Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism

Abstract

HER2+ breast tumors have abundant immune-suppressive cells, including M2-type tumor-associated macrophages (TAMs). Although TAMs consist of the immune-stimulatory M1 type and immune-suppressive M2 type, the M1/M2-TAM ratio is reduced in immune-suppressive tumors, contributing to their immunotherapy refractoriness. M1- versus M2-TAM formation depends on differential arginine metabolism, where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- versus M2-TAMs is attributed to different availability of BH₄ (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently, we reported that sepiapterin (SEP), the endogenous BH₄ precursor, elevates the expression of M1-TAM markers within HER2+ tumors. Here, we show that SEP restores BH₄ levels in M2-like macrophages, which then redirects arginine metabolism to NO synthesis and converts M2 type to M1 type. The reprogrammed macrophages exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in the metabolic shift of the HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy.

Figure 1.. M1- versus M2-TAMs are distinguished by the preferential production of NO versus PAs through differential arginine metabolism.

(A, B) Schematic representation of the polarization protocols of TAMs derived from the THP-1 human monocytic cell line (A) and PBMC (B). THP-1 monocyte (Mn) was treated with phorbol myristate acetate for differentiation into inactive macrophages (M₀). PBMC-derived Mn was treated with SCF+GM-CSF (leading to M1) or M-CSF (leading to M2) for differentiation into M₀. For M1 polarization, M₀-TAMs were treated with LPS and IFNγ; for M2 polarization, M₀-TAMs were treated with IL4 plus IL13. (C) Representative images of phalloidin staining (left, n = 3) and scanning electron microscopy (SEM, right, n = 3) imaging of M0-, M1-, and M2-TAMs. Green: phalloidin; blue: DAPI. SEM images are shown at different magnifications: M0: x1,300; M1: x3700; and M2: x500. (D) Immunofluorescence images of TAM subsets (n = 3) stained for M1 (green, TNFα) versus M2 markers (red, CD206) and counterstained with DAPI (blue). Scale bars: 50 μm. (E) Western blot analysis (n = 3) of THP-1–derived Mn-, M0-, M1-, and M2-TAMs for the expression of TLR2 (M1 marker) versus CD206 (M2 marker). β-Actin was used as the internal loading control. (F) Quantification of the Western blot results of the expression of TLR2 (left) and CD206 (right) normalized against β-actin and presented as fold differences. (G) NO-to-PA ratios in THP-1–derived TAM subsets (n = 6) measured with ELISA. Error bars: ±SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05.

Figure 2.. SEP elevates M1 marker expression in M2-TAMs.

(A) Levels of BH₄ produced by THP-1–derived M0-, M1-, and M2-TAMs after being treated with vehicle (DMSO) or SEP (100 μM) for 3 d (n = 6). BH₄ levels were measured with ELISA and normalized against the total protein levels. One-way ANOVA with a post hoc test (Tukey’s test) was performed to measure the significance of the mean difference between treatment groups. (A, B) Phalloidin staining and SEM imaging of THP-1–derived M0-, M1-, and M2-TAMs treated as in (A) (n = 3). SEM images are shown at different magnifications. (A, C) Western blot analysis of TAM subsets treated with a vehicle or SEP as in (A), and β–actin was used as the internal loading control (n = 5). (D) Quantification of the Western blot results based on the expression of TLR2 (M1 marker) versus CD206 (M2 marker) normalized against β–actin signal and presented as fold differences. (E) Immunofluorescence imaging of THP-1–derived TAMs after treatments as shown above, and stained for an M1 marker (green, TNFα) versus M2 marker (red, CD206) and counterstained with DAPI (blue) (n = 3). (F) Levels of secreted cytokines, type 1: TNFα (top left), IL1β (top right), and IL6 (bottom left) versus type 2: TGFβ (bottom right), for M1- versus M2-TAMs treated with a vehicle versus SEP (n = 6) measured with ELISA. Error bars: ±SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05.

Figure 3.. SEP redirects arginine metabolism from PA to NO synthesis in M2-TAMs, while rendering them an M1-TAM phenotype.

(A) Levels of NO (left), PAs (middle), and NO/PA ratios for THP-1–derived M0-, M1-, and M2-TAMs after being treated with DMSO (vehicle) and SEP (100 μM) for 3 d (n = 5). (B) One-way ANOVA with post hoc Tukey’s test was used for statistical analysis. Error bars: ±SEM. GraphPad Prism version 9.5.1. was used to perform all statistical analyses. (B) Levels of NO (left), PAs (middle), and NO/PA ratios for THP-1–derived Mn-, M0-, M1-, and M2-TAMs after being treated with DMSO (vehicle), NOS2 inhibitor, 1400W (50 μM), or arginase 1 (Arg1) inhibitor, nor-NOHA (50 μM), for 3 d (n = 5). Note the significant decrease of the NO level in 1400W-treated M1-TAMs and the significant decrease of the PA level in nor-NOHA–treated M2-TAMs. (C) Immunofluorescence imaging of THP-1–derived M0-, M1-, and M2-TAMs stained for an M1 marker (green, TNFα) versus M2 marker (red, CD206) and counterstained with DAPI (blue). M1-TAMs were treated with DMSO (control: Ctrl) or NOS2 inhibitor (100 μM 1400W), whereas M2-TAMs were treated with DMSO (Ctrl) or ARG1 inhibitor (50 μM nor-NOHA) for 3 d (n = 3). (D) Levels of type 1 cytokine IL12 (left) and type 2 cytokine IL10 (middle), as well as IL12/IL10 ratios for THP-1–derived TAM subsets measured with ELISA. M1-TAMs were treated with DMSO or NOS inhibitors, 1400W (50 μM) and L-NAME (2.5 mM). M2-TAMs were treated with DMSO, SEP (100 μM), or positive control LPS (5 ng/ml) plus IFNγ (20 ng/ml) for 3 d (n = 6). The cytokine levels were measured using ELISA and normalized against the total protein levels. Error bars: ±SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05. (E) Working scheme for the induction of M1 versus M2 polarization by activation of NOS2 versus ARG1/OCD1 pathways and M2-to-M1 reprogramming by SEP.

Figure 4.. Arginine metabolites, nitric oxide and polyamines, drive M1- and M2-TAM polarization, respectively.

(A) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1_S727 versus M2 marker: CD206 in THP-1–derived M0-TAMs treated with DMSO (vehicle control), SEP (100 μM), or NO donor [GSNO (100 and 200 μM) and SNAP (10 and 20 μM)] in comparison with M1- and M2-TAMs (n = 4). GAPDH was used as the internal loading control. (B) Western blot analysis on M1 markers: STAT1 and TLR2 versus M2 marker: CD206 in M0-TAMs treated with DMSO (vehicle control), SEP (100 μM), or PAs (5 or 7.5 mM spermine) in comparison with M1- and M2-TAMs (n = 4). (A, B) β-Actin was used as the internal loading control. (For quantification of (A, B), see Appendix Fig S1.) (C) Ratios of IL12 to IL10 secreted by THP-1–derived M0-TAMs treated with DMSO, SEP, NO donors, and PAs in comparison with M1- and M2-TAMs. (D) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1_S727 in M1-TAMs treated with NO scavenger (50 μM cPTIO) with and without NO donor (100 μM GSNO) and M2-TAMs treated with DMSO or SEP (n = 4). GAPDH was used as the internal loading control. (E) Western blot analysis on M1 markers: TLR2, STAT1, and pSTAT1_S727 versus M2 marker: CD206 in THP-1–derived M2-TAMs treated with a PA analog (50 and 100 μM DENSPM) and PAs (5 mM spermine) and M1-TAMs treated with DMSO or SEP (n = 4). (D, E) β-Actin was used as the internal loading control. (For quantification of (D, E), see Appendix Fig S2.) (D, E, F) Ratios of IL12 to IL10 secreted by THP-1–derived M1- and M2-TAMs with treatment combinations shown in (D, E). Error bars: ± SEM. GraphPad Prism version 9.5.1. was used to perform all statistical analyses. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05. (G) Scheme for the induction of M1 versus M2 polarization by NO versus PAs and M2-to-M1 reprogramming by SEP.

Figure S1.. Arginine metabolites, nitric oxide and polyamines, drive M1 and M2 macrophage polarization, respectively.

(A) Quantification of Western blots (Fig 4A) detecting M1 markers, TLR2, pSTAT1, STAT1, and M2 marker, CD206, in different subtypes of THP-1–derived macrophages (M0, M1, and M2 types) treated with DMSO (vehicle control), SEP (100 μM), or NO donor (GSNO [100 and 200 μM] and SNAP [10 and 20 μM]) (n = 4). GAPDH was used as the internal loading control. (B) Quantification of Western blots (Fig 4B) detecting M1 markers: STAT1 and TLR2, versus M2 marker: CD206 in THP-1–derived macrophages (M0, M1, and M2 types) treated with DMSO (vehicle control), SEP (100 μM), or PAs (5 or 7.5 mM spermine) (n = 4). β-Actin was used as the internal loading control.

