Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy treatments. In this study, single-cell RNA sequencing and protein analyses uncovered a subpopulation of myeloid cells in pancreatic lesions associated with an immune‐excluded tumor phenotype and effector T-cell exhaustion exclusively in females. This myeloid subpopulation was positively correlated with poor survival and genetic signatures of M2-like macrophages and T-cell exhaustion in females. The G-protein coupled receptor formyl peptide receptor 2 (FPR2) mediated these immunosuppressive effects. In vitro, treatment of myeloid cells with a specific FPR2 antagonist prevented exhaustion and enhanced cytotoxicity of effector cells. Proteomic analysis revealed high expression of immunosuppressive secretory proteins PGE2 and galectin-9, enriched integrin pathway, and reduced proinflammatory signals like TNFα and IFNγ in female M2-like macrophages upon FPR2 agonist treatment. In addition, myeloid cells treated with FPR2 agonists induced TIM3 and PD-1 expression only in female T cells. Treatment with anti-TIM3 antibodies reversed T-cell exhaustion and stimulated their ability to infiltrate and kill pancreatic spheroids. In vivo, progression of syngeneic pancreatic tumors was significantly suppressed in FPR2 knockout (KO) female mice compared with wild-type (WT) female mice and to WT and FPR2 KO male mice. In female mice, inoculation of tumors with FPR2 KO macrophages significantly reduced tumor growth compared with WT macrophages. Overall, this study identified an immunosuppressive function of FPR2 in females, highlighting a potential sex‐specific precision immunotherapy strategy.

Significance:

FPR2 is a sex-dependent mediator of macrophage function in pancreatic cancer and can be targeted to reprogram macrophages and stimulate antitumor immunity in females.

Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal, with a prognosis typically between 4 and 6 months. Although surgery and adjuvant chemotherapy have brought therapeutic benefits to ca. 20% of patients, where 5-year survival reaches up to 30%–58%, PDAC remains among the mostly deadly form of cancer types. Unfortunately, most patients with pancreatic cancer are diagnosed with advanced-stage disease, where treatment options are limited and a high unmet need exists (1).

Although immunotherapy has revolutionized the management of numerous cancer types, including melanoma, lung, renal, and liver cancers, it remains ineffective for PDAC (2). This is considered to be a result of the immunosuppressive tumor microenvironment (TME) in PDAC (3). The dysfunction of cytotoxic immune cells is linked to the accumulation of tumor-associated macrophages (TAM), contributing to a dense stroma, accompanied by immune cell exhaustion and limited effector cell infiltration (4). Although there have been numerous reports describing the TME in PDAC, very little is known regarding sex-specific differences and how this contributes to immune response variability.

In this study, we sought to define the role of formyl peptide receptor-2 (FPR2) in contributing to sex-immune dimorphism in the TME. The FPRs are a subtype of G-protein coupled receptors (GPCR). These are expressed in particular on myeloid cells, where unique ligand-binding features enable molecular pattern recognition receptors to bind bacteria-derived peptides. This results in the secretion of chemoattractants for neutrophils and mononuclear/macrophage cells, which are important effector cells in the immune response (5, 6).

Initially, the understanding of FPRs is mainly focused on the regulation of inflammation, given that ligand binding of FPRs results in a series of cell signaling events, leading to the migration of myeloid cells, the release of inflammatory mediators, and increased phagocytosis (7, 8). However, there is increasing evidence that FPRs are involved in many pathophysiological processes in addition to controlling inflammation. FPRs can promote the host-defense process, regulate the activation of neutrophils and dendritic cells (DC; refs. 9, 10), and participate in response to bacterial infection, tissue damage, and wound healing (8). At the same time, FPRs can participate in cancer growth, invasion, and metastasis (11, 12). It has been reported that FPR2 activation promotes M2 macrophage polarization in the ovarian cancer microenvironment, results in downregulation of E-cadherin and upregulation of vimentin, implying the involvement of epithelial–mesenchymal transition, and confers an invasive phenotype of human glioblastoma cells (13–15).

Herein, we report that FPR2 regulates immune cells in the TME in a sex-dependent manner. It has been reported that estradiol can regulate the expression of FPR2’s ligands (16, 17). Our data reveal that FPR2 is mainly expressed in neutrophils and macrophages, but the sex-differential FPR2 expression mainly occurs in macrophages. This FPR2-dependent sex-immune dimorphism regulates the phenotype and function of macrophages, thereby playing a central role in defining gender imbalance in the immune TME in PDAC.

Human patient and healthy samples

Pancreatic tumor collection was approved by the regional ethics review board in Stockholm (Dnr. 2020–06587). Written informed consent was obtained from all subjects, all samples were deidentified before use, and the studies were conducted in accordance with the Declaration of Helsinki. Cryopreserved or fresh peripheral blood mononuclear cells (PBMC) from healthy blood donors (HD) were obtained after Ficoll-Hypaque density gradient purification. Blood samples from HD were procured from the Stockholm Blood Bank. CD14+ monocytes, CD3+ T from HD were isolated by positive selection and natural killer (NK) cells were isolated by negative selection, based on the manufacturer's protocol (Miltenyi Biotec). T and NK cells were frozen for later analysis.

Cell culture

Isolated monocytes were cultured in RPMI-1640 (Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco). CD14+ monocytes were stimulated by M-CSF (50 ng/mL) for 6 days and refreshed medium supplemented with M-CSF on the fourth day. Then medium was completely changed with supplementation of LPS (200 ng/mL) + IFNγ (20 ng/mL) or IL4 (20 ng/mL) + IL10 (20 ng/mL), to polarize macrophages into proinflammatory or anti-inflammatory type, respectively. In addition, 6 days differentiated macrophages were conditioned by pancreatic cancer cell lines for 2 days, tumor-conditioned macrophages (TCM). Tumor cells were cocultured in 6-well-plate Transwell inserts and separated from macrophages to allow for soluble factors exchange only. After induction, macrophages were collected by cell dissociation buffer (Thermo Fisher Scientific) for flow cytometry analysis or effector cell suppression assay. T cells or NK cells were rested overnight before the suppression assay. In the presence of IL2 (1,000 IU/mL, PeproTech), NK cells were cocultured with macrophages for 4 days before flow cytometry analysis. Alternatively, in the presence of anti-CD3/CD28 beads (1 μL/mL, Thermo Fisher Scientific), T cells were cocultured with macrophages for 3 days until flow cytometry analysis. Both NK and T cells (5 × 104) were cocultured at a 1:1 ratio with macrophages in 96-well plates.

In macrophage suppression experiments, macrophage–T-cell co-cultures were treated with FPR2 agonists/antagonists, immune checkpoint antibody blockade in cell-to-cell contact experiments. FPR2 agonists ACT and Cpd43 (2 μmol/L), WRW4 (100 nmol/L), anti-PD1 (10 μg/mL), and anti-Tim3 (10 μg/mL) were used. Transwell experiments were only used to separate macrophages and T cells and allow for soluble factors exchange only to test whether macrophages suppress T cells in a cell-to-cell contact-dependent way. We seeded 5 × 105 polarized macrophages in the upper chamber of a 24-well plate and 5 × 105 T cells in the lower chamber. Corresponding cells were mixed and seeded. The cultures were conditioned with IL2 and anti-CD3/CD28 beads as mentioned above.

Cell lines and cell culture

Pancreatic cancer cell lines [human BXPC3, CFPAC (ATCC), and murine KPC] were all tested for Mycoplasma (Lonza) before use. Cells were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin. For induction of TCM, 5×105 pancreatic cancer cells were cultured in the upper Transwell inserts of 6-well plate or directly mixed with macrophages in 6-well plate.

The Cancer Genome Atlas analysis

All data were retrieved from The Cancer Genome Atlas (TCGA) pancreatic adenocarcinoma (PAAD) dataset (https://portal.gdc.cancer.gov/projects/TCGA-PAAD). mRNA expression levels in PDAC and matched normal pancreatic tissue controls were downloaded from UCSC Xena (UCSC Xena, RRID:SCR_018938). Then FPR2 expression levels from males and females were compared and visualized in GraphPad Prism 8.0. Survival analysis for FPR2 and T-cell exhaustion–related genes were performed with top and bottom expression values or separated by sex as indicated in respective figure legends and plotted as Kaplan–Meier curves. Gene Expression Profiling Interactive Analysis 2 (GEPIA2; Gene Expression Profiling Interactive Analysis, RRID:SCR_018294) integrates 33 types of tumor and matched nontumor samples from TCGA and Genotype-Tissue Expression (GTEx), providing differential expression analysis, correlation analysis, and patient survival analysis online. Our study used GEPIA2 to analyze the expression correlation of M2 macrophage signature genes and T-cell exhaustion–related genes in a variety of solid tumors. The Spearman correlation coefficient was used for correlation testing.

Single-cell analysis

Gene expression counts were downloaded from project PRJCA001063 in the Genome Sequence Archive. Then the counts data were imported into the Seurat (V4.0) R toolkit for quality control and further analysis of single-cell RNAseq data. All functions were run with default alignment. Low-quality cells (<500 genes/cell, >8,000 genes/cell, <10 cells/gene, and >20% mitochondrial genes) were excluded. The doublet cells were removed.

t-SNE and clustering

The Seurat package was used to identify major cell types in R. LogNormalize was used to compute comparable expression values. Highly variable genes were generated and used to perform principal component analysis. The parameters for computing the visualized dimensionality reduction coordinates and performing unsupervised clustering were as follows (reduction = "rpca,” at res  =  0.1 for PDAC samples and 1 for pooled cells from PDACs and controls). We characterized the identities of cell types of these groups based on previously reported method (18).

Flow cytometry

Detailed information of antibodies used in this study is summarized in Supplementary Table S1. A variety of fluorophore-conjugated antibodies were used to evaluate the phenotype and function of tumor cells, macrophages, T, NK cells, cultured in vitro, and cells obtained from spleens and tumor environment in vivo. Cell surface marker staining was first performed according to the manufacturer's instructions, followed by the fixation and permeabilization of (eBiosciences) cells for the detection of intracellular cytokines or transcription factors (Ki67, CD107a, IFNγ, TNFα, and FOXP3). Fixable live/dead cell dye (Thermo Fisher Scientific) was used to determine viable cells. Cells were treated with protein transport inhibitors Golgistop and Golgiplug (BD Biosciences) for 6 hours before staining. All cells were acquired via BD FACSAria (BD Biosciences) and analyzed by Flowjo 10.8 (FlowJo, RRID:SCR_008520).

Quantitative RT-PCR

To quantify gene expression, RNA was isolated from macrophages using TRizol (Invivogene). Synthesized cDNA from RNA using iScript reverse transcription (Bio-Rad, Reliance Select cDNA Synthesis Kit). qRT-PCR reactions were performed using SYBR Green Supermix (Bio-Rad, iQ SYBR Green Supermix). The primers were used to measure mRNA quantification for IL1RN, IL2, IL4, IL10, CD274, LGALS9, IL12p40, TGFB, and PTGS2 (Supplementary Table S2). All reactions were carried out in the Bio-Rad thermal cycler (Applied Biosystems 7500 Real-Time PCR System).

Immunofluorescence microscopy

Sequential multiplex immunofluorescence staining was performed on 4 μmol/L glass sections of paraffin-embedded human PDAC (n = 20) and murine tissues. Murine tissues were obtained from the KrasLSL-G12D / +; Trp53LSL-H172R / +; Pdx1-Cre (KPC) mouse model (19). These genetically engineered mice develop PDAC similar to human PDAC. Here, PDAC tumor tissues from 10 mice, six males and four females (Linköping's ethical committee, Dnr S31–15 and Dnr ID 1776) were investigated. Slides were baked in 60℃ for 1–2 hours and sections were dewaxed in xylene and subsequently dehydrated in ethanol at various concentrations. Later, the slides were microwaved in citrate buffer solution on high heat for 20 minutes and allowed to cool down naturally for antigen retrieval. Sections were subsequently blocked with 5% goat serum (DAKO) in PBS for 30 minutes at room temperature. All antibodies were incubated in a humidified chamber for 60 to 120 minutes at room temperature. After blocking sections with antifade mounting media (Thermo Fisher Scientific), they were imaged on a Leica LSM 880 confocal microscope and images were analyzed in ImageJ. The images were analyzed with ImageJ (Fiji, RRID:SCR_002285). Color balance was adjusted on the basis of negative control images stained with secondary antibodies, to account for unspecific binding. Nuclear Hoechst-stained particles were identified. The "Cell Counter" function was used to determine single- and double-positive signal in nucleated cells, for including FPR2, pan-CK, CD11b, CD3, and CD45. To study the cell distance between immune cells and the tumor, measurements were made in ImageJ, defining the distance between FPR2-positive cells and panCK tumor marker or other immune cells.

ROS detection assays

The oxidation-sensitive dye DCFDA (Molecular Probes, Invitrogen) was used to measure reactive oxygen species (ROS) produced by macrophages. Cells were incubated in RPMI-1640 at 37°C for 30 minutes in the presence of different treatments. Macrophages were first polarized by related cytokines and washed before the assay. Cells were treated for 30 minutes with FPR2 agonists (The Act-389949 compound; ref. 20), synthesized by Ramidus AB are generous gift from ProNoxis AB, or Cpd43 (Tocris Bioscience; ref. 21). Analysis was then performed by flow cytometry as described above.

Viability assay

CD14+ monocytes isolated from male and female human blood HD were first cytokine polarized and washed before the assay. A titration of FPR2 antagonist WRW4 (R&D Systems) or agonist ACT-389949 or CpD43 were added to cultures for 3 days and assessed for viability by using Fixable live/dead cell dye and analyzed by flow cytometer.

LC/MS-MS

Protein extraction and quantification

Cell pellet was lysed with 2% SDS 100 mmol/L Tris HCL 100 mmol/L Ammonium Bicarbonate with complete protease inhibitor cocktail. To completely solubilize the protein and break DNA, sonication was performed on ice bath. Supernatant was collected for protein quantification after centrifugation at 15,000 RCF for 10 minutes at 4°C. Protein quantification was performed using bicinchoninic acid (BCA; Sigma) and Bradford (Bio-Rad). Absorbances were read using Tecan Infinite M200Pro plate reader at 562 and 595 nm, respectively. Quantified proteins were aliquoted and stored in −20°C before proteomic analysis.

Proteomic sample preparation and LC/MS-MS analysis

All chemicals were obtained from Thermo Fisher Scientific unless stated. An equal amount of proteins was pooled together from each condition consisting of 4 biological replicates in each sex. In-solution digestion was performed as previously published (22). Trypsin (Promega, mass spectrometry grade) at 1:100 was used at 0 and 2 hours later and incubated at 37°C overnight. Digestion was stopped with final 2% acetic acid, followed by desalting with Sep-Pak Vac 1cc 100-mg C18 Solid Phase Extraction (SPE) cartridge (Waters) in accordance with the manufacturer's guidance. High pH reverse phase fractionation was performed with buffer A—0.02% ammonium hydroxide (NH₄OH) and buffer B—80% ACN 0.02% NH₄OH using XBridge BEH C18 column (4.6 × 250 mm; Waters Corporation). Fractionated peptides were reconstituted in 0.1% formic acid and 3% ACN for LC/MS-MS analysis in an UltiMate 3000 RSLCnano system coupled to a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) as previously described. Each sample was injected in triplicate into EASY-Spray column (75 μm x 10 cm ID Acclaim PepMap RSLC C18, 3 μm, 100 A°; Thermo Fisher Scientific).

Proteomic bioinformatic analysis

Raw output files from LC/MS-MS were processed using Proteome Discoverer software version 2.1.1.21 (PD2.1; Thermo Fisher Scientific). Protein identification was done by mapping against the UniProt KnowledgeBase (UniProtKB) Homo sapiens protein database (downloaded on February 6, 2017, 1,586,247 sequences and 61,972,042 residues) and using SEQUEST-HT and Mascot search engines. For target FDR, a semi-supervised machine learning, Percolator, was used with q value <0.01 strict and <0.05 relaxed validation on identified peptides. Quantification of identified protein within the sample uses exponentially modified protein abundance index (emPAI) and spectral area. A protein list generated from PD2.1 was sorted to include only high protein FDR confidence (q value <0.01), is a Master Protein, ascending experimental q value and descending sum posterior error probability. The identified proteins were present in at least two out of three technical replicates before converting to human genome organization (HUGO) gene nomenclature committee (HGNC) gene symbol for the downstream gene ontology (GO) analysis. Normalization with average was performed within each condition and sex. Functional Enrichment Analysis Tool (FunRich) version 3.1.3 (23) for its quantitative gene enrichment analysis in cellular component, biological process, and pathway as well as transcription factor were used.

Three-dimensional tumor cultures and T-cell infiltration

Three-dimensional (3D) cocultures were performed as previously described (24). Briefly in BXPC3 and CFPAC cells were seeded together with human primary monocytes in the presence of M-CSF (100 ng/mL) macrophage differentiation for 5 days at 37 and 5% CO2 until spheroid formation. On day 5, T cells labeled with CellTracker Deep Red (Thermo Fisher Scientific) to assess tumor infiltration. The cultures were treated with ACT (2 μmol/L) and anti-PD1 (10 μg/mL) or anti-Tim3 (10 μg/mL), and combination, or control IgG. All wells were incubated with CellEvent Caspase-3/7 Detection Reagents (Thermo Fisher Scientific) to detect tumor killing. Spheroid size and NK-cell infiltration were assessed with IncuCyte S4 Live-cell analysis system (Sartorius) for at least 30 hours.

