Therapeutic targeting of macrophage plasticity remodels the tumor-immune microenvironment

Abstract

Although the majority of patients with advanced lung adenocarcinoma (LUAD) are eligible to receive immune checkpoint blockade, approximately 80% of these tumors are resistant to this therapeutic approach. Insights at the single-cell level into mechanisms that drive LUAD tumorigenesis and the relationship of LUAD histologic heterogeneity to response to immune checkpoint blockade could help identify biomarkers and potential combinational approaches to improve immunotherapy efficacy. Here we used a genetically engineered mouse model that replicates the development of human LUAD through a spectrum of pre-invasive to invasive adenocarcinoma histologic subtypes. A systems onco-immunology approach of integrating the analytical power and unique, complementary capabilities of time-of-flight mass cytometry (CyTOF) and imaging mass cytometry was leveraged to identify cellular and spatial immune contextures in LUAD. Comprehensive investigation of mouse and human LUAD using these single-cell proteomics platforms showed that LUAD progression is associated with spatiotemporal evolution of tumor-associated macrophages in the tumor-immune microenvironment, which governs tumor response to immunotherapy. PD-1 was expressed in a highly plastic tumor-promoting subtype of tumor-associated macrophages that develops during tumor progression from pre-invasive to invasive adenocarcinoma, controls the lymphocyte-depleted niche of invasive tumors, and protects tumor cells in the solid histologic components of the tumor. Longitudinal, multi-dimensional single-cell analyses of LUAD tumorigenesis revealed dynamic alteration of immunoregulatory PD-1-expressing tumor-associated macrophages that can be targeted to overcome resistance to checkpoint immunotherapy.

INTRODUCTION

Lung cancer is responsible for the most cancer-related deaths in the United States and only 21% of patients who receive this diagnosis are alive five years later.(1) Immune checkpoint inhibitors (ICIs) against PD-1 and PD-L1 have shown encouraging clinical results and tolerable safety in patients with advanced non-small cell lung cancer (NSCLC). (2–8) Unfortunately, the majority of NSCLCs are resistant to ICI therapy and the rate of response is only about 20%.(8,9)

Lung adenocarcinoma (LUAD) is the most common form of lung cancer. In recent years it has become evident that LUAD is actually comprised of a number of histologic subsets with variable biologic behavior. Among them, several share lepidic pattern growth in which tumor cell proliferation occurs along the pre-existing framework of lung alveolar structures, initially without destruction of its architecture. Pre-invasive lesions called adenocarcinoma in situ (AIS) progress to minimally invasive adenocarcinoma (MIA) and to other invasive subtypes including lepidic predominant adenocarcinoma (LPA) and solid subtype adenocarcinoma.(10,11) A number of studies have reported better prognosis in patients with AIS and MIA forms of LUAD, and worse prognosis in patients with invasive subtypes of LUAD including LPA, solid, and others.(10,12,13) There remains a lack of deep insight into mechanisms that drive progression of AIS to invasive adenocarcinoma at the single-cell level, and the relationship of the histologic heterogeneity of LUAD to response of ICI therapy is poorly understood.

To address these gaps, we used a genetically engineered mouse model that replicates the development of human lung adenocarcinoma through a spectrum of pre-invasive (AIS) to invasive adenocarcinoma histologic subtypes.(11) We leveraged a systems onco-immunology approach of integrating the analytical power and unique, complementary capabilities of time-of-flight mass cytometry (CyTOF) and imaging mass cytometry (IMC) to identify cellular and tissue architectural determinants of an immunologic state that is conducive to productive anti-tumor immunity in LUAD. Through longitudinal analyses, we discovered the emergence of population of immuno-regulatory, tumor-protective macrophages identified by their expression of PD-1, and that are phenotypically, functionally, and spatially dynamic. We show that these cells are intricately related to the tumor architecture of LUAD subtypes, compromise anti-tumor immunity, and are associated with maintenance of the immune-depleted milieu of invasive LUAD. Targeting tumor-associated macrophages (TAMs) decreased the extent of invasive lepidic and solid LUAD histology, and combining the macrophage-targeting agent with PD-1 blockade further improved these therapeutic responses, particularly for solid subtype LUAD. These data provide a novel spatiotemporal immunologic and histologic framework to approach the treatment of patients suffering from LUAD.

MATERIALS and METHODS

Mouse model

Animal experiments were performed in accordance with the Institutional Animal Care and Use Committees and in accordance with all relevant animal use guidelines and ethical regulations at Baylor College of Medicine (IRB. No. AN-6685). We utilized a doxycycline-inducible EGFR^L858R bi-transgenic mouse model of lung adenocarcinoma driven by an organ-specific human EGFR gene mutation as previously described.(11,14) Both strains of mice were gifts from Dr. Katerina Politi, Yale School of Medicine (New Haven, CT) and are of FVB background. The mutant human EGFR (L858R) transgene was activated by feeding male bitransgenic (CCSP/EGFR) mice with doxycycline-impregnated food pellets (625 ppm; Harlan Laboratories). This mouse model results in progressive development of lung adenocarcinomas with preinvasive lepidic histology that progress to invasive adenocarcinoma in both whole lungs,(11,14) and is very similar to the progression of these tumors in humans. Histological changes are present in the lungs as early as two weeks after starting their doxycycline diet, resembling human lepidic type of lung adenocarcinomas with mainly diffuse thickening of the alveolar walls.(11) Invasive adenocarcinomas develop five weeks after doxycycline feeding, and discontinuing the doxycycline diet induces tumor regression.(11)

Human samples

This study was performed in accordance with an institutional review board protocol at Baylor College of Medicine (IRB. No. H-42636). A prospectively maintained single institution database was retrospectively reviewed. Twenty LUAD patients who received nivolumab or pembrolizumab from 2015 to 2019 were evaluated. All anonymous human samples were collected with written informed patient consent.

Single-cell preparations

Tumor-bearing lungs were finely minced and digested in unsupplemented RPMI 1640 (without glutamate) using a mouse Tumor Dissociation Kit (Miltenyi Biotec Inc., Auburn, CA, USA, Cat.No.130-096-730) in 50 mL Falcon tubes for 20 minutes in the 37°C rotating incubator. Cells were then filtered through a 70 μm cell strainer (Corning Life Sciences Plastic, Cat.No.07201431), washed, and lysed in ACK lysing buffer (Life Technologies, Cat.No.A049201). After centrifuging cells with unsupplemented RPMI media for 5 min at 400×g in 20°C and washing two times, supernatants were carefully suctioned off. The cell pellet underwent a final washing with 40 mL supplemented cell culture media (RPMI with FBS), and cells were refiltered through a 70 μm cell strainer. After centrifuging the cells for 5 min at 400×g in 20°C, the supernatant was carefully suctioned off. Cells were placed in freezing media (FBS with 7% DMSO) and cryopreserved in −80°C freezer storage after cell counting with a cell counter (Countess II FL, Life Technologies, Cat.No.AMQAF1000). For long-term preservation, the cryovials were transferred into a liquid nitrogen tank at −196°C.

Flow cytometry and cell sorting

To sort cancer cells, PD-1^HIGH TAMs, and PD-1^LOW TAMs from single-cell suspensions of mouse lung adenocarcinoma, cells were incubated with TruStain FcX^™ (anti-mouse CD16/32) antibodies (BioLegend, San Diego, CA, Cat.No 422301) as an Fc Receptor Blocking Solution. The panel of antibodies used in the fluorescence-activated cell sorting (FACS) of macrophages included anti-mouse CD45-BUV-395 (clone 30-F11, BD Bioscience, San Jose, CA, Cat.No 564279), CD11b-BV711 (clone M1/70, BioLegend, Cat.No 101242), F4/80-APC (clone BM8, BioLegend, Cat.No 123116), CD3-AF488 (clone 17A2, BioLegend, Cat.No 100212), CD19-AF488 (clone 6D5, BioLegend, Cat.No 115524), and PE-conjugated PD-1 antibody. To generate PE-conjugated PD-1 antibody, unconjugated anti-mouse PD-1 antibodies (clone 29F.1A12, BioXCell, Lebanon, NH, Cat.No BE0273) were conjugated to DIBO-modified R-phycoerythrin (R-PE) using the SiteClick^™ Antibody Labeling Kit (Invitrogen, Carlsbad, CA, Cat.No S10467) following the manufacturer’s protocol. Dead cells were excluded using 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, St. Louis, MO). Anti-human EGFR was purchased from Biolegend (352906). Cell viability assay was performing with Annexin V (Biolegend, Cat. No 640922) and DAPI (Life Technologies, Cat. No D1306) staining. Flow cytometry was performed by an LSR Fortessa cytometer equipped with FACS Diva software (BD Biosciences). Data were analyzed using FlowJo (FlowJo, LLC.). PD-1^HIGH TAMs (CD45+F4/80+CD11b+PD-1+), PD-1^LOW TAMs(CD45+F4/80+CD11b+PD-1-), and EGFR-mutant epithelial cells (CD45-human EGFR+) were sorted from single lung suspensions of bitransgenic mice fed doxycycline for 7 weeks using a FACSAria II Cell Sorter (BD Biosciences).

CyTOF experiments

Single cells were stabilized for 6 hours in media at 37°C. Five hundred thousand cells were resuspended in Maxpar Cell Staining Buffer (Fluidigm, Cat.No.201068) in individual 5 mL tubes for each sample to be barcoded. Mass-tag cellular barcoding using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm, Cat.No. 201060) was performed as previously described (15). Cell-ID^™ Intercalator-Ir is a cationic nucleic acid intercalator that contains naturally abundant Iridium (191Ir and 193Ir) and is used for identifying nucleated cells in CyTOF analysis according to standard protocol. For measurement of intracellular cytokines by CyTOF, cells from lungs with tumor-bearing mice were incubated in 1μl/mL Golgistop (BD cell analysis, Cat.No.BDB554724A), 2 μl/mL PMA/Ionomycin (eBioscience Cell Stimulation Cocktail, Cat.No.00-4970-93) for 6 hours at 37°C, according to standard protocol. The samples were then washed and incubated with cell surface antibodies for 30 minutes at RT and washed. After overnight at 4°C with resuspension in 1× Fix I buffer, the samples were stained with intracellular antibodies against cells cytokines for 30 minutes at RT and washed. Stained cells were analyzed on a mass cytometer (CyTOF3^™ mass cytometer, Fluidigm) at an event rate of 400 to 500 cells per second. All mass cytometry files were normalized together using the mass cytometry data normalization algorithm,(16) which uses the intensity values of a sliding window of these bead standards to correct for instrument fluctuations over time and between samples.

CyTOF analysis

The scheme of data analysis is illustrated in Supplementary Figure 1. Total live nucleated cells were used for all analyses and visualized using Spanning-tree Progression Analysis of Density-normalized Events (SPADE)-based SCAFFOLD (Single-Cell Analysis by Fixed Force- and Landmark-Directed) map generation as a mixture of human guided knowledge and automated clustering algorithm.(17,18) To characterize all cells obtained from harvested lungs, all 733 nodes, or clusters of single cells with similar phenotypes, were organized in 17 phenotypes. Eighteen cellular phenotypes were manually defined by a panel of 38 antibodies (Supplementary Figure 2): B cells (CD45+CD19+B220+), CD4 T cells (CD45+CD3+TCR-β+CD4+), CD8 T cells (CD45+CD3+TCR-β+CD8+), double negative (DN) T cells, (CD45+CD3+TCR-β+CD4-CD8-), γδT cells (CD45+CD3+ TCR-β-TCR-γδ+), monocytes (CD45+CD3-CD11b+CD115+), tumor-associated macrophages (TAMs; CD45+CD3-CD64+F4/80+SiglecF-), alveolar macrophages (AMs: CD45+CD3-CD64+F4/80+SiglecF+), plasmacytoid dendritic cells (CD45+CD3-CD19-CD11c+B220+CD317+), conventional DC (CD45+CD3-CD19-CD11c+B220-MHCII+), neutrophils (CD45+CD3-CD19-CD11b+Ly-6G+), NK cells (CD45+CD3-CD19-CD64-CD49b+), cancer cells (CD45-Pan-cytokeratin(CK)+Ki-67+), cancer-associated fibroblasts (CAFs; CD45-Pan-CK-Vim+), epithelial cells (CD45-Pan-CK-EpCAM+), endothelial cells (CD45-Pan-CK-CD31+), and stromal cells (CD45-Pan-CK-Vim-EpCAM-CD31-). Furthermore, through deep phenotyping of lung macrophages, AMs and TAMs were distinctly separated by SiglecF expression. AMs were divided into typical SiglecF+CD11c+CD11b- AMs with PD-1^low and monocyte-derived AMs (SiglecF+CD11c+CD11b+). TAMs were divided into SiglecF-CD11b+CD11c- MHCII^high TAMs with PD-1^low (PD-1^LOW TAMs) and SiglecF-CD11b+CD11c-MHCII^low TAMs with PD-1^high (PD-1^HIGH TAMs).

Based on SPADE results, we generated hierarchical inferences combined with the representative node. A cluster of similar patterns of protein expression was regarded as the same phenotype, and the most fitted node to each phenotype was regarded as the representative node. After assignment of all nodes to each phenotype, nodes were connected and displayed with single-cell analyses by fixed force and landmark directed (SCAFFOLD) maps for creating a reference map from high-dimensional single-cell data, facilitating comparisons across samples.(19) SCAFFOLD maps were generated as previously reported.(19) Briefly, a graph is constructed by connecting together the nodes representing the manually gated landmark populations and then connecting to them the nodes representing the cell clusters as well as connecting the clusters to one another. Each node is associated with a vector containing the median marker values of the cells in the cluster (unsupervised nodes) or gated populations (landmark nodes). Edge weights are defined as the cosine similarity between these vectors after comparing the results from the implementation of several distance metrics. Each circle represents a node, a population of cells with the same pattern of expression, and the size of the node represents the percentage of cells in each experiment. The clusters for all the tissues are combined in a single graph with edge weights defined as the cosine similarity between the vectors of median marker values of each cluster. All the pair-wise distances are calculated. The graph is then laid out using the ForceAtlas2 algorithm in Gephi 0.9.1 (https://gephi.org). In this representation of the cellular immune system of the mouse lung, each node is a population of cells with a similar pattern of protein expression, and the size of the node correlates with the average number of cells in the corresponding cell population. To overlay the additional samples on the SCAFFOLD map, the position and identity of the landmark nodes were fixed and the clusters of each sample were connected to the landmark nodes as described above.

Imaging mass cytometry (IMC)

The FFPE tissues were sectioned at a 5-μm thickness for IMC. FFPE tissues on charged slides were stained with 1:100 diluted antibody cocktails (concentration of each antibody = 0.5mg/mL) as recommended by the user’s manual. The slides were scanned in the Hyperion Imaging System (Fluidigm). They were scanned at least four regions of interest in > 4mm² width at 200 Hz. By applying the novel approach of IMC to complex immunological systems such as the tumor microenvironment, multiplexed measurements of up to 35 protein expressions in formalin-fixed paraffin-embedded (FFPE) tissue samples can be analyzed all at once in a single experiment. The value in IMC with FFPE as a multiplex platform lies in its efficiency and practicality in displaying tissue architecture. We are able to investigate the cellular phenotypes and biomarkers of a single tissue sample, observe each components’ spatial distribution simultaneously, and compare the qualities of each. This unique and comprehensive analysis of a single tumor sample affords us to draw conclusions between the identification and spatial relationships of the significant cellular components within a tumor microenvironment. The samples targeted for study are FFPE tissues that make this kind of architectural analysis easy to handle and highly convenient for use in the lab. We require some other image processing and computational methods in order to quantify the .mcd imaging files (Fluidigm) yielded from IMC and to determine the overall spatial architecture of the tumor immune microenvironment (20). We introduce the free, open-source, image-analyzing platform FIJI as the best resolution to fulfill this need. The advantages offered by Fiji (21) include a fast, robust, unsupervised, automated cell segmentation method requiring minimal expertise in computers. 32-bit TIFF stacked images were loaded in Fiji and we applied our novel method of automated cell segmentation that estimates cell boundaries by expanding the perimeter of their nuclei, identified by Cell ID Intercalator-iridium (191Ir). Once images from the IMC methodology are acquired, they can be quantified through FIJI’s threshold and watershed tools. Protein expression data were then extracted at the single-cell level through mean intensity multiparametric measurements performed on individual cells (22), and acquired single-cell data were transferred into additional cytometric analysis in FlowJo and Cytosplore (23,24), a software for interactive immune cell phenotyping for large single-cell datasets.

SPACE map (SPAtial Cancer Evolution)

Quantified IMC data are adjusted with 191Ir and 193Ir intensities and normalized with CytoNorm (25) across IMC ROIs. CytoNorm is a normalization method for cytometry data applicable to large clinical studies. CytoNorm allows reducing mass cytometry signal variability across multiple batches of barcoded samples. Normalized IMC data are combined by using FlowJo. To visualize temporal trajectories during tumor progression, the Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction is applied.(26)

Three-dimensional co-culture system of tumor-associated macrophages and cancer cells

We performed the three-dimensional co-culture system of TAMs and cancer cells to form spheroid structures. For in vitro spheroid formation assay using a 3D co-culture system with Nunclon^™ Sphera^™ 96-Well, Nunclon Sphera-Treated, U-Shaped-Bottom Microplate (Thermo Scientific^™, Cat.No. 174925), single cells obtained from invasive adenocarcinoma were cultured in medium containing 2% Matrigel and seeded onto the plate precoated with Matrigel. In this model, we detected spheroid formation at 7 days. Spheroid size was evaluated by measuring spheroid perimeter using Fiji software (NIH, Bethesda, Maryland, USA), which area was converted into % relative area based on the size of the intact spheroid. Results are expressed as mean pixel measure ± S.E.M. from three independent experiments in four replicates. Pictures for spheroid size evaluation were taken with a Zeiss Axiovert 200/M-based phase-contrast microscope.

Treatment

Trabectedin (Yondelis®, Manufactured by Baxter Oncology GmbH, Halle/Westfalen Germany) was purchased for research and was dissolved in 0.9% Sodium Chloride, USP to 2mM and kept at −20°C. Bitransgenic mice carrying human EGFR transgene were fed doxycycline impregnated food pellets and trabectedin (0.15 mg/kg/body weight) was administrated intravenously 3 times biweekly for adenocarcinoma in situ or weekly for invasive adenocarcinoma after starting their doxycycline diet. After completion of the trabectedin treatment, the doxycycline diet was continued until Day 21 for measurement of the tumor burden. A total of 50ul of anti-mouse PD-1 Abs (10mg/kg, InVivoMAb anti-mouse PD-1 (CD279), clone 29F.1A12, BioXCell, Cat.No BE0273) diluted with InVivoPure pH 7.0 Dilution Buffer (Cat.No IP0070, BioXcell) are injected intraperitoneally. As a control group, mouse IgG isotype (InVivoMAb rat IgG2a isotype control, anti-trinitrophenol, Clone 2A3, BioXCell, BE0089) was used.

MTT assay

All isolated cells were cultured in RPMI media as recommended by the providers in a humidified incubator at 37°C and 5% CO₂. The cytotoxicity of trabectedin in all three types of cells was determined by MTT assay (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega). Compactly, cells (1 × 10⁴ cells/well) were seeded in a 96-well plate with fresh medium and incubated overnight. MTT solution was added into each well at the indicated time points for 24 hours with 9 different concentrations of trabectedin diluted in fresh medium. As a positive control, cells were cultured in fresh medium, and medium only was used as a negative control. Cell viability was evaluated at different time points following the manufacturer’s instructions through the spectrophotometric reading at two different wavelengths (570 and 650 nm). All experiments were repeated in at least triplet.

Detailed experimental methods of protein profiling by mass spectrometry are described in the supplementary documents.

Statistical analysis

Student’s t-tests, chi-square tests, and Fisher exact tests were used to compare the data. All in vitro experiments were performed at least in triplicate. Differences between treatment groups were analyzed by the two-tailed Student t-test using Prism^® 5.0 (GraphPad Software, Inc. CA) or SPSS 28.0 (SPSS, Inc., IL). Mean mass intensities (MMI) of proteins were compared with t-tests accordingly among phenotypes before and after treatment. To generate the statistical values of phenotypic differences between two subsets, the internodal differences in the same phenotypes were analyzed using Significance Analysis of Microarrays according to the percentages of cell frequency in corresponding nodes (27). Biometric Research Branch (BRB)-Array Tools 4.3.0 (National Cancer Institute, Bethesda, MD, USA) were used for statistical analysis of the internodal differences. To compare the longitudinal alteration of protein markers between macrophage subtypes, we performed analysis of variance (ANOVA) test of difference of proteins of interest. CyTOF data were additionally analyzed with FlowJo^® V10 software (FlowJo, LLC, OR) to confirm consistency of results by CyTOF analysis. Statistical significance was accepted for P<0.05, and all tests were two-tailed.

Data availability

Expression profile data analyzed in this study were obtained from NCBI’s Gene Expression Omnibus (GEO) database at GSE52248 (48), GSE58772(49), GSE93157 (51), and GSE136961(52). Protein profiling data generated in this study are available within the article and its supplementary data files. The CyTOF and IMC data generated in this study are available upon request from the corresponding author.

RESULTS

The tumor-immune microenvironment transforms during histologic progression of invasive lung adenocarcinoma.

Lung adenocarcinoma exists along a continuum of pre-invasive to invasive histology (10). We hypothesized that the evolving histologic stages of LUAD harbor distinct tumor-immune microenvironments (TiMEs) that differentially regulate response to immunotherapy. To test this hypothesis, we used a genetically engineered mouse model that replicates the development of human LUAD (Figure 1A)(11) and compared the TiME of progressing tumors through early lepidic (largely pre-invasive), invasive lepidic, and invasive solid adenocarcinoma, using a 38-channel CyTOF platform (Figures S1–S2 and Table S1). Ten major phenotypes of immune cells were overlaid by T-distributed Stochastic Neighbor Embedding (t-SNE)(28) and demonstrated striking differences between the TiME of these tumors (Figure 1B). B cell and T cell populations diminished steadily over the course of tumor development (Figure 1C). With progression to invasive LUAD, CD8 T cells acquired an immunoregulatory phenotype with high expression of TIM-3 and PD-1 and became functionally exhausted, decreasing production of interferon-γ and TNF-α in response to in vitro stimulation (Figure S3); while the frequency of regulatory T cells steadily increased (Figure S4).

Figure 1. Transformation of the tumor-immune microenvironment occurs with progression to invasive lung adenocarcinoma.

A. Serial histologic evaluation of tumorigenesis in lung adenocarcinoma showing gradually increased tumor burden with solid invasive carcinoma. Early lepidic adenocarcinoma developed 2 weeks after feeding doxycycline. Lungs at 5 weeks of tumorigenesis showed late lepidic adenocarcinoma. Pulmonary nodules implying invasive solid adenocarcinomas (dotted black circles) were developed at 7 weeks of tumorigenesis.

B. Immune atlas of early lepidic and invasive lung adenocarcinoma. The t-distributed stochastic neighbor embedding (t-SNE) plots were generated with pooled single-cell data of five mice from each group.

C. Longitudinal changes of cytometric data in CD19+ B and CD3+ T lymphocytes. During the tumorigenesis of lung adenocarcinoma, B and T lymphocytes contracted after 5 weeks of doxycycline feeding. We performed CyTOF with single cell suspension obtained from five mice in each timepoint.