Figure S2.. Scavengers of arginine metabolites, nitric oxide and polyamines, prevent M1 and M2 macrophage polarization, respectively.

(A) Quantification of Western blots (Fig 4D) detecting M1 markers: TLR2, STAT1, and pSTAT1S727 in M1 macrophages treated with NO scavenger (50 μM cPTIO) with and without NO donor (100 μM GSNO) and M2 macrophages treated with DMSO or SEP (n = 4). GAPDH was used as the internal loading control. (B) Quantification of Western blots (Fig 4E) detecting M1 markers: TLR2, STAT1, and pSTAT1S727 versus M2 marker: CD206 in THP-1–derived M2 macrophages treated with a PA analog (50 and 100 μM DENSPM) and PAs (5 mM spermine) and M1 macrophages treated with DMSO or SEP (n = 4). β-Actin was used as the internal loading control.

Figure 5.. M2-TAMs treated with SEP show increased antigen presentation activities.

(A) Scheme of measuring antigen presentation activities of TAMs. Once the M1 macrophage is pulsed with an OVA_323–339 peptide, it phagocytoses and presents the epitope through the cell surface MHC II. The level of cell surface MHC II, representing antigen presentation activity, is detected by FACS. (The presented epitope is then recognized by TCR on Th1 T cells to trigger immunogenic responses.) (B) Mean fluorescence intensity of cell surface HLA-DR (MHC II) after being pulsed with or without an OVA_323–339 peptide (20 μg/ml for 2 h) on THP-1–derived M₀-, M1-, and M2-TAMs pretreated with DMSO or 100 μM SEP. Two-sample t tests (unpaired) were performed for pairwise comparison. (B, C, D) Percentages of TAM subsets treated as in (B) that expressed cell surface HLA-DR (bound by an OVA peptide) presented as histograms (C) and quantification (D). Unstained (Unst) and isotype (Iso) controls were used (n = 5). Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05.

Figure 6.. SEP-treated M2-TAMs activate cytotoxic T cells.

(A) Scheme of detection methods for activation of cytotoxic CD8⁺ T cells, based on IFNγ production, proliferation, and degranulation indicated by cell surface CD107a expression. (B) Schemes of the TAM–T-cell–cancer cell co-cultures (2:2:1 ratio) using the Transwell system (top) and direct system (bottom). (Top) THP-1–derived TAMs, PBMC-derived T cells, and BT474 breast cancer cells co-cultured using Transwell. (Bottom) PBMC-derived autologous TAMs and T cells were directly co-cultured with BT474 cells. (C) FACS-detected IFNγ expression levels in CD8⁺ T cells after being co-cultured with BT474 cells and PBMC-derived TAM subsets pretreated with DMSO or 100 μM SEP (n = 6). Positive control: M2-TAMs treated with LPS and IFNγ. Negative control: M1-TAMs treated with 1400W and L-NAME. T cells were gated for CD3 and CD8 expression. IFNγ levels are shown as MFU. (C, D) Detection of proliferation of cytotoxic T cells co-cultured as in (C) based on the dilution of CFSE signals through cell doubling (n = 6). Percentages of proliferating T cells (CFSE low) are highlighted in the histogram. (E) Cytotoxic T-cell proliferation shown as fold change of proliferating cells (CFSE low) with respect to non-proliferating cells (CFSE high). (B, F) Cell surface CD107a (degranulation marker) expression on cytotoxic T cells directly co-cultured as in (B). Percentages of CD107a+ CD8⁺ cells are shown in the plots. (G) Quantification of the percentages of CD107a+ CD8⁺ cells in co-cultures. Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05.

Figure 7.. SEP-treated M2-TAMs induce T cells to kill HER2+ breast cancer cells.

(A) Cell cycle analyses of BT474 cancer cells (CMFDA-labeled) co-cultured with PBMC-derived TAM subsets, pretreated with DMSO (vehicle), SEP (100 μM), or LPS + IFNγ (positive control), along with T cells. Adherent cells (TAMs+BT474 cells) were dissociated, fixed in 70% ethanol for 3 h, and stained with PI. BT474 cells were gated based on the CMFDA signal and analyzed for the PI-stained DNA contents. (A, B) Cell cycle distribution of BT474 cells co-cultured with TAMs and T cells as in (A). Note the dramatic increase in sub-G1 population co-cultured with SEP-treated M2-TAMs. (For quantification of sub-G1 and G1/G0 populations, see Appendix Fig S3A). (C) Annexin V/PI staining of co-cultured BT474 cells to measure cell deaths (n = 6). Viable cells: Annexin V -ve, PI -ve; early apoptotic cells: Annexin V +ve, PI -ve; late apoptotic cells: Annexin V +ve, PI +ve; and necrotic cells: Annexin V -ve, PI +ve. (D) Percentage of total apoptotic (Annexin V +ve) cancer cells. (E) Early and late apoptotic cancer cells. (F) Levels of ATP secreted by BT474 cells in co-cultures. Secretion of ATP indicates immunogenic cell death of cancer cells. Note the dramatic increase in ATP secretion by cancer cells in co-culture with SEP-pretreated M2-TAMs. Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; and ns, P > 0.05.

Figure S3.. SEP-treated M2 macrophages induce T cells to kill HER2+ breast cancer cells.

(A) Quantification of sub-G1 (top) and G1/G0 (bottom) populations of BT474 cancer cells subjected to cell cycle profiling (Fig 7A). BT474 cells were co-cultured with PBMC-derived macrophage subsets (M0, M1, and M2 types), pretreated with DMSO (vehicle), SEP (100 μM), or LPS + IFNγ (positive control), along with T cells. Adherent cells (macrophages+BT474 cells) were dissociated, fixed in 70% ethanol for 3 h, and stained with PI. BT474 cells were gated based on the CMFDA signal and analyzed for the PI-stained DNA contents. (B) (Top) Cell cycle distribution of SKBR3 cancer cells subjected to cell cycle profiling. SKBR3 cells were co-cultured with PBMC-derived macrophage subsets (M0, M1, and M2 types) and T cells as for BT474 cells. Quantification of sub-G1 (bottom left) and G1/G0 (bottom right) populations of SKBR3 cells analyzed by cell cycle profiling.

Figure 8.. Oral SEP treatment promotes the immunogenicity of TAMs and suppresses the growth of spontaneous MMTV-neu (HER2) mammary tumors.

(A) Scheme of the experiment where MMTV-neu (unactivated) mice were allowed to develop palpable mammary tumors (tumor latency of 6–14 mo) and given DMSO or SEP (10 mg/kg) in drinking water ad libitum for 6 wk (n = 7). (B) Tumor growth was measured by caliper, and the volume was determined (V = (W(2) x L)/2). (C) Pictures of exercised tumors. (D) Exercised tumors were analyzed for M1- versus M2-TAM profiles (CD80 versus CD163; IL12 versus IL10; and IFNγ) by FACS. (Top row) DMSO-treated tumors; (bottom row) SEP-treated tumors. (D, E) Quantification of the expression of M1- versus M2-TAM markers as in (D) in exercised tumors (n = 6) in comparison with spleens (n = 6) of the same animals. Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001.

Figure 9.. Suppressive effects of oral SEP treatment on MMTV-neu (HER2) tumors are linked to the elevation of anti-tumor lymphocytes in the circulation and within tumors.

(A) Mammary tumor growth of NT2.5 cells (derived from MMTV-neu tumors (57)) orthotopically transplanted into FVB/NJ mice (n = 6) and treated with DMSO or SEP (1 mg/kg) in drinking water ad libitum. Tumor growth was measured by caliper, and the volume was determined (V = (W(2) x L)/2). Statistical significance was evaluated with a two-way ANOVA test. Error bars: ± SEM. **P ≤ 0.01. (B) Pictures of exercised tumors. (C) Eosin-and-hematoxylin–stained tumor sections. Note the presence of numerous mammary gland–like structures in SEP-treated tumors, but not in DMSO-treated tumors. (D) (Top) Tumor sections were stained for M1- versus M2-TAMs (CD80 versus CD163); CD8⁺ versus CD4⁺ T cells, and Treg cells (CD4+/FOXOP3) by immunohistochemistry. (Bottom) The numbers of positive cells for each marker were counted per field (10x objective, n = 8–10). Error bars: ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; and ****P ≤ 0.0001. (E) Percentages of tumor-free mice after DMSO versus SEP treatment. Four-week-old female MMTV-neu/FVB (n = 20) mice were treated with DMSO versus SEP (1 mg/kg) in acidified drinking water for over 7 mo. (F) At the end of the experiment, PBMCs were isolated from four groups (DMSO with tumors, DMSO without tumors, SEP with tumors, and SEP without tumors) and analyzed by single-cell sequencing—tSNE images comparing cell clusters among the four groups. Differentially represented clusters for each group are marked with arrows. (G) For each group, the major cell type and the subcell types of the former are shown. Note the predominance of T cells in the SEP without tumor group in contrast to the predominance of granulocytes in the DMSO with tumor group.