Animal experiments and animal tissue isolation

All animal experiments were performed in accordance with national laws and institutional guidelines and were carried out with the approval of the local ethical committees: (Linköping's ethical committee, Dnr. 10970–2021). Following the Swedish government's ethical guidelines, FPR2−/− knockout (KO) mice kindly provided by Dr. Jiming Wang (NIH) and wild-type (WT) c57BL/6N from in house breading, were maintained under pathogen-free conditions.

Murine macrophages were differentiated from the bone marrow in M-CSF (20 ng/mL), medium was refreshed on day 3, and the adherent macrophages were harvested after 6 days. Murine leukocytes were collected from the FPR2 WT and KO spleens, and then sorted using a CD8-positive cell sorting kit. The obtained CD8+ T cells were cryopreserved for future use.

Syngeneic tumor model

Four- to 6-weeks-old FPR2−/− and WT female and male mice (n = 4–6 per group) were injected with either control PBS or 0.5×106 KPC cells in 100 μL PBS per mouse subcutaneously in the right or left flank. Seven days after tumor injection, the tumor length and width were measured by caliper every 2 to 3 days. Volume calculation was based on LW^2/2. At day 15 to 18 after tumor inoculation, mice were euthanized to harvest tumors and spleens. Experiment was repeated twice.

Macrophage transfer in syngeneic tumor model

Macrophage collection and induction methods were the same as in vitro experiments. Subsequently, a total of 5×105 M-CSF inducted macrophages were mixed with 5×105 KPC cells and injected subcutaneously into WT mice (follow the same-sex principle). Experiment was repeated twice.

Statistical analysis

All data were first analyzed in the software mentioned above and summarized by Prism Version 9 software (GraphPad Prism, RRID:SCR_002798). All data were first tested for normal distribution. Among the parameters compared between the two groups, statistical differences among groups were analyzed by parametric or nonparametric t test or multiple comparison tests and P values were corrected using FDR for multiple comparisons (FDR < 0.05 was considered significant) as indicated in the figure legends. In between-group comparisons of multiple datasets, statistical differences among groups were analyzed by one-way or two-way ANOVA test. The Wilcoxon rank-sum test was used to examine unpaired data in single-cell RNA by wilcox.test function in R package stats and multiplicity is controlled using FDR correction. Representative histograms or images were chosen based on the average values. Statistically significant outcomes P < 0.05 are shown as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 or no stars for not significant data.

Data availability

Data generated in this study for proteomic analysis are available upon request and transcriptomic data analyzed in this study were obtained from publicly available data in the GSA: Project #: PRJCA001063, at https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA001063, and in TCGA-PAAD, at https://portal.gdc.cancer.gov/projects/TCGA-PAAD. R scripts are available at GitHub https://github.com/sarhanlab/FPR2.git. Other raw data are available on request from the corresponding author.

Characterization of FPR2+ cells in patients with PDAC and their clinical relevance

Despite TAMs being closely associated with the occurrence and development of pancreatic cancer, there are only limited reports on the involvement of sex differences in the regulation of their function. With this in mind, we first examined FPR2 expression levels in PDAC and normal tissues (n = 180 vs. n = 425), and later compared male (TCGA n = 99, GTEx n = 193) and female (TCGA n = 81, GTEx n = 153) patients. We found a 2.7-fold increase in FPR2 mRNA expression levels in whole-tumor tissues compared with normal tissues and significantly higher levels in female tumor tissues compared with male (mean RPKM: 13.15 vs. 12.49, P = 0.03; Fig. 1A). However, such sex-bias was not observed in normal tissues (Fig. 1B). Notably, survival outcomes in patients with pancreatic cancer revealed a significant association with FPR2 expression in female patients only, where high expression results in worse survival (Fig. 1C).

Figure 1.

Macrophage FPR2 expressions reveal a sex-biased TME that correlates with pro-cancer immune cell signatures and worse clinical outcomes. A, FPR2 gene expression in patients with pancreatic adenocarcinoma tumor from TCGA database (n = 180) and normal pancreatic tissues from GTEx database (n = 425). B, Left, FPR2 gene expression in TCGA-PAAD patients performed with male (n = 99) versus female (n = 81). Right, FPR2 gene expression in normal tissues GTEx performed with male (n = 193) versus female (n = 153). C, Kaplan–Meier survival curves comparing FPR2 low and high mRNA expression levels in total PAAD cohort (left), in female (middle), and male (right) patients, respectively. Survival curves based on overall survival (OS) are shown. D, The t-distributed stochastic neighbor embedding (t-SNE) plot demonstrates main cell types in PDAC. E, Expression levels of FPR2 from male and female patients are plotted onto the t-SNE map (left). Color key from gray to red indicates relative expression levels from low to high (right). Violin plots displaying the statistics analysis of FPR2 PDAC male (M) and female (F) patents. The y-axis shows the normalized read count. F, Bubble plot showing functional enrichment of FPR2-positive versus FPR2-negative macrophage upregulated genes. G, Bubble plot showing functional enrichment of male versus female macrophage upregulated genes. H, Proportion of cells derived from male and female PDAC samples that contain the 11 subgroups defined in D. I, Correlation of FPR2 with M1/M2 signature score (MRC1+CD163/ CD83+IL12B+TNF) in TCGA-PAAD female and male cohort, respectively. *, P < 0.05; ***, P < 0.001.

Figure 1.

Macrophage FPR2 expressions reveal a sex-biased TME that correlates with pro-cancer immune cell signatures and worse clinical outcomes. A, FPR2 gene expression in patients with pancreatic adenocarcinoma tumor from TCGA database (n = 180) and normal pancreatic tissues from GTEx database (n = 425). B, Left, FPR2 gene expression in TCGA-PAAD patients performed with male (n = 99) versus female (n = 81). Right, FPR2 gene expression in normal tissues GTEx performed with male (n = 193) versus female (n = 153). C, Kaplan–Meier survival curves comparing FPR2 low and high mRNA expression levels in total PAAD cohort (left), in female (middle), and male (right) patients, respectively. Survival curves based on overall survival (OS) are shown. D, The t-distributed stochastic neighbor embedding (t-SNE) plot demonstrates main cell types in PDAC. E, Expression levels of FPR2 from male and female patients are plotted onto the t-SNE map (left). Color key from gray to red indicates relative expression levels from low to high (right). Violin plots displaying the statistics analysis of FPR2 PDAC male (M) and female (F) patents. The y-axis shows the normalized read count. F, Bubble plot showing functional enrichment of FPR2-positive versus FPR2-negative macrophage upregulated genes. G, Bubble plot showing functional enrichment of male versus female macrophage upregulated genes. H, Proportion of cells derived from male and female PDAC samples that contain the 11 subgroups defined in D. I, Correlation of FPR2 with M1/M2 signature score (MRC1+CD163/ CD83+IL12B+TNF) in TCGA-PAAD female and male cohort, respectively. *, P < 0.05; ***, P < 0.001.

Close modal

Given that FPR1–3 are highly homologous, we first examined the expression of these in normal and tumor tissues, where FPR2 as well as FPR3 were highly expressed in the majority of different types of cancers compared with normal tissues (Supplementary Fig. S1A). However, FPR3 had no sex-bias effect on the clinical outcome of PDAC (Supplementary Fig. S1B). Assessing the different cells that might express FPR2 from 24 PDAC tumor (13 females and 11 males) samples and 11 control pancreases (nontumor, not inflamed, from different individuals) from treatment naïve patients (six female and five male) by single-cell RNA sequencing (scRNAseq), using publicly available data (18) with cell clusters shown (Fig. 1D), we found that female TAMs exhibited differential FPR2 expression compared with all other cell populations in tumor tissues (Fig. 1E). Differentially expressed genes from FPR2-positive and FPR2-negative macrophage were extracted from the single cells and subjected to pathway enrichment analysis; this showed a clear cytokine regulation preference and specially the anti-inflammatory M2 driving IL4–IL13 cytokine pathway was upregulated in FPR2-positive versus FPR2-negative macrophages (Fig. 1F). Pathway enrichment analysis revealed that female macrophages were different to male macrophages in pathways involving pancreatic secretion, invasion, hypoxia, and lipid metabolism, among others (Fig. 1G). In addition, T cells from female patients accounted for 54.1% of the total T-cell frequency, whereas males accounted for 45.9%. Meanwhile macrophages in female tumors accounted for 56.9% whereas males accounted for 43.1% (Fig. 1H). Furthermore, we investigated the expression of common genes related to the protumoral M2 and antitumoral M1 macrophage signatures assessed from the TCGA database. Our results demonstrated that M2 macrophage signature in six different tumor types associated closely with FPR2 expression, with pancreatic cancer showing the most pronounced association (Supplementary Fig. S1C and S1D). The data showed that FPR2 and the M2 cell signatures were highly correlated (r>0.6); however, the correlation between the scores of FPR2 and M2/M1 was only significant in females, not males (Fig. 1I). Data from Primary Cell Atlas in BioGPS Dataset Library showed that FPR2 expression was concentrated in monocytes and neutrophils compared with all other immune cells (Supplementary Fig. S2A). To further clarify the distribution of FPR2 in immune cells, we collected peripheral blood from HDs and isolated PBMCs for flow cytometry. Flow cytometry results showed that FPR2 was highly expressed in CD11b+CD14+ and CD11b+CD16+ cells in peripheral blood. In CD11b+CD14+ cells representing classical monocytes, the expression in females was significantly higher than that in males (Supplementary Fig. S2B). Altogether, our data suggest that FPR2 expression on myeloid cells is associated with a suppressive myeloid profile and worse tumor progression in female patients with PDAC.

Presence of FPR2+ macrophages in the TME uncover a female skewed immune distribution and correlation with pro-cancer immune cell signatures in pancreatic cancer

Next, we investigated the density of FPR2-expressing TAMs and their spatial association with immune effector cells in primary pancreatic cancer tumors from mice and human patients. We performed multiple immunofluorescence staining and multispectral imaging to first investigate the abundance and location of FPR2+ TAM (FPR2+CD206+F4/80+), FPR2 TAMs (F4/80+CD206+). We found very few FPR2 TAMs, on the other hand FPR2+ TAMs were highly abundant in murine PC (Fig. 2A, left). These TAMs were located close to the tumors both in male and females; however, the T-cell proximity to tumors is closer in female murine tumors compared with male (Fig. 2A, right) Furthermore, we observed a trend toward a higher proportion of FPR2+ TAMs (FPR2+CD45+CD11b+) in women with PDAC in comparison with men; more FPR2+ TAMs were found in the tumor stroma in female patients and were located more closely to tumor cells (Pan-CK+; Fig. 2B and C). We determined the signal of FPR2 expression to be solely from immune cells (CD45+) but not tumor cells in both sexes (Fig. 2D). Given the observed trend in abundance of FPR2+ TAMs, we found the number to be significantly higher in the TME of female human tumors, in comparison with males. However, we found no difference between T-cell numbers in the two sexes (Fig. 2E). Next, we examined the spatial distribution of FPR2+ TAMs and T cells with regard to tumor cells and found that FPR2+ TAMs were in close proximity to tumor cells in both sexes, when compared with T-cell tumor proximity. Nevertheless, T cells and FPR2+ TAMs were closer to tumor cells in female patients, in comparison with the distance in male patients (Fig. 2F).

Figure 2.

Macrophage-derived FPR2 correlates with sex-dimorphic immune features. A, Multispectral imaging of KPC tumor sections (40×) stained for FPR2 (orange), CD206 (green), F4/80 (red), and nucleus marker Hoechst (blue), and quantification of cell distance was performed in ImageJ. KPC-tumor sections 3 out of 10 are shown. B and C, Immunofluorescence staining for FPR2, CD11b, CD3, and Pan-CK of paraffin-embedded PDAC sections of female (n = 10; B) and male (n = 10; C) patients. Representative images are shown (20×). D, Total FPR2-positive cells, CD45+FPR2-positive cells, and Pan-CK+FPR2-positive cells were counted by ImageJ in immunofluorescence sections. E, FPR2+ TAMs and CD3+ cells were counted by ImageJ in immunofluorescence sections. F, Average cell-to-cell distances were counted by ImageJ in immunofluorescence sections. G, Purified monocytes from HD were differentiated in M-CSF and later polarized into M0, M1, and M2 or polarized into TCM with different pancreatic cancer cell lines (BXPC3 and CFPAC). Macrophage feature markers (FPR2, CD163. MARCO, CD206, CD86, and HLA-DR) were measured by flow cytometry analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

Macrophage-derived FPR2 correlates with sex-dimorphic immune features. A, Multispectral imaging of KPC tumor sections (40×) stained for FPR2 (orange), CD206 (green), F4/80 (red), and nucleus marker Hoechst (blue), and quantification of cell distance was performed in ImageJ. KPC-tumor sections 3 out of 10 are shown. B and C, Immunofluorescence staining for FPR2, CD11b, CD3, and Pan-CK of paraffin-embedded PDAC sections of female (n = 10; B) and male (n = 10; C) patients. Representative images are shown (20×). D, Total FPR2-positive cells, CD45+FPR2-positive cells, and Pan-CK+FPR2-positive cells were counted by ImageJ in immunofluorescence sections. E, FPR2+ TAMs and CD3+ cells were counted by ImageJ in immunofluorescence sections. F, Average cell-to-cell distances were counted by ImageJ in immunofluorescence sections. G, Purified monocytes from HD were differentiated in M-CSF and later polarized into M0, M1, and M2 or polarized into TCM with different pancreatic cancer cell lines (BXPC3 and CFPAC). Macrophage feature markers (FPR2, CD163. MARCO, CD206, CD86, and HLA-DR) were measured by flow cytometry analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Close modal

Compared with multiple classical M2 macrophage markers, FPR2 showed a unique sex dimorphism. Following induction of M2-type macrophages in vitro, FPR2 protein expression was about 2 times higher in female-derived macrophages, and similar differential expression was found in TCM, when using pancreatic cancer cell lines (Fig. 2G; Supplementary Fig. S3).

FPR2-activated human macrophages upregulate proteins involved in secreting immunosuppressive molecules–PGE2 and galectin9

Motivated by the above-mentioned finding, we investigated whether targeted inhibition of FPR2 can selectively affect M2 macrophages in female patients with pancreatic cancer. We used pharmacological-specific agonists ACT-389949 (20), henceforth referred to as ACT and Cpd43 (25), and the antagonist WRW4 (26) of FPR2. These were used to treat macrophages derived from monocytes (from PBMC). These three selective small-molecular compounds bound the FPR2-active site and interfered with its activity, while not inducing apoptosis (Supplementary Fig. S4A). ACT treatment induced ERK phosphorylation, whereas WRW4 diminished the pERK signaling, confirming the site binding of FPR2 by these small molecules (Supplementary Fig. S4B). Furthermore, we found that the agonist ACT induced the production of ROS in M2 macrophages compared with M1 and compared with another agonist CpD43 (Supplementary Fig. S4C).

Under the hypothesis that macrophages play a differential role in the immune suppressive TME of different sexes, we compared macrophages between males and females under different treatment conditions using shotgun proteomics to identify and quantify proteins involved in this process. The anti-inflammatory M2 macrophages displayed a differential proteome profile when comparing the two sexes, with the FPR2 agonist ACT treatment further enhancing these differences (Fig. 3A; Supplementary Fig. S5A and S5B). Female untreated M2 macrophages upregulated the expression of several proteins compared with male, where these differences were more clearly observed under ACT treatment (Supplementary Fig. S5C and S5D). These were likewise observed within female M2 after ACT treatment (Supplementary Fig. S5E).

Figure 3.

FPR2+ macrophages upregulate proteins involved in secreting immunosuppressive molecules. A, Heatmap of proteins identified in M2 with and without FPR2 Agonist, ACT treatment. B and C, FunRich quantitative GO analysis in cellular component (B) and its corresponding fold change (C). D, Protein abundance of FPR, PTGES2, LGALS9, TGFB, and ANAXA1 between the sexes among the M2 only and with ACT. Specific q value from n = 4 biological replicates are indicated, respectively. E–G, Biological processes (E), biological pathways (F), and transcription factor quantitative GO analysis (G). All proteins (n = 4 biological replicates each condition and each sex) used in all proteomic analysis are identified with FDR≤0.01 confidence and are a master protein that is present in at least two of three technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

FPR2+ macrophages upregulate proteins involved in secreting immunosuppressive molecules. A, Heatmap of proteins identified in M2 with and without FPR2 Agonist, ACT treatment. B and C, FunRich quantitative GO analysis in cellular component (B) and its corresponding fold change (C). D, Protein abundance of FPR, PTGES2, LGALS9, TGFB, and ANAXA1 between the sexes among the M2 only and with ACT. Specific q value from n = 4 biological replicates are indicated, respectively. E–G, Biological processes (E), biological pathways (F), and transcription factor quantitative GO analysis (G). All proteins (n = 4 biological replicates each condition and each sex) used in all proteomic analysis are identified with FDR≤0.01 confidence and are a master protein that is present in at least two of three technical replicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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To understand how FPR2 could play a role in anti-inflammatory macrophage suppression, we examined the proteins identified and their corresponding abundances (Supplementary Fig. S5F and S5G) using quantitative GO (qGO). The proteins were identified with HUGO HGNC gene symbol for downstream GO analysis. We first inspected the cellular components in qGO, in which female M2 had higher percentages of quantified proteins in the cytosol and lysosome components, with and without ACT treatment (Fig. 3B). Subsequent fold-change qGO analysis indicated cytosol, secretory components, and recycling endosomes to be upregulated in female M2 macrophages (Supplementary Fig. S5H). These findings were supported by the observation that the recycling endosomes were upregulated over 20-fold in female M2 macrophages upon ACT treatment compared with their male counterparts (Supplementary Fig. S5I) and compared with no ACT treatment in females (Fig. 3C). Correspondingly, we also found that female TCM had highly expressed proteins in these cellular components (Supplementary Fig. S5J). These data may suggest that FPR2 involvement in secreting immunosuppressive factors is occurring via the recycling endosomes and exosomes secretion. This observation is consistent with recent report regarding hypoxia-induced hypersecretion of immunosuppressive exosomes in TME (27, 28).

As FPR1 and FPR2 share high sequence homogeneity (29), it was not possible to differentiate the two receptors in shotgun proteomics. Similar to the gene expression investigated from patients with PDAC and the protein expression from in vitro generated M2 macrophages and TCM, high FPR protein abundance was also found in female M2 compared with male M2, with the difference being further increased when treated with ACT (Fig. 3D). Moreover, expression of immunosuppressive secretory proteins such as PGE2 (PTGES2) and galectin-9 (LGALS9) were upregulated in female M2 upon ACT treatment. In addition, TGFβ, and the FPR2 endogenous ligand ANXA1 were also highly abundant and exclusively upregulated in female M2 macrophages following ACT treatment, suggesting a sex-specific FPR2 regulation of these proteins.

To dissect the potential FPR2 immunosuppressive signaling, biological processes and pathways were also studied. We found that female M2 macrophages had approximately 20% more expression in genes involved in cell growth and maintenance upon ACT treatment (Fig. 3E), which was also observed among female TCMs to be mainly involved in cell growth and maintenance (Supplementary Fig. S5K). The integrin pathway was also highly upregulated in female M2 macrophages. Subsequently, qGO demonstrated that female M2 macrophages had a higher percentage of several integrin signaling proteins, as well as CXCR4 and GMCSF-mediated signaling events essential for macrophages (Fig. 3F). We also found about 1.5-fold reduction in proinflammatory signaling like TNF and IFNy upon ACT treatment. Transcription factor qGO analysis then revealed Kruppel-like factor 7 (KLF7) and/or activating transcription factor 3 (ATF3) to be mainly involved in the anti-inflammatory M2 macrophages profile (Fig. 3G). In summary, these data suggest that FPR2 drives the sex-bias in macrophages through upregulation of proteins involved in immunosuppressive pathways.

Targeting FPR2 inhibits the induction of sex-biased antitumor immunity in vitro

Given our transcriptomic and proteomic findings, indicating that FPR2+ macrophages exhibit immunosuppressive properties, we next evaluated their ability to inhibit immune effector cells. To investigate whether FPR2 activation in TME affects immune effector cells and overall antitumor immunity, we constructed a coculture model of either cytokine induced macrophages or TCM (resembling immunosuppressive myeloid cells) and effector killer cells in vitro. To determine the extent to which FPR2-activated macrophages affect T-cell IFNγ production and proliferation, we cocultured CD3+ T cells, also from human peripheral blood, with either M1 or M2 macrophages treated with vehicle, ACT, or WRW4-treated. We found that in these experiments, both male and female anti-inflammatory macrophages displayed high suppression capacity by inhibiting T-cell activities as previously described by our group and others. Notably, inhibition of T-cell activation was associated with increased macrophage FPR2 expression and activities, where agonists and antagonists of FPR2, further regulated macrophage specific suppression of cytotoxic cell IFNγ production and proliferation exclusively in female immune cells (Fig. 4A and B). We observed similar sex-immune dimorphism in T cells cocultured with TCM, where only female cells were highly inhibited (Fig. 4C and D).

Figure 4.

Specific activation of FPR2 in macrophages amplifies immunosuppression of T cells. A and B, A representative flow cytometry overlay plot of male and female T-cell, and cumulative data of quantification of IFNγ+ (A) and Ki67+ (B) in T cells cocultured with cytokine-differentiated macrophages isolated from male and female HD PBMC (male, n = 10; female, n = 10). C and D, A representative flow cytometry plot and cumulative data of quantification of IFNγ+ (C) and Ki67+ (D) in T cells cocultured with TCMs isolated from male and female HD PBMC (males, n = 8; females, n = 8). E and F, A representative flow cytometry plot and cumulative data of quantification of IFNγ+ (E) and CD107a+ (F) in NK cells cocultured with cytokine-differentiated macrophages isolated from male and female HD PBMC (males, n = 8; females, n = 8). G and H, Quantified Ki67+, percentage, and IL10+ of Tregs in total T cells cultured with or without macrophages. Tregs was identified as CD4+CD25+FOXP3+ cells in CD3+ T cell. I, Frequency and IFNγ production of CD8+ Tc and CD4+ Tc frequency and proliferation (Ki67) of total T cells assessed in cultures with or without macrophage. All macrophages first underwent differentiation and were subsequently polarized by different combinations of cytokines or tumor cells before cocultures. Addition of ACT or WRW4 to activate or inhibit FPR2 during coculture of T cells and macrophages. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

Specific activation of FPR2 in macrophages amplifies immunosuppression of T cells. A and B, A representative flow cytometry overlay plot of male and female T-cell, and cumulative data of quantification of IFNγ+ (A) and Ki67+ (B) in T cells cocultured with cytokine-differentiated macrophages isolated from male and female HD PBMC (male, n = 10; female, n = 10). C and D, A representative flow cytometry plot and cumulative data of quantification of IFNγ+ (C) and Ki67+ (D) in T cells cocultured with TCMs isolated from male and female HD PBMC (males, n = 8; females, n = 8). E and F, A representative flow cytometry plot and cumulative data of quantification of IFNγ+ (E) and CD107a+ (F) in NK cells cocultured with cytokine-differentiated macrophages isolated from male and female HD PBMC (males, n = 8; females, n = 8). G and H, Quantified Ki67+, percentage, and IL10+ of Tregs in total T cells cultured with or without macrophages. Tregs was identified as CD4+CD25+FOXP3+ cells in CD3+ T cell. I, Frequency and IFNγ production of CD8+ Tc and CD4+ Tc frequency and proliferation (Ki67) of total T cells assessed in cultures with or without macrophage. All macrophages first underwent differentiation and were subsequently polarized by different combinations of cytokines or tumor cells before cocultures. Addition of ACT or WRW4 to activate or inhibit FPR2 during coculture of T cells and macrophages. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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Here, we have determined that FPR2 is predominantly expressed by the CD11b+ cell population, with little or no expression of FPR2 in CD3+ T cells. To further exclude the possible influence of FPR2 treatment on the function of T cells themselves, and to investigate the influence of other types of macrophages, we also set up a T-cell–only culture control, and a further control coculture with T cells and M1 macrophages. We confirmed that the activation and blocking of FPR2 by ACT and WRW4 did not affect the functions of T cells and M1 macrophages (Supplementary Fig. S6A and S6B).

Considering the partial similarity in NK-cell and CD8+ T-cell signaling pathways, we also treated NK cells from peripheral blood of HD in the same way and assessed for IFNγ production and cytolytic activity measured by degranulation (CD107a). We observed that female NK cells also displayed stronger immunosuppression following the functional induction of FPR2 on M2 macrophages, and that NK cells themselves were not affected by low concentrations of agonists and antagonists (Fig. 4E and F; Supplementary Fig. S6C and S6D). Following CD3+T-cell coculture with M2 macrophages, the number of cells and the proportion of various T-cell subtypes were assessed. We detected an increased trend in both the proliferation and proportion of female regulatory T cells (Treg) in the T-cell population following ACT treatment (Fig. 4G; Supplementary Fig. S7A). In addition, Tregs expressed significantly higher IL10 in the ACT treatment group compared with controls only in women (Fig. 4H). At the same time, we observed a significant reduction of the frequency and function of CD8+ and CD4+ T cells in the ACT treatment group (Fig. 4I; Supplementary Fig. S7B and S7C). Therefore, female-derived macrophages expressing FPR2 can effectively block effector cell cytotoxicity and inhibit cytokine production and proliferation. In addition, these immunosuppressive FPR2+ macrophages not only inhibited effector cells but extended to promoting the activation of immunosuppressive components.

T-cell exhaustion induced by macrophage FPR2 activation

Because our results have shown that T-cell function is highly correlated with the regulation of FPR2, we paid particular attention to whether the process of T-cell exhaustion is affected by the sex-specific regulation of FPR2. From the TCGA data of patients with pancreatic cancer, we found that higher expression of T-cell exhaustion defined by TIM3 and PD1 signatures, is likely to predict worse prognosis in women, but not in men (Fig. 5A and B). In the pancreatic cancer cohort, the survival characteristics of T-cell exhaustion markers were similar to FPR2. Having found immuosuppressive ligands that can affect Tim3 and PD1 expression, namely PGE2 and Gal9, we suspected that T-cell exhaustion may be an important pathway through which FPR2-mediated macrophages inhibit T-cell function. Indeed, in a variety of TCGA tumors, we found a strong correlation between the molecular signature of M2 macrophages (MRC1, CD163, CD68, FPR2, and ARG1) and that of T-cell exhaustion (HAVCR2, TIGIT, PDCD1, LAG3, LAYN, CXCL13; Supplementary Fig. S7D). We used our in vitro coculture model to assess T-cell exhaustion in male and female cells. These cultures revealed that both CD4+ and CD8+T cells showed characteristics of T-cell exhaustion. With ACT treatment, anti-inflammatory macrophages induced upregulation of PD1 and TIM3 expression levels in CD8+T cells. Either PD1 or TIM3 expression were significantly increased in female-derived T cells following ACT treatment (Fig. 5C and D). In addition, we also found that female TCM had the ability to induce upregulation of the expression of PD1 and TIM3 in T cells compared with male T cells (Fig. 5E).

Figure 5.

PGE2- and galectin9-mediated T-cell exhaustion is essential for FPR2-mediated sex-biased immunosuppression. A, Kaplan–Meier survival curves comparing Tc exhaustion-related gene (PD1&Tim3) mRNA high and low expression levels in PAAD. B, Kaplan–Meier survival curves comparing Tc exhaustion-related gene (PD1&Tim3) mRNA high and low expression levels in PAAD in women (left) and men (right), respectively. C and D, %PD1 (C) or %Tim3 (D)-positive CD8 and CD4 T cells cocultured with polarized macrophages or alone. E, PD1+ Tcs (left) and Tim3+ Tcs (right) were analyzed by flow cytometry following coculture with macrophages or TCM. F, Female and male Tc function was evaluated following coculture with macrophages in a direct cell-to-cell contact indirectly separated by Transwell inserts. G, RT-qPCR screen for representative immune regulatory factors indicated in male and female human macrophages in presence or absence of ACT and WRW4 treatment. H, Frequency of IFNγ+ (left), TNFα+ (middle), and Ki67+ Tc (right) of CD3+ T cells cultured with M2 macrophages performed in the presence or absence of FPR2 agonist (ACT) and immune checkpoint therapy (PD1 and Tim3 block antibodies). T cells were CellTrace labeled and added to the tumor–macrophage spheroids in the presence of indicated treatments, and the relative tumor killing (I) and T-cell infiltration (J) were monitored by hourly live imaging over 30 hours using IncuCyte Live-Cell Analysis System. Pooled data from either female or male immune cells (n = 4) are presented as mean + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

PGE2- and galectin9-mediated T-cell exhaustion is essential for FPR2-mediated sex-biased immunosuppression. A, Kaplan–Meier survival curves comparing Tc exhaustion-related gene (PD1&Tim3) mRNA high and low expression levels in PAAD. B, Kaplan–Meier survival curves comparing Tc exhaustion-related gene (PD1&Tim3) mRNA high and low expression levels in PAAD in women (left) and men (right), respectively. C and D, %PD1 (C) or %Tim3 (D)-positive CD8 and CD4 T cells cocultured with polarized macrophages or alone. E, PD1+ Tcs (left) and Tim3+ Tcs (right) were analyzed by flow cytometry following coculture with macrophages or TCM. F, Female and male Tc function was evaluated following coculture with macrophages in a direct cell-to-cell contact indirectly separated by Transwell inserts. G, RT-qPCR screen for representative immune regulatory factors indicated in male and female human macrophages in presence or absence of ACT and WRW4 treatment. H, Frequency of IFNγ+ (left), TNFα+ (middle), and Ki67+ Tc (right) of CD3+ T cells cultured with M2 macrophages performed in the presence or absence of FPR2 agonist (ACT) and immune checkpoint therapy (PD1 and Tim3 block antibodies). T cells were CellTrace labeled and added to the tumor–macrophage spheroids in the presence of indicated treatments, and the relative tumor killing (I) and T-cell infiltration (J) were monitored by hourly live imaging over 30 hours using IncuCyte Live-Cell Analysis System. Pooled data from either female or male immune cells (n = 4) are presented as mean + SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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Targeting FPR2+ macrophages prevents T-cell exhaustion and immunosuppression

Our previous study found that targeting TAM is an efficient way to activate TAM reprogramming in mice, and reduced tumor growth has been observed in several tumor models (30). To further clarify the possible pathway that anti-inflammatory macrophages may mediate in T-cell dysregulation, we used Transwell inserts to separate T cells from macrophages for coculture, and at the same time compared with direct contact coculture. As showed above, we found that M2 macrophages statistically inhibited IFNγ expression of T cells in both male and female cultures and that ACT further augmented the M2 suppression capacity that was restored by WRW4 treatment in females. Here, we show that such suppression capacity was not influenced by separating the T cells to only allow for soluble factor exchange with M2 macrophages (Fig. 5F). The results suggest that the anti-inflammatory macrophages mediate T-cell inhibition, mainly through secreted factors. To verify our findings, we further screened the soluble molecules that have been reported to induce T-cell dysfunctionality and that were identified in our proteomic analysis. Following the induction of macrophages in vitro, and treatment with either ACT or WRW4, we assessed candidate gene expression using quantitative PCR. It was found that genes encoding for COX2 and galectin9 were regulated by FPR2 activation, whereas the expression was significantly different between the two sexes, confirming the sex-dimorphism effect (Fig. 5G). Subsequently, exogenous Galectin-9 and PGE2 were used to treat T cells cultured in vitro at different concentrations. The results showed that PGE2 and Galectin-9 had the ability to induce T-cell exhaustion (Tim3) in a concentration-dependent manner (Supplementary Fig. S7E). Considering clinical applicability, we designed coculture experiments with macrophages and T cells where we blocked PD1 and Tim3 pathways. These experiments uncovered that blocking PD1 and Tim3 alone or combined, restored T-cell activation to varying degrees. Given that ACT treatment induced stronger T-cell inhibition in females, we combined ACT with anti-PD1 treatment and found that male, but not female T cells were activated. On the other hand, when ACT was combined with anti-Tim3 treatment or double-blocking with PD1-TIM3, the T-cell activation in females was restored (Fig. 5H). Furthermore, we investigated the T-cell capacity to infiltrate and kill 3D tumor spheroids from male and female donors over a time course of 36 hours in live imaging. We found that anti-Tim3 alone or in combination with anti-PD1 was able to restore T-cell infiltration and antitumor activity only in females in ACT treated cultures. Anti-PD1 and anti-TIM3 alone or in combination induced a transient restoration of T-cell antitumor activity (Fig. 5I and J). In summary, our results suggest that elevated Tim3 expression on T cells mediated by FPR2 activation of macrophages is the primary cause of higher T-cell exhaustion in women in contrast with men.

Bone marrow–derived macrophages from WT c57BL/6N mice show greater immunosuppression than macrophages from FPR2−/− mice, especially in females

TAMs have long been thought to play a role in accelerating immune escape, leading to tumor progression and metastasis. To study the function of FPR2-expressing macrophages in vivo, FPR2 KO mice were used. Monocytes were isolated from bone marrow and CD8+ T cells were isolated from spleens for coculture. Polarized macrophages were then cultured in vitro with autologous T cells (Fig. 6A). In both male and female mice, anti-inflammatory macrophages showed clear immunosuppressive characteristics, where IFNγ and TNFα production, and Ki67 expression (proliferation) were downregulated in T cells. Female-derived FPR2−/− anti-inflammatory macrophages were not able to induce immunosuppression in female T cells, whereas male cells were capable of inducing immunosuppression (Fig. 6BD). Importantly, female-derived FPR2−/− anti-inflammatory macrophages induce lower PD1 and Tim3 in T cells (Fig. 6E and F). These results replicate findings from human peripheral blood and in FPR2−/− murine-derived immune cells, where female FPR2+ macrophages mediate a sex-specific immunosuppression and T-cell exhaustion.

Figure 6.

FPR2−/− c57BL/6 mice bone marrow–derived macrophages showed greater immunosuppression, especially in females. A, Schematic diagram of a coculture model of bone marrow macrophages and splenic lymphocytes of FPR2−/− and WT c57BL/6N mice in vitro. B–D, Splenic T-cell activation of IFNγ (B) and TNFα (C) and proliferation of Ki67 (D) were measured following coculture with macrophages from WT and FPR2−/− mice. Cumulative data are shown as mean ± SEM. E and F, Splenic T-cell exhaustion markers PD1 and TIM3 were analyzed by flow cytometry. Mean ± SEM of percentage and mean fluorescence intensity-isotype control (MFI) is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

FPR2−/− c57BL/6 mice bone marrow–derived macrophages showed greater immunosuppression, especially in females. A, Schematic diagram of a coculture model of bone marrow macrophages and splenic lymphocytes of FPR2−/− and WT c57BL/6N mice in vitro. B–D, Splenic T-cell activation of IFNγ (B) and TNFα (C) and proliferation of Ki67 (D) were measured following coculture with macrophages from WT and FPR2−/− mice. Cumulative data are shown as mean ± SEM. E and F, Splenic T-cell exhaustion markers PD1 and TIM3 were analyzed by flow cytometry. Mean ± SEM of percentage and mean fluorescence intensity-isotype control (MFI) is shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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FPR2+ macrophages promote tumor growth in vivo

Although the tumor-promoting role of TAMs in TME is well known, data from targeted TAM reprogramming and the resulting effects on the pancreatic cancer microenvironment are still ambiguous especially in different sexes. Some studies have shown that FPR2 is highly expressed in a variety of human cancers through immunohistochemical staining and is associated with tumor progression (14, 31). We evaluated the growth of the pancreatic cancer cells subcutaneously injected in FPR2−/− and WT mice. To validate our observations in a more clinically relevant experimental system, we used the murine KPC cell line, which was derived from a genetically engineered PDAC mouse model, in syngeneic, that is, immunocompetent mouse model (Fig. 7A). A significant reduction of tumor growth was observed in female FPR2−/− mice compared with WT female mice (Fig. 7B). Notably, there was no significant difference in tumor growth between male FPR2−/− and WT mice (Fig. 7C).

Figure 7.

FPR2 regulates myeloid function and suppresses antitumor T-cell response in the tumor microenvironment. A, Schematic representation of subcutaneous tumor models in the FPR2−/− and WT C57BL/6N mice, including subcutaneous injection of KPC cells and FPR2−/− or WT macrophages in WT mice. B and C, Tumor growth curves of female (WT, n = 16; FPR2−/−, n = 16; B) and male (WT, n = 16; FPR2−/−, n = 14; C) mice are shown separately. D–G, The percentage of Treg (D), CD4+ T (E), CD8+ T (F), and CD206+ macrophages in total F4/80+CD11b+ macrophages (G) in spleens or in tumors. H, The percentage of IFNγ, Ki67, and TNFα detected in KPC tumor-infiltrating CD8+ T cells. I, The percentage of PD1 and Tim3 detected in KPC tumor-infiltrating CD8+ T cells. Accumulative data are shown as mean ± SEM. J and K, Tumor growth curves following inoculation of tumors and FPR2−/− or WT macrophages in male (WT, n = 10; FPR2−/−, n = 10; J) and female (WT, n = 10; FPR2−/−, n = 10; K) mice are shown separately. *, P < 0.05; **, P < 0.01.

Figure 7.

FPR2 regulates myeloid function and suppresses antitumor T-cell response in the tumor microenvironment. A, Schematic representation of subcutaneous tumor models in the FPR2−/− and WT C57BL/6N mice, including subcutaneous injection of KPC cells and FPR2−/− or WT macrophages in WT mice. B and C, Tumor growth curves of female (WT, n = 16; FPR2−/−, n = 16; B) and male (WT, n = 16; FPR2−/−, n = 14; C) mice are shown separately. D–G, The percentage of Treg (D), CD4+ T (E), CD8+ T (F), and CD206+ macrophages in total F4/80+CD11b+ macrophages (G) in spleens or in tumors. H, The percentage of IFNγ, Ki67, and TNFα detected in KPC tumor-infiltrating CD8+ T cells. I, The percentage of PD1 and Tim3 detected in KPC tumor-infiltrating CD8+ T cells. Accumulative data are shown as mean ± SEM. J and K, Tumor growth curves following inoculation of tumors and FPR2−/− or WT macrophages in male (WT, n = 10; FPR2−/−, n = 10; J) and female (WT, n = 10; FPR2−/−, n = 10; K) mice are shown separately. *, P < 0.05; **, P < 0.01.

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Subsequently, spleens and tumors from the subcutaneous tumor experiments described above were harvested and analyzed with multicolor flow cytometry for immune infiltration and characteristics. In WT compared with FPR2−/− female mice, Treg, CD4+ T cells were significantly increased, the percentage of CD206+ macrophages in total macrophages was upregulated, whereas CD8+ T cells were significantly downregulated, within the tumor environment. In male mice, FPR2−/− compared with WT only showed a significant increase in CD8+ T (Fig. 7DG). The immune cells in the spleen were also collected as a control, and the results showed that there was no significant difference in the composition of immune cells in the spleen confirming the importance of the local environment. To further examine the functional inhibition of T cells by FPR2+ macrophages in vivo, we examined the functional status of tumor-infiltrating T cells and the characteristics of T-cell exhaustion. Female FPR2−/− mice expressed higher levels of IFNγ in CD8+ T cells than male FPR2−/− mice, whereas TNFα and Ki67 only had a marginal increase. However, compared with WT mice, FPR2−/− mice exhibited significantly stronger CD8+ T-cell activity, in females but not males (Fig. 7H). Unlike in vitro assays, CD4+ T-cell exhaustion did not appear within the tumor environment (Supplementary Fig. S8A and S8B). Similar to in vitro, T-cell exhaustion was significantly prevented in FPR2−/− female mice (Fig. 7I). In addition, FPR2−/− mice had increased granulocytic (g)MDSC/neutrophils and decreased monocytic (m)MDSC ratios in both females and males (Supplementary Fig. S8C).

Later, we isolated monocytes from bone marrow of WT and FPR2−/− mice, induced macrophages in vitro, mixed with KPC tumor cells, and performed cross-paired adoptive transfer in the same-sex of WT mice, to create a subcutaneous injected tumor model. In this experiment, we considered whether the sex-dependent immunosuppressive characteristics of FPR2+ macrophages were depended on the systemic or local environment. Compared with WT, FPR2−/− macrophages did not impair tumor growth in male mice, whereas FPR2−/− macrophages did in female WT mice (Fig. 7J and K; Supplementary Fig. S8D). Notably, CD8 T-cell activity in form of IFNγ production reverse correlated with the tumor growth (Supplementary Fig. S8E), confirming our hypothesis that targeting FPR2 results in T-cell activation and impairment of tumor progression.

Overall, the subcutaneous tumor experiments in FPR2−/− mice showed that FPR2−/− female mice had a strong antitumor immune response.

Immunotherapy has developed rapidly in the last two decades and has gradually become an effective treatment method that can be operated in the clinic by regulating the switch of the immune system to fight tumors. Because of the diversity and specificity of tumors themselves, the characteristics of the tumor immune environment vary greatly. In the face of advanced cancers, immunotherapy is often used as a standard therapy for patients who cannot undergo surgery or radical radiotherapy. In pancreatic cancer, more than 70%–80% of the patients cannot undergo surgery; however, immunotherapy did not show any advantage. This observed immunotherapy disappointment in pancreatic cancer has been associated with a complex immunosuppressive environment. Therefore, the development of new targets for immunotherapy and the promotion of effective combination therapies are key and difficult points in PDAC research.

There remains a huge knowledge gap about the differential response to immunotherapy between sexes. We have previously identified pancreatic cancer's ability to regulate myeloid cells, transforming them into MDSC and TAMs. Here, in female myeloid-suppressor cells, we found high expression of a GPCR. In addition, we found that in in vivo and in vitro experiments, such macrophages with high expression of FPR2 showed a strong immunosuppressive property and promoted the inhibition of T cells and NK cells through IL10 and PGE2 release, ROS activation, and other mechanisms. We visualized the spatial relationships between anti-inflammatory TAM subsets and effector T cells, and tumor cells in human pancreatic cancer tumors and found differences in the spatial structure and cellular composition of the TME itself between males and females. Using cytokine generated and TCM, we demonstrate that FPR2+ macrophages were associated with an immunosuppressive phenotype. Through the above in vitro experiments, we investigated the male and female human peripheral blood derived macrophages. We found that FPR2 activation caused functional and phenotypic differences in macrophages of the two sexes associated with immunosuppressive characteristics. This indicates that FPR2 is an important sex-difference "switch" in macrophages, and that it can be targeted for reprogramming to alter macrophage suppressive function.

Targets of T cells in the TME are considered promising immunotherapy approaches. At present, it is known that many immunotherapies will encounter T-cell or NK-cell exhaustion in clinical treatment, resulting in poor prognosis of patients with cancer and failure to achieve the predicted therapeutic effect (32). Human pancreatic cancer is a typical immune "cold" tumor, with poor differentiation and hypoxia in the central region of the tumor, few blood vessels observed in histological examination, possibly local nerve infiltration, abundant interstitial tissue in the tumor, and high stiffness of the tumor extracellular matrix (33). The complex TME results in a very poor immune microenvironment for human advanced pancreatic cancer. The highly infiltrating immunosuppressive cells (MDSC, M2-type macrophages, N2 neutrophils, Treg, etc.) and exhausted T cells (Tex) in cold tumors have extensive biological relationship (34). Mechanistically, here we show that sex-differential activation of FPR2 in macrophages in the TME mediates sex-dimorphic T-cell exhaustion gene and protein expression of the PGE2 and galectin9.

The expression of FPR2 has been linked to tumor progression, metastatic dissemination, activation of cancer stem cell compartment, and activation of TME components in pancreatic cancer (35, 36). FPR2 is also known to be expressed in human inflammatory cells, including DCs and monocytes. It is noteworthy mentioning that Belvedere and colleagues (37) have proposed that the phenotype of ANXA1-driven pancreatic cancer progression is dependent of the FPR2 pathway. In contrast, Liu and colleagues showed that FPR2-KO mice bearing subcutaneously implanted Lewis lung carcinoma cells exhibited significantly worse survival. These discrepancies between the mentioned studies can be explained by the type of cancer studied and notably the sex differences. Although Liu and colleagues used male mice for their investigations, all the other studies, including ours observed the FPR2 differential functional in the TME of female subjects. Our data suggest that FPR2 is more likely to play an important regulatory role in the TME of female patients with pancreatic cancer. FPR2 is not a sex-related gene linked to the sex chromosome, rather located on chromosome 19q. Previous studies have suggested that sex-driven immune system differences are not necessarily linked to sex chromosomes but are regulated through a combination of hormones and genetic and epigenetic factors. It is worth mentioning that FPR2 is highly expressed in hepatocytes and healthy livers from females than males and is downregulated with estrogen reduction. FPR2 deletion exacerbated liver damage in female mice, highlighting the anti-inflammatory function of FPR2 (38). FPR2 was first recognized to mediate extracellular ROS release from neutrophils, whose activator, WKYMVM hexapeptide activates NADPH-oxidase in murine neutrophils much stronger than the formylated peptide, formyl-Met-Leu-Phe (39). Previously, Lind and colleagues (20) have verified in human peripheral blood-derived neutrophils that the FPR2-selective agonist, ACT, can induce an FPR2-dependent and Gαq-independent transient rise in intracellular Ca2+ and recruitment of β-arrestin (20, 40). Here, we first show that the FPR2 high-affinity agonist, ACT, stimulates the release of ROS and induction of PGE2 and galectin9 in TCM. We also propose several immunosuppressive pathways that can account for the sex differences in macrophages.

The hostile TME is a major challenge for the treatment of solid tumors, reducing the efficacy of immunotherapies, including adoptive cell therapy and immune checkpoint inhibitors (41, 42). In recent years, more studies have found that the complex immunosuppressive environment of tumors may mediate T-cell dysfunction and exhaustion by upregulating or/and activating immune checkpoint receptors on T cells (43). Meanwhile, some inflammatory cytokines (including IL10, TGFβ, IL12, IL2, galectin9, etc.) have been found to stimulate the exhaustion of CD8 T cells to varying degrees, but the source of cytokine expression is not clear (44–48). PGE2/EP2 signaling has been reported to directly upregulate the level of PD-1 in infiltrating CD8+ T cells in patients with lung cancer (49). In addition, PGE2 has also been repeatedly reported to increase PD-L1 expression in the TME (50, 51). Here, our results clearly show that FPR2+ macrophages are the source of these immunosuppressive factors. As a ligand of Tim3, galectin9 has the function of directly activating T-cell inhibitory receptors. Yang and colleagues (48) proposed that galectin9 also binds PD1, thereby attenuating the persistent apoptosis signaling caused by interaction with TIM3 and contributes to the persistence of PD-1+TIM-3+ T. Here, we stimulated activated T cells using human recombinant proteins in vitro and found that PGE2 and galecin9 induced T-cell exhaustion in a dose-dependent manner. T-cell exhaustion was rescued by blocking the macrophage FPR2 pathway in combination with blocking the PD1 and Tim3 pathways. Also, the FPR2+ macrophages were predominantly/completely dependent on the Tim3 pathway to induce female T-cell exhaustion.

A recent article from our group, elicits the impact of sex-specific characterization of the immune component of the TME on disease characterization and prognosis (52). Although clinicians and researchers have long recognized that sex-specific factors broadly and deeply affect disease progression, it has been difficult to find sex-specific targets because of the susceptible gender and confounding factors. After neoadjuvant chemotherapy in patients with PDAC, the degree of CD204+ TAM declined in female compared with male patients (53). These results suggest that there are differences in the composition of the immune microenvironment in male and female patients with pancreatic cancer. Here, we used multicolor immunofluorescence staining to evaluate immune component infiltration, demonstrating the complexity of the immune environment of pancreatic cancer. Although they were all cold tumors, the immune infiltration characteristics of males were closer to "desert cold tumor" than females, characterized by low T-cell and TAM infiltration.

Conclusion

Our study indicates that reprogramming of TAMs represents a strategy to rescue T-cell exhaustion in the tumor immune microenvironment and to convert the immunosuppressive milieu. These mechanisms may include consideration of immunotherapy strategies for pancreatic cancer by stratifying according to patient sex. In addition, our results suggest that modulation of the immune environment by synthetic drugs may reprogram women's immunosuppressive TME and confer potential immune checkpoint therapeutic opportunities. Meanwhile, our results suggest that immune checkpoint therapy for PD1 and TIM3 may have a strong sex-bias due to the sex-differential immunosuppressive microenvironment of pancreatic cancer. In summary, our study revealed evidence that sex-driven differences in immune surveillance affect the determinants of tumor progression and the landscape of the TME, and likely mediate the dimorphic outcomes with immunotherapy. Deeper understanding of the selective pressures and mechanisms of immune escape in tumors in males and females can inform patient selection strategies and can be used to further improve immunotherapy approaches in cancer.

P. Olofsson-Sahl reports grants and nonfinancial support from Redoxis AB, Lund Sweden during the conduct of the study and also outside the submitted work, as well as grants from Pronoxis AB. P. Olofsson-Sahl reports a patent application pending and is a co-founder and employee of Pronoxis AB, which has a commercial interest in developing FPR agonists. D. Sarhan reports grants from Region Stockholm CIMED, Swedish Cancer Society, KI Stiftelser och Fonder, and China Scholarship Council during the conduct of the study, as well as nonfinancial support from Pronoxis outside the submitted work. D. Sarhan reports a patent for anti-FPR2 antibodies pending. No disclosures were reported by the other authors.

F. He: Conceptualization, data curation, software, formal analysis, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. A.H.M. Tay: Data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. A. Calandigary: Data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. E. Malki: Data curation, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. S. Suzuki: Data curation, formal analysis, validation, writing–review and editing. T. Liu: Software, formal analysis, validation, investigation, visualization, writing–review and editing. Q. Wang: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. C. Fernández-Moro: Data curation, software, formal analysis, validation, investigation, writing–review and editing. M. Kaisso: Data curation, formal analysis, writing–review and editing. P. Olofsson-Sahl: Resources, validation, writing–review and editing. M. Melssen: Data curation, formal analysis, writing–review and editing. S.K. Sze: Resources, data curation, software, validation, writing–review and editing. M. Björnstedt: Resources, investigation, writing–review and editing. M.J. Löhr: Resources, investigation, writing–review and editing. M.C.I. Karlsson: Resources, data curation, supervision, writing–review and editing. R. Heuchel: Resources, data curation, formal analysis, funding acquisition, validation, investigation, writing–original draft, writing–review and editing. D. Sarhan: Conceptualization, resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

The authors would like to thank Dr. I. Moore at NIH/NIAID/Infectious Disease Pathogenesis Section for providing guidance in immune fluorescence staining of the paraffin-embedded tumor tissues. The authors thank Dr. A. Samson for critical feedback and corrections of the article. This study was supported by Centrum for Innovative Medicine, FoUI-963251, KI Stiftelser och Fonder, 2020–01829, and Swedish Cancer Society 200169F (to D. Sarhan), China Scholarship Council 201906280459 (to F. He), Swedish Cancer Society (to M.C.I. Karlsson), Swedish Cancer Society 201356Pj (to R. Heuchel), and Horizon 2020 NEUTROCURE 861878 (to P. Olofsson-Sahl).

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

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

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