D. Deeper phenotyping of lung macrophages. We show that this expanding macrophage population is comprised predominantly of a discrete population of tumor-associated macrophages (TAMs, CD45+CD64+F4/80+SiglecF−CD11b+CD19-B220-CD3-Ly-6G-), and that a separate population of normal lung alveolar macrophages (AMs, CD45+CD64+F4/80+SiglecF+CD11c+CD19-B220-CD3-Ly-6G-) contracts during tumor development. Compared with normal lung, which consists of 94% AMs in lung macrophages, tumor-bearing mice had only 12% AM in lung macrophages.

E. SCAFFOLD immune atlas of mouse lung adenocarcinoma. The SCAFFOLD maps were generated with pooled single-cell data of five mice from each group.

SCAFFOLD denotes single-cell atlases by fixed force- and landmark-directed.

We found the majority of macrophages in the normal lung to be alveolar macrophages (AMs, CD64+F4/80+SiglecF+CD11c+) and the remaining macrophages to be of the interstitial phenotype (IMs, CD64+F4/80+SiglecF-CD11c-)(29–32). As we have previously shown in this model(14), the total macrophage population (CD45+CD64+F4/80+) more than doubled during the development of invasive LUAD. The proportion of AMs, however, contracted, and the majority of macrophages were replaced by CD11b-expressing macrophages not present in the non-tumor-bearing lung, referred to hereafter as tumor-associated macrophages (TAMs, CD64+F4/80+CD11b+SiglecF-) (Figure 1D).

To understand these changes within the context of the evolving TiME of LUAD, we applied a semi-supervised approach and constructed single-cell atlases of early lepidic and invasive solid adenocarcinoma by fixed force- and landmark-directed (SCAFFOLD) maps (19,27) (Figure 1E and Figures S1–S2). Briefly, the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) unsupervised clustering algorithm(17) was applied to enumerate 1,472 cellular subpopulations, each defined by its unique protein expression and mapped based on their relative similarities to 14 landmark populations defined by prior knowledge of the immune system (Figure 1E). Early lepidic histology adenocarcinoma contained abundant CD4 effector T cells (CD4 Teff), CD8 effector T cells (CD8 Teff), and B cells, which contracted in invasive solid adenocarcinoma that contained TAMs predominantly (Figure 1E). These data indicate that a series of potential immune evasive mechanisms evolve in parallel to histologic tumor progression in LUAD, including reduction of lymphocytes, increase in the number of dysfunctional CD8 T cells, and expansion of a potential tumor-promoting macrophage population present only in tumors.

PD-1 distinguishes highly plastic TAM subtypes in lung adenocarcinoma.

Given the striking increase in TAMs in invasive LUAD, we scrutinized the TAM phenotype by hierarchical unsupervised clustering of the 449 nodes within the TAM gate (CD64+F4/80+CD11b+SiglecF-). Two subtypes of TAMs were segregated by expression of PD-1 (Figure 2A and Figure S5). Compared with PD-1^HIGH TAMs, PD-1^LOW TAMs demonstrated high expression of MHC-II and PD-L1. PD-1^HIGH TAMs highly expressed the proliferation markers, Ki-67 and IdU (5-Iodo-2′-deoxyuridine) (Figure 2A) and also highly expressed the chemokine receptor CX3CR1 (Figure 2B), suggesting a proliferative, tissue-resident ontology (33–35).

Figure 2. PD-1 distinguishes the highly plastic TAM subtype in lung adenocarcinoma.

A. Unsupervised clustering of TAMs according to protein expression by CyTOF showed two distinct clusters. One cluster showed high expression of PD-1, Ly-6C, and Ki67, and the other had high expression of PD-L1 and MHC-II. Thus, we divided TAMs into PD-1^HIGH and PD-1^LOW TAMs. Compared with PD-1^HIGH TAMs, PD-1^LOW TAMs expressed higher levels of MHC-II and PD-L1. PD-1^HIGH TAMs were proliferative with higher expression of the proliferative marker, Ki-67, and increased IdU incorporation.

B. PD-1^HIGH TAMs have a high expression of CX3CR1, suggesting a tissue-resident ontology.

C. Dynamic alteration of PD-1^HIGH TAMs during tumor progression. The Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction were generated with pooled single-cell data of five mice from each group.

D. Dynamic alteration of the functional markers of PD-1^HIGH and PD-1^LOW TAMs during tumor progression. We performed functional CyTOF including cytokines and phosphosignals with single cell suspension obtained from five mice at each timepoint. P-values were generated by ANOVA test of difference of proteins of interest between two groups.

E. Pathway analysis with proteins differentially expressed between early PD-1^HIGH and late PD-1^HIGH TAMs. Global protein profiling of PD-1 TAMs was triplicated.

In normal, non-tumor-bearing lungs, PD-1^HIGH macrophages (interstitial macrophages) were rare (≤ 3% of all macrophages) and PD-1^LOW macrophages comprised 17.3 ± 15.4% of all macrophages. In tumor-bearing lungs, both PD-1^HIGH and PD-1^LOW TAMs increased during tumor progression to invasive LUAD, and PD-1^HIGH TAMs constituted 37.5 ± 10.9 % of all macrophages at 6 weeks of tumorigenesis when invasive lepidic and solid invasive histology is present (Figure S6). To visualize longitudinal changes of PD-1^HIGH TAMs and PD-1^LOW TAMs during tumor progression, we used Uniform Manifold Approximation and Projection (UMAP) for dimensional reduction (26). Early in tumorigenesis during the development of lepidic histology LUAD, PD-1^HIGH TAMs and PD-1^LOW TAMs had divergent phenotype. Late in tumorigenesis during the development of invasive solid histology LUAD, PD-1^HIGH TAMs were juxtaposed with PD-1^LOW TAMs on UMAP plots, implying more similar protein expression (Figure 2C). To more completely understand the evolution of phenotype and function of PD-1^HIGH TAMs and PD-1^LOW TAMs during LUAD progression, we investigated cytokine production and phospho-signaling in TAMs using CyTOF. In early tumorigenesis, PD-1^HIGH TAMs increased their expression of Arg1, cMyc, Ki-67, vimentin, mTOR, and phospho-NF-κβ, then decreased expression of these proteins as tumor histology became invasive. PD-L1, MHC-II, CD44, and CD80 expression on PD-1^HIGH also declined dramatically at later time points of tumor progression (Figure 2D).

We next performed protein profiling of sorted PD-1^HIGH TAMs and PD-1^LOW TAMs early and late in tumorigenesis using mass spectrometry (Table S2). In early lepidic LUAD, PD-1^HIGH TAMs upregulated signaling pathways associated with mitochondrial dysfunction, activated oxidative phosphorylation, the TCA cycle, and the JAK/Stat pathway (Figure 2E), similar to the metabolic profile seen in IL-4-activated macrophages (36). In contrast, the protein profile of PD-1^HIGH TAMs isolated in later tumorigenesis were engaged in different metabolic pathways including fatty acid β-oxidation, protein synthesis, and branched-chain amino acids catabolism (Figure 2E). These data demonstrate that high PD-1 expression is associated with a TAM subtype in lung LUAD that has a plastic, immune-regulatory phenotype, and that undergo functional and metabolic transformation during tumor development.

PD-1^HIGH TAMs are enriched in the solid component of invasive lung adenocarcinoma.

We investigated the spatial contexture of PD-1^HIGH and PD-1^LOW TAMs in invasive LUAD at 7 weeks of tumorigenesis, a time when both invasive lepidic and invasive solid histologic components are present, using imaging mass cytometry (IMC) (Table S3). In these experiments, CD11b was used to identify macrophages, as greater than 90% of CD45+CD11b+ cells were TAMs according to our CyTOF data (Figure S7). From the same invasive LUAD tumors, we compared the immune composition of invasive lepidic tumor architecture with that of invasive solid tumor architecture by selecting separate regions of interest (ROIs) (Figure 3A). From each ROI, we extracted cellular information at the single-cell level using Fiji software-based cell segmentation (21) (Figure S8). Density plots were generated using data from both lepidic and solid ROIs (9427 cells) by hierarchical stochastic neighbor embedding (HSNE) within the single-cell analysis tool, Cytosplore (24). Using unsupervised Gaussian mean shift (GMS) clustering (37), we identified three clusters, based upon local probabilistic cell density (Figure 3B). Cells from invasive solid LUAD (ROI-2) contained high densities of PD-1^HIGH TAMs (CD11b+PD-1+) (Cluster 3) and high densities of cancer cells (Pan-cytokeratin+PD-L1+) (Cluster 1) (Figure 3B). In contrast, cells from invasive lepidic LUAD (ROI-1) contained abundant PD-1^LOW TAMs (Cluster 2) and cancer cells (Cluster 1) (Figure 3B). Consistent with our CyTOF data, PD-1^LOW TAMs expressed PD-L1 and the phagocytic marker, CD44 (38) (Figure 3C). These analyses indicate that PD-1^HIGH TAMs are localized to the solid components of LUAD tumors and PD-1^LOW TAMs are distributed mainly within their lepidic components.

Figure 3. Spatiotemporal evolution of tumor-supporting PD-1^HIGH TAMs occurs through PD-1/PD-L1 interaction.

A. PD-1^HIGH TAMs are enriched in the solid component of invasive lung adenocarcinoma. Spatial characterization of PD-1^HIGH and PD-1^LOW TAMs within lung adenocarcinoma using imaging mass cytometry to compare cell subpopulations between lepidic and solid components. PD-1^HIGH TAMs were mainly localized to the solid component of invasive tumor and PD-1^LOW TAMs were spread over the lepidic area.

B. Distinct clustering of cancer cells and TAMs.

C. Functional marker expressions in invasive lung adenocarcinoma. The solid component contained PD-1^HIGH TAMs, and the lepidic component had PD-1^LOW TAMs, expressing with PD-L1 and a phagocytic marker, CD44.

D. Spatial dynamics of the tumor-immune microenvironment during the tumorigenesis of lung adenocarcinoma.

E. PD-1^HIGH TAMs interact with cancer cells expressing PD-L1 to form a solid fortress in invasive adenocarcinoma.

F. IMC-based SPACE (SPAtial Cancer Evolution) maps to show cellular interactions of cancer cells, PD-1^HIGH TAMs, and PD-1^LOW TAMs during the tumorigenesis.

G. Cytokine release on SPACE map. TGF-β and IL-10 were released from cancer cells, PD-1^HIGH TAMs, and PD-1^LOW TAMs on late-stage tumors.

H. Effect of PD-1^HIGH TAMs on cancer cells. PD-1^HIGH and PD-1^LOW TAMs from mice at a 4-week timepoint were sorted and cultured with sorted tumor cells (CD45-human EGFR+). After 72 hours, the number of apoptotic tumor cells was significantly fewer in the co-culture with PD-1^HIGH TAMs. All in vitro experiments in triplicate were repeated.

I. Scheme of 3D-spheroid formation assay to test cell-to-cell interaction.

J. Disruption of spheroid formation by inhibiting PD-1 and PD-L1 interaction in spheroid formation assay. Single cells obtained from invasive adenocarcinoma at a 7-week timepoint formed a spheroid. The spheroid formation assay showed that blocking PD-1/PD-L1 interaction disrupted a spheroid formation with the wider spheroid area. The spheroid size was evaluated by measuring the spheroid perimeter using Fiji software, which area was converted into % relative area based on the size of the intact spheroid. Results are expressed as mean pixel measure ± S.E.M. from three independent experiments in four replicates. The IgG2a isotype antibody was the control isotype for both the anti-PD-1 and anti-PD-L1 antibodies.

PD-1^HIGH TAMs evolve through space and time to protect cancer cells.

Longitudinal IMC analyses were performed to track the spatiotemporal determinants of PD-1^HIGH TAMs during the histologic progression of LUAD. During early tumor development, PD-1^HIGH TAMs were diffusely distributed among these tumors of pre-invasive lepidic histology, however at later time points, PD-1^HIGH TAMs localized predominantly within the invasive solid histologic components that developed in these tumors (Figure 3D). Similar to our CyTOF findings, IMC showed that PD-1^HIGH TAMs highly expressed the proliferation marker Ki67, which additionally demonstrated its expression on PD-1^HIGH TAMs during all stages of tumor development. Given the congregation of PD-1^HIGH TAMs with high densities of cancer cells in the solid components of invasive LUAD tumors, we used IMC to dissect this spatial relationship. At later time points in tumor development, PD-1^HIGH TAMs co-localized with cancer cells, displaying an appearance similar to hook and loop fasteners within invasive lepidic and solid components of the tumor (Figure 3E). We leveraged the nuances of cell segmentation during IMC to develop a method to quantify the proximity of two defined cell populations to each other within a defined area of tumor, based on signal interference of protein expression at the points of contact of their cell borders. We created a series of SPACE maps (SPAtial Cancer Evolution) at incremental time points of tumor progression to define the regional relationship of PD-1^HIGH TAMs with cancer cells. These SPACE maps showed the steady emergence of PD-1^HIGH TAMs during progression to invasive LUAD and demonstrate their progressive co-localization with cancer cells (Figure 3F). Shown also in these SPACE maps is an increase in cellular TGF-β towards later time points in tumor development (Figure 3G), similar to the trends in cellular cytokine production by PD-1^HIGH TAMs shown in our longitudinal CyTOF analyses (Figure 2C).

Given the intimate spatial network of PD-1^HIGH TAMs and cancer cells in invasive LUAD, we investigated whether PD-1^HIGH TAMs were tumor-promoting. Co-culture of TAMs and cancer cells sorted from 4-week point tumors demonstrated that PD-1^HIGH TAMs protected cancer cells from apoptosis and to a significantly greater degree than PD-1^LOW TAMs (Figure 3H). To investigate PD-1/PD-L1 interactions between PD-1^HIGH TAMs and PD-L1 expressing cancer cells, we set up a three-dimensional spheroid culture system (39–41). PD-1^HIGH TAMs, PD-1^LOW TAMs, and PD-L1-expressing cancer cells sorted from 7-week point invasive solid adenocarcinoma tumors (at the 7 week time point) were cultured at a ratio of 1:2:2 for 7 days to allow cells to form spheroid structures (42). Spheroid size was measured by its cross-sectional area on the 7^th day after seeding, and smaller size was considered to have better spheroid integrity, reflecting stronger cell-to-cell interactions (43). In an effort to disrupt the interaction of PD-1 on TAMs and PD-L1 on tumor cells, we added anti-PD-1 or anti-PD-L1 antibodies to the cultures, and each compromised spheroid formation (Figure 3I and 3J). These results indicate that PD-1^HIGH TAMs co-localize with PD-L1-expressing tumor cells, particularly in the solid histologic components of LUAD, and that PD-1^HIGH TAMs interact with PD-L1(+) tumor cells and support their survival.

PD-1^HIGH TAMs and PD-1^LOW TAMs have differential impact on PD-L1-expressing cancer cells.

To further investigate the interaction between PD-1^HIGH TAMs and PD-L1-expressing cancer cells, we performed functional CyTOF to detect cytokines and phosphoproteins. PD-1^HIGH TAMs, PD-1^LOW TAMs, and PD-L1(+) cancer cells were sorted from lungs harboring invasive adenocarcinoma at the 7-week time point and cultured at a ratio of 1:1:1 for 24 hours (Figure 4A and Figure S9). Sorted PD-L1-expressing cancer cells were cultured alone or with either PD-1^LOW TAMs or PD-1^HIGH TAMs, in the presence or absence of blocking antibodies to PD-1. In the presence of PD-1^HIGH TAMs, cancer cells increased their expression of EPCAM and phospho-STAT3 (Figure 4A), molecules that are related to initiation and maintenance of cancer stem cells (44,45). UMAP plots demonstrated that cancer cell phosphosignaling and cytokine production were drastically and differentially affected by PD-1^HIGH TAMs compared with PD-1^LOW TAMs. Expression of EPCAM and pSTAT3 in cancer cells were abrogated by the presence of blocking antibodies to PD-1 (Figure 4B). Further, the addition of PD-1 blocking antibodies to the cultures resulted in increases in cancer cell expression of pSTAT1, pSTAT5, phosphor-p38, pS6, and IL-10, implying loss of stemness with concomitant gain of proliferative properties (Figure 4B). In contrast, in the presence of PD-1^LOW TAMs, cancer cells had relative high expression of pSTAT1, pSTAT5, pS6, and phospho-p38 and production of IL-10 compared with PD-1^HIGH TAMs, but anti-PD-1 treatment did not induce functional changes of cancer cells (Figure 4A). These data demonstrate that PD-1^HIGH TAMs have a distinct impact on PD-L1-expressing tumor cells, compared with PD-1^LOW TAMs.

Figure 4. PD-1^HIGH TAMs and PD-1^LOW TAMs have differential impact on cancer cells.

A. High expression of EPCAM and pSTAT3 in cancer cells in the presence of PD-1^HIGH TAMs. PD-1^HIGH TAMs, PD-1^LOW TAMs, and PD-L1(+) cancer cells sorted from 7-week point invasive solid adenocarcinoma tumors were cultured at a ratio of 1:1:1 for 24 hours. Sorted PD-L1-expressing cancer cells were cultured with either PD-1^LOW TAMs or PD-1^HIGH TAMs, in the presence or absence of PD-1 blockade. The properties of cancer cells were dynamically altered in the presence of PD-1^HIGH TAMs and PD-1^LOW TAMs. UMAP plots visualize similarities among cytokines and phosphosignals. Anti-PD-1 treatment led to a dramatic change of cellular properties in cancer cells cocultured with PD-1^HIGH TAMs.

B. Alteration of functional proteins in cancer cells after anti-PD-1 treatment. EPCAM and pSTAT3 in cancer cell in the presence of PD-1^HIGH TAMs was abrogated by the presence of blocking antibodies to PD-1. Anti-PD-1 treatment led to increase of Ki67, pSTAT1, pSTAT5, phosphor-p38, pS6, and IL-10, implying loss of stemness with concomitant gain of proliferative properties. In contrast, in the presence of PD-1^LOW TAMs, cancer cells had relatively high expression of pSTAT1, pSTAT5, pS6, and phospho-p38 and released IL-10 compared with PD-1^HIGH TAMs, but anti-PD-1 treatment did not induce functional changes of cancer cells.

Targeting PD-1^HIGH TAMs reduces tumor burden and restores immunity in lung adenocarcinoma.

We evaluated the efficacy of TAM-targeting strategies in LUAD using trabectedin, an FDA-approved alkylating agent with macrophage/monocyte-depleting properties.(46) We administered trabectedin to tumor-bearing mice on two different schedules: 1) beginning at 2 weeks of tumorigenesis when lungs have early lepidic (predominantly pre-invasive) histology (Figure 5A), and 2) at 6 weeks of tumorigenesis when lungs contain tumors with invasive solid components (Figure 5B). At the early time point, trabectedin significantly decreased the extent of lepidic tumor formation and prevented the development of invasive solid components (Figure 5C–D). At the late time point, trabectedin also reduced the extent of lepidic tumor formation, but had only a modest effect on reducing invasive solid foci (Figure 5E–F). Tumor-immune SCAFFOLD maps revealed that trabectedin reduced TAMs and recovered AM populations when administered at either early (Figure 5G) or late timepoints (Figure 5H). Treatment with trabectedin also resulted in the maintenance of B cells and CD4 T cells when delivered at the early timepoint, and recruitment of B cells, CD4 T cells, CD8 T cells, and NK cells when delivered at the late time point (Figure 5G–H). To determine whether TAMs were directly affected by trabectedin, we cultured sorted TAMs and cancer cells with trabectedin or vehicle for 24 hours. We found that trabectedin had a greater direct effect on PD-1^HIGH TAMs than PD-1^LOW TAMs (Figure 5I). PD-1^HIGH TAMs underwent a higher degree of apoptosis than PD-1^LOW TAMs (Figure 5J) and trabectedin downregulated the expression of Arg1, c-myc, and Ki67 on PD-1^HIGH TAMs (Figure 5K). These data indicate that trabectedin directly targets PD-1^HIGH TAMs in vitro and reduces the extent of lepidic and invasive solid components of LUAD.

Figure 5. Targeting PD-1^HIGH TAMs reduces tumor burden and restores immunity in lung adenocarcinoma.

A. Treatment scheme of lepidic adenocarcinoma with a macrophage-depleting agent, trabectedin. Trabectedin was administrated intravenously starting at 2 weeks of tumorigenesis and then biweekly to treat pre-invasive adenocarcinoma (untreated n=9 vs. treated n=7).

B. Treatment scheme of invasive adenocarcinoma with trabectedin. Trabectedin was administrated intravenously starting at 6 weeks of tumorigenesis and then weekly to treat invasive adenocarcinoma (untreated n=6 vs. treated n=6).

C. Histologic evaluation of lepidic adenocarcinoma showed a dramatically reduced tumor burden in the lungs of trabectedin-treated mice.

D. Comparison of the percentage of lepidic component and the number of solid foci more than 2mm before and after trabectedin treatment on early tumorigenesis.

E. Histologic evaluation of invasive adenocarcinoma showed reduced tumor burden in the lungs of trabectedin-treated mice.

F. Comparison of the percentage of lepidic component and the number of solid foci more than 2mm before and after trabectedin treatment in late tumorigenesis.

G. Tumor-immune atlas of lepidic adenocarcinoma. Comprehensive mass cytometric analysis revealed the alteration of the tumor-immune microenvironment before and after trabectedin treatment for AIS. The SCAFFOLD maps were generated with pooled single-cell data of all mice from each group. Each circle represents a node (a cluster of cells with a similar phenotype). Seven hundred thirty-three nodes were identified and organized into 18 major phenotypes, which are represented by the black landmark node. The size of node represents the number of cells, and the distance from nodes to one and other (lines) represents the degree of similarity of their complement of protein expression. Tumor cells, TAMs, CAF were significantly decreased, but AMs, CD4 T cells, B cells, and stromal cells were significantly increased after trabectedin treatment. P-values were calculated by t-tests.

H. Tumor-immune atlas of invasive adenocarcinoma. Comprehensive mass cytometric analysis revealed the alteration of the tumor-immune microenvironment before and after trabectedin treatment for invasive lung adenocarcinoma. The SCAFFOLD maps were generated with pooled single-cell data of all mice from each group. 1,412 nodes were identified and organized into 13 major phenotypes. Tumor cells, TAMs, and CAF were significantly decreased, but remained after trabectedin treatment. AMs, CD4 T cells, CD8 T cells, B cells, NK cells, and stromal cells were significantly increased after trabectedin treatment. P-values were calculated by t-tests.

I. Cell viability of cancer cells and TAMs isolated from tumor-bearing mice at a 4-week timepoint after trabectedin incubation for 24 hours. Trabectedin was more cytotoxic to PD-1^HIGH TAMs than cancer cells and PD-1^LOW TAMs.

J. The in vitro effect of macrophage depletion treatment on PD-1^HIGH TAMs from mice at a 4-week timepoint. in vitro trabectedin (10nM) treatment for 24 hours increased apoptosis in PD-1^HIGH TAMs compared with PD-1^LOW TAMs. All in vitro experiments in triplicate were repeated.

K. The in vitro functional alteration of TAMs from mice at a 4-week timepoint after trabectedin treatment. Trabectedin treatment on PD-1^HIGH TAMs led to an immediate decrease of Arg1 and cMyc, and a significant decrease of Ki-67 expression by 24 hours. All in vitro experiments in triplicate were repeated.

AM, alveolar macrophages; CAF, cancer-associated fibroblast; cDC, conventional dendritic cells; NK cells, natural killer cells; pDC, plasmacytoid dendritic cells; TAMs, tumor-associated macrophages.

Combining anti-PD-1 therapy with trabectedin eradicates invasive LUAD.

We reasoned that concomitant boosting of the innate and adaptive immune system by combining trabectedin with a PD-1 blockade, respectively, could improve anti-tumor efficacy over single-agent therapy. We treated mice with trabectedin and anti-PD-1 antibodies beginning at 6 weeks of tumorigenesis when tumors contained invasive lepidic and solid adenocarcinoma histologies (Figure 6A). PD-1 blockade alone reduced invasive solid foci but did not decrease the extent of lepidic adenocarcinoma (Figure 6B–C). Interestingly, PD-1 blockade alone increased the proportion of proliferative (Ki-67 expressing) TAMs (Figure 6D) and resulted in a spatial remodeling of the tumor-immune architecture from one in which proliferative TAMs were localized predominantly within the solid component of tumors before treatment to one in which they were diffusely disturbed throughout lepidic components after treatment (Figure 6E). The majority of these proliferative TAMs but were quantified here based on their expression of Ki-67 to eliminate the confounding effects of anti-PD-1 antibody therapy on PD-1 expression, however the majority did express PD-1 (Figure S10). Whereas anti-PD-1 monotherapy did not increase lymphocyte infiltration into invasive adenocarcinoma tumors, combination therapy resulted in a marked increase in B cells, CD4 effector T cells, and CD8 effector T cells (Figure 6F and Figure S11), decreased the fraction of exhausted phenotype (PD-1+TIM-3+) CD8 T cells, and increased the fraction of INF-γ and TNF-α releasing CD8 T cells (Figure 6G).

Figure 6. Combining anti-PD-1 therapy with trabectedin eradicates invasive lung adenocarcinoma.

A. Treatment scheme of invasive adenocarcinoma with trabectedin and anti-PD-1 antibodies. At 6 weeks of tumor development, mice began treatment with weekly trabectedin and anti-PD-1 antibodies.

B. H&E staining images in each treatment group.

C. Comparison of the percentage of lepidic component and the number of solid foci more than 2mm before and after each treatment.

D. Alteration of proliferative TAMs after treatment of invasive adenocarcinoma. Proliferative TAMs were significantly increased after anti-PD-1 monotherapy, but reduced after combination therapy in invasive adenocarcinoma.

E. Spatial changes of the tumor-immune microenvironment after anti-PD-1 treatment.

F. Tumor-immune SCAFFOLD map of invasive adenocarcinoma. Single trabectedin treatment for invasive adenocarcinoma showed TAMs were significantly decreased, but AMs, B cells, and CD4 effector, and CD8 effector T cells were significantly increased. Anti-PD-1 treatment showed no alteration of lymphocytes and significantly increased conventional dendritic cells. The combination treatment of trabectedin with anti-PD-1 led to a dramatic increase of CD4 and CD8 effector T cells, B cells, and AMs with decreased PD-1^HIGH and PD-1^LOW TAMs. The SCAFFOLD map was generated from the pooled CyTOF data of five or six mice in each group. P-values were calculated by t-tests.

G. Targeting TAMs by trabectedin led to the recruitment of active cytotoxic T cells releasing IFN-γ and TNF-α. Dysfunctional CD8 T cells expressing PD-1 and TIM-3 were significantly decreased after trabectedin or combination treatment. Mean metal intensities (MMI) of IFN-γ and TNF-α after PMA/Ionomycin stimulation were compared with ANOVA test.

PD-1^HIGH TAMs in human LUAD are associated with unfavorable clinical outcomes to PD-1 blockade.

To evaluate whether the enrichment of PD-1^HIGH TAMs in invasive LUAD was relevant to humans, we generated new macrophage gene signatures based on the differential protein profiles obtained from early PD-1^HIGH TAMs from mouse early pre-invasive lepidic adenocarcinoma, late PD-1^HIGH TAMs from invasive solid adenocarcinoma, and PD-1^LOW TAMs (Figure 2E, Figure S12, and Table S2). Using CIBERSORT (http://cibersort.stanford.edu/)(14,47) we deconvoluted mRNA microarray datasets from laser-captured microdissected fresh-frozen tumor samples obtained from 60 patients with LUAD, which were deposited in the NCBI’s Gene Expression Omnibus database (GEO GSE52248 and GSE58772)(48,49). These datasets include transcriptome data from normal lung (n=6), adenocarcinoma in situ (AIS, n=6), lepidic (n=10), acinar (n=10), papillary (n=9), micropapillary (n=9), and solid histology LUAD (n=10). We replaced the original M0/M1/M2 macrophage populations in CIBERSORT with compositions of PD-1^HIGH TAMs and PD-1^LOW TAMs (Figure S13). We found that PD-1^LOW TAMs were present among all histologic LUAD subtypes and that early PD-1^HIGH TAMs were abundant in AIS and lepidic LUAD subtypes. Late PD-1^HIGH TAMs were more frequent in invasive LUAD including solid, acinar, micropapillary, and papillary subtypes, when compared with AIS and lepidic subtypes (P=1.2×10⁻⁷) (Figure 7A).

Figure 7. PD-1^HIGH TAMs in human lung adenocarcinoma are associated with unfavorable clinical outcomes to PD-1 blockade.

A. PD-1^HIGH TAMs are dominant in micropapillary, papillary, and solid histologic subtypes in human lung adenocarcinoma. TAMs proportion is calculated with CIBERSORT based on our protein profiling of PD-1^HIGH and PD-1^LOW TAMs in early and late tumorigenesis. These datasets include transcriptome data from normal lung (n=6), adenocarcinoma in situ (AIS, n=6), lepidic (n=10), acinar (n=10), papillary (n=9), micropapillary (n=9), and solid histology LUAD (n=10).

B. Spatial aggregation of PD-1^HIGH TAMs in human lung invasive adenocarcinoma. Quantification of IMC data showed co-expression of CD68, CD11b, and PD-1.

C. Representative IMC images of abundant PD-1^HIGH TAMs in solid components in human lung invasive adenocarcinoma consistent with our in vivo findings.

D. Inverse correlation of PD-1^HIGH TAMs and the infiltration of B and T lymphocytes in lung adenocarcinoma (n=20). Most patients with PD-1^HIGH TAMs had disease progression.

E. Strong association between progressive disease and high PD-1^HIGH TAM in the tumor immune microenvironment. While the proportion of PD-1^HIGH TAM was calculated based on IMC data in BCM LUAD cohort (n=20), that of PD-1^HIGH TAM in publicly available cohorts (n=32: GSE93157 and GSE136961) treated with PD-1blockade was deconvoluted with mRNA-based in silico analysis tool, CIBERSORT.

F. Distinct overall survival according to the abundance of PD-1^HIGH TAMs in patients with lung adenocarcinoma treated with anti-PD-1 monotherapy (n=20).

To investigate tissue distribution patterns of PD-1^HIGH TAMs in human LUAD, and the relationship of these cells to responses to immunotherapy in patients, we performed IMC on pre-treatment tumor specimens from 20 patients with metastatic LUAD who underwent PD-1 blockade by either nivolumab (n=13) or pembrolizumab (n=7) (Table S4). Consistent with our findings in mouse LUAD, representative regions from core needle biopsies of these tumors showed that PD-1^HIGH TAMs (PD-1+CD68+CD11b+) aggregated in the solid components of human LUAD (Figure 7B and Table S3) and that lepidic-predominant LUAD histology demonstrated a lower proportion of PD-1^HIGH TAMs. Also, in line with our mouse data, invasive solid histology had a paucity of CD3 T cell infiltration, while lepidic histology showed abundant stromal infiltration of B cells and T cells (Figure 7C). We next quantified TAM and lymphocyte populations in this cohort using IMC (Figure S8) and found an inverse correlation between PD-1^HIGH TAM density and tumor infiltration of T cells and B cells (Figure 7D), as seen in other human tumors in which decreased infiltration of cytotoxic T cells is accompanied by increased numbers of immunosuppressive myeloid cells.(50) Patients whose tumors contained abundant PD-1^HIGH TAMs were more likely to have disease progression during PD-1 blockade (Figure 7D), validated with an independent cohort (n=32: GSE93157(51) and GSE136961(52))(Figure 7E), as well as decreased overall survival (Figure 7F). These findings indicate that PD-1^HIGH TAMs are enriched in the solid histologic components of human LUAD, are associated with a non-inflamed TiME that is resistant to PD-1 blockade, and may be potential targets for therapies combining macrophage-targeting agents and ICIs.

DISCUSSION

Lung cancer is a complex and dynamic ecosystem of epithelial, stromal, and immune cells that regulate its progression and response to therapy (53–55). Improvement in our understanding of the immunobiology of lung cancer can be expected to result in improved therapeutic options for the many patients suffering from this malignancy. Our comprehensive single-cell analytic approach revealed that the progression of LUAD is associated with a macrophage plasticity-dependent spatiotemporal evolution of the TiME that governs response to immunotherapy. We discovered that PD-1 is expressed on a highly plastic and tumor-promoting subtype of TAMs that are intricately associated with cancer cells and that may play a role in the formation of invasive tumor histology. We targeted PD-1^HIGH TAMs with the FDA-approved agent, trabectedin, and found that this approach favorably remodels its TiME and improves response to PD-1 inhibition.

PD-1 is an inhibitory surface protein found on a variety of intratumoral immune cells including T cells, B cells (43), natural killer cells (56), dendritic cells (57). and macrophages (58). Macrophages have previously been reported to express PD-1 following pathogen infection (59), however PD-1 expression on tumor macrophages has only recently been reported. Gordon and colleagues demonstrated that murine TAMs can express surface PD-1 and that this is associated with impaired phagocytic activity (58). In their model of murine colon cancer, the majority of PD-1(+) TAMs displayed an M2-like, pro-tumorigenic phenotype, whereas PD-1(−) TAMs had an M1-like, phagocytic phenotype. Our data demonstrate the novel roles of PD-1^HIGH TAMs in mediating the histologic progression of cancer and in promoting resistance to checkpoint blockade by their maintenance of an immune-depleted tumor milieu. We show that TAMs highly expressing PD-1 are not present in normal lung tissue and develop over the course of tumor progression from pre-invasive to invasive adenocarcinoma. Further, our data demonstrate that these PD-1^HIGH TAMs have the plastic phenotype, function, and tissue distribution. We describe a striking spatiotemporal evolution in these cells that can be modified by therapy, and that includes dynamic changes in expression of MHC and immunoregulatory molecules, proliferation and phagocytic activity, release of cytokines and enzymes, and tissue architectural location. Interestingly, PD-1^HIGH TAM had a proliferative phenotype and expressed CX3CR1, and consistent with our understanding that cellular proliferation can sustain certain populations of tissue-resident macrophages (60,61), could play an important role in supporting a niche for TAM maintenance in LUAD tumors (Figure S14).

Trabectedin is an anti-neoplastic agent that is active against several human tumors including sarcoma, ovarian, and breast adenocarcinoma in phase II clinical trials, and it is FDA approved for use in patients with advanced soft tissue sarcoma. Trabectedin is a DNA minor groove binder that blocks cell cycle and has been shown to suppress monocyte recruitment to tumor sites, inhibit macrophage differentiation, and initiate caspase-8-dependent apoptosis in tumor macrophages in transplantable mouse models of cancer (46,62). In our genetically engineered mouse model of LUAD, treatment of tumor-bearing mice with trabectedin resulted in TAM depletion and decrease in tumor burden of lepidic and solid adenocarcinoma histology. In vitro, PD-1^HIGH TAMs were directly killed by trabectedin and were more sensitive to killing than PD-1^LOW TAMs. These findings are consistent with our finding that PD-1^HIGH TAM had a highly proliferative phenotype, and with the known ability of trabectedin to block cell cycle on cells with rapid turnover including myeloid bone marrow progenitor cells (46). Combining trabectedin with PD-1 inhibition resulted in improved anti-tumor immunity, and decreased both the solid and lepidic components of LUAD, likely by several mechanisms (Figure S15). PD-1 blockade alone resulted in a striking increase in the number of proliferative TAMs, potentially increasing their susceptibility to trabectedin. Possibly as a result of its effects on immunoregulatory TAM, trabectedin remodeled the TiME of LUAD to an environment rich in lymphocytes and INF-γ-producing T cells, which would be expected to improve responses to PD-1 inhibition from an “inflamed” tumor (50,63). Furthermore, our data show a trajectory of spatiotemporal development of PD-1^HIGH TAMs during the progression of LUAD resulting in their clustering within the invasive solid components of advanced LUAD and intimate co-localization with PD-L1 expressing tumor cells. PD-1 blockade caused a disassociation of this solid tumor architecture in vivo with a decrease in the number of solid invasive foci. We explored this further in a spheroid culture system of TAMs and PD-L1+ tumor cells within which the addition of PD-1 or PD-L1 antibodies decreased the integrity of tumor spheroid structures. This suggests that PD-1 inhibition can interrupt the cell-cell interactions important for tumor integrity, and could also potentially facilitate trabectedin delivery within these tumors. In line with these findings, combination therapy with trabectedin and PD-1 blockade was found to have efficacy against a mouse model of fibrosarcoma (64). By way of its pleiotropic anti-tumor activity that includes suppression of tumor-promoting immune cells and direct cytotoxicity against lung cancer cells, trabectedin or its derivative, lurbinectedin, combined with checkpoint immunotherapy may be a viable therapeutic option for immunoresistant NSCLC tumors, alone or in combination with PD-1 blockade.

This mouse model shares features with human AIS, MIA, LPA, and solid subtype LUAD, and shares several similarities with our human data that support its translational relevance. Broadly, the TiME of mouse LUAD steadily became lymphocyte-depleted and enriched for phenotypically and functionally exhausted T cells with increasing elements of histologic invasion. Reciprocally, PD-1^HIGH TAMs were minimally present in pre-invasive mouse tumors (AIS), were highly enriched in mouse tumors with invasive solid histology, and localized most densely within solid histologic components. In human datasets, we found a similar inverse correlation between the number of tumor-infiltrating lymphocytes and the number of PD-1^HIGH TAM. We also found a lower frequency of PD-1^HIGH TAMs in pre-invasive and early invasive human LUAD tumors (AIS and MIA) and a higher frequency of PD-1^HIGH TAMs in the solid histology subtype. Further, IMC of human LUAD tumors additionally showed increased density of PD-1^HIGH TAM in solid components of human LUAD tumors. Importantly, an abundance of PD-1^HIGH TAMs was associated with resistance or hyperprogression, disease progression, and decreased overall survival in LUAD patients who received anti-PD-1 therapy. These findings are highly suggestive that PD-1^HIGH TAMs, and adenocarcinoma subtype histology are important determinants of response to immunotherapy in human LUAD.

In summary, our spatiotemporal investigation of lung adenocarcinoma with single-cell proteomic platforms identified histologic subtype-specific elements of its TiME that have important considerations for immunotherapy. We identified an immunoregulatory subtype of TAM in LUAD that expressed PD-1, that expanded during tumor progression from pre-invasive to invasive adenocarcinoma histology, that dominated the lymphocyte-depleted niche of invasive tumors, and that co-localized with tumor cells in the solid histologic components of the tumor. These tumor-protecting TAMs were sensitive to killing by trabectedin, which remodeled the TiME and which improved response to PD-1 blockade. Ultimately, our data support pursuing PD-1^HIGH TAMs targeting therapies in patients with immunoresistant lung cancer.

STATEMENT OF SIGNIFICANCE.

Comprehensive single-cell proteomics analyses of lung adenocarcinoma progression reveal the role of tumor-associated macrophages in resistance to PD-1 blockade therapy.