Glufosinate constrains synchronous and metachronous metastasis by promoting anti‐tumor macrophages
2020; Springer Nature; Volume: 12; Issue: 10 Linguagem: Inglês
10.15252/emmm.201911210
ISSN1757-4684
AutoresAlessio Menga, Marina Serra, Simona Todisco, Carla Riera‐Domingo, Ummi Ammarah, Manuel Ehling, Erika M. Palmieri, Maria Antonietta Di Noia, Rosanna Gissi, Maria Favia, Ciro Leonardo Pierri, Paolo E. Porporato, Alessandra Castegna, Massimiliano Mazzone,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle4 September 2020Open Access Source DataTransparent process Glufosinate constrains synchronous and metachronous metastasis by promoting anti-tumor macrophages Alessio Menga Alessio Menga orcid.org/0000-0002-2827-5298 Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Marina Serra Marina Serra Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Simona Todisco Simona Todisco Department of Sciences, University of Basilicata, Potenza, Italy Search for more papers by this author Carla Riera-Domingo Carla Riera-Domingo Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Ummi Ammarah Ummi Ammarah Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Manuel Ehling Manuel Ehling Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Erika M Palmieri Erika M Palmieri Cancer & Inflammation Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Maria Antonietta Di Noia Maria Antonietta Di Noia Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Rosanna Gissi Rosanna Gissi Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Maria Favia Maria Favia Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Ciro L Pierri Ciro L Pierri Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Paolo E Porporato Paolo E Porporato Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Alessandra Castegna Corresponding Author Alessandra Castegna [email protected] orcid.org/0000-0003-0235-6847 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy IBIOM-CNR, Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy Search for more papers by this author Massimiliano Mazzone Corresponding Author Massimiliano Mazzone [email protected] orcid.org/0000-0001-8824-4015 Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Alessio Menga Alessio Menga orcid.org/0000-0002-2827-5298 Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Marina Serra Marina Serra Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Simona Todisco Simona Todisco Department of Sciences, University of Basilicata, Potenza, Italy Search for more papers by this author Carla Riera-Domingo Carla Riera-Domingo Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Ummi Ammarah Ummi Ammarah Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Manuel Ehling Manuel Ehling Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Erika M Palmieri Erika M Palmieri Cancer & Inflammation Program, National Cancer Institute, Frederick, MD, USA Search for more papers by this author Maria Antonietta Di Noia Maria Antonietta Di Noia Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Rosanna Gissi Rosanna Gissi Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Maria Favia Maria Favia Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Ciro L Pierri Ciro L Pierri Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy Search for more papers by this author Paolo E Porporato Paolo E Porporato Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Alessandra Castegna Corresponding Author Alessandra Castegna [email protected] orcid.org/0000-0003-0235-6847 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy IBIOM-CNR, Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy Search for more papers by this author Massimiliano Mazzone Corresponding Author Massimiliano Mazzone [email protected] orcid.org/0000-0001-8824-4015 Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy Search for more papers by this author Author Information Alessio Menga1,2,3,4, Marina Serra1,2, Simona Todisco5, Carla Riera-Domingo1,2, Ummi Ammarah3, Manuel Ehling1,2, Erika M Palmieri6, Maria Antonietta Di Noia4, Rosanna Gissi4, Maria Favia4, Ciro L Pierri4, Paolo E Porporato3, Alessandra Castegna *,4,7 and Massimiliano Mazzone *,1,2,3 1Laboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology (CCB), VIB, Leuven, Belgium 2Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium 3Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Centre, University of Torino, Torino, Italy 4Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy 5Department of Sciences, University of Basilicata, Potenza, Italy 6Cancer & Inflammation Program, National Cancer Institute, Frederick, MD, USA 7IBIOM-CNR, Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, National Research Council, Bari, Italy *Corresponding author. Tel: +39 080 5442322; E-mail: [email protected] *Corresponding author. Tel: +32 16 37 3213; E-mail: [email protected] EMBO Mol Med (2020)12:e11210https://doi.org/10.15252/emmm.201911210 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Glutamine synthetase (GS) generates glutamine from glutamate and controls the release of inflammatory mediators. In macrophages, GS activity, driven by IL10, associates to the acquisition of M2-like functions. Conditional deletion of GS in macrophages inhibits metastasis by boosting the formation of anti-tumor, M1-like, tumor-associated macrophages (TAMs). From this basis, we evaluated the pharmacological potential of GS inhibitors in targeting metastasis, identifying glufosinate as a specific human GS inhibitor. Glufosinate was tested in both cultured macrophages and on mice bearing metastatic lung, skin and breast cancer. We found that glufosinate rewires macrophages toward an M1-like phenotype both at the primary tumor and metastatic site, countering immunosuppression and promoting vessel sprouting. This was also accompanied to a reduction in cancer cell intravasation and extravasation, leading to synchronous and metachronous metastasis growth inhibition, but no effects on primary tumor growth. Glufosinate treatment was well-tolerated, without liver and brain toxicity, nor hematopoietic defects. These results identify GS as a druggable enzyme to rewire macrophage functions and highlight the potential of targeting metabolic checkpoints in macrophages to treat cancer metastasis. Synopsis The GS inhibitor glufosinate is shown to significantly reduce metachronous and synchronous metastasis with no detectable toxicity. This occurs by selective rewiring of both TAMs and MAMs to an antitumoral function, reducing immunosuppression and angiogenesis with a consequent decrease of metastasis. Glutamine synthetase expression is a typical feature of "M2-like" macrophages and tumor-associated macrophages (TAMs), and its pharmacological targeting with glufosinate elicits therapeutic effects in murine models of highly metastatic melanoma, breast and lung cancer. Upon treatment with glufosinate in tumor-bearing mice, TAMs are reprogrammed from a protumoral to an antitumoral phenotype, as evidenced by changes in molecular markers and biological functions. Treatment of tumor-bearing mice with glufosinate stimulates cytotoxic T cells while decreasing vessel sprouting, leading to reduction of metachronous and synchronous metastasis through TAM reprogramming. Therapeutic doses of glufosinate do not display overt signs of brain, liver, kidney, or hematological toxicities. Introduction Tumor-associated macrophages (TAMs) constitute the main stromal compartment in many tumors. TAMs support different crucial functions for cancer progression by shaping adaptive immune responses and by contributing to vessel and matrix remodeling, cancer cell proliferation, survival, and metastasis (Noy & Pollard, 2014; Singh et al, 2017). At the metastatic site, macrophages (that are metastasis-associated macrophages, MAMs) can also favor the preparation of the pre-metastatic niche and cancer cell extravasation (Gil-Bernabé et al, 2012; Sharma et al, 2015; Lin et al, 2019). For these reasons, targeting macrophage functions is now considered a promising strategy to harness primary and metastatic tumor growth. More evidence on TAMs suggests but does not always prove that these diverse functions are driven and sustained by distinct metabolic states (Mazzone et al, 2018; Flerin et al, 2019; Castegna et al, 2020). The classical pro-inflammatory and antitumoral macrophage phenotype can be resembled by in vitro stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ), whereas the alternative, pro-tumor function is achieved by IL-4 and IL-10. These cytokines are responsible for metabolic shifts that underline defined functional states in macrophages (Biswas & Mantovani, 2012; Mills & O'Neill, 2016). More information is awaited regarding the role of specific metabolic features affecting TAM behavior in vivo, the strategies to rewire their protumoral phenotype toward their original protective, anti-tumor function, and their impact on disease outcome (Wenes et al, 2016; Palmieri et al, 2017b). In the effort to dissect which pathways underline the phenotypic switch in TAMs, we have discovered that targeting glutamine synthetase (GS; a.k.a. glutamate ammonia ligase, GLUL, EC 6.3.1.2) in macrophages represents an effective way to reprogram in vitro IL10-stimulated macrophages and TAMs toward a desirable "M1-like function." GS-inhibited IL10 macrophages display typical M1-like features both metabolically and functionally, with an increased glycolytic flux partly linked to HIF1α stabilization, enhanced ability to impair cancer invasion, reduced capacity to induce angiogenesis, and enhanced propensity for T-cell activation and recruitment. This ultimately results in metastasis inhibition in tumor-bearing mice (Palmieri et al, 2017b). These encouraging results have prompted us to pursue inhibition of GS in macrophages as an immunometabolic strategy to reduce metastasis in cancer. Methionine sulfoximine (MSO) is the classical GS inhibitor through irreversible binding to the glutamate site, but many studies have been performed on different classes of molecules able to inhibit GS, with particular attention to plant GS, in which inhibition of glutamine synthesis has been proved particularly effective in killing weeds (Occhipinti et al, 2010). Glufosinate ammonium is the ammonium salt of phosphinothricin, a non-proteinogenic amino acid that inhibits GS in bacteria (Bayer et al, 1972), algae (Hall et al, 1984), and higher plants (Leason et al, 1982). Studies in rats have shown that pharmacological inhibition of GS by glufosinate ammonium is lethal at very high doses (LD50 1,500–2,000 mg/kg); however, our understanding on the systemic toxicity of this molecule (e.g., TD50 studies) remains sparse and not conclusive (Cox, 1996). Glufosinate is a glutamate analogue and competes with glutamate for the binding in the active site (Logusch et al, 1989). The effect of glufosinate on human GS has never been proven, but the specificity of its inhibitory action in plant GS (Occhipinti et al, 2010) prompted us to test its effect on the human recombinant GS as well as in macrophages both in vitro and in vivo. Our results point to a significant inhibitory capacity of glufosinate toward human GS. Furthermore, