R.A. NemenoffAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.Y. Li, M. McSharry, B. Bullock, J. Kwak,R.A. NemenoffStudy supervision: H.Y. Li

AcknowledgmentsThe authors gratefully acknowledge use of the services and facilities of the

Flow Cytometry and the Genomics and Microarray Shared Resources at theUniversity of Colorado Cancer Center.

Grant SupportThis workwas supported by theUnited States Department of Veterans Affairs

Biomedical Laboratory Research and Development Service (Career Develop-ment Award IK2BX001282 to H.Y. Li), the NIH/NCI (R01 CA162226 and R01CA108610 toR.A.Nemenoff), Colorado Lung SPOREP50 (CA058187 toH.Y. Liand R.A. Nemenoff), and the Cancer League of Colorado, Inc. (research grantto H.Y. Li). The Flow Cytometry and the Genomics and Microarray SharedResources at the University of Colorado receive direct funding support from theNCI through the Cancer Center Support Grant P30CA046934.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 9, 2016; revised April 21, 2017; accepted July 28, 2017;published OnlineFirst August 17, 2017.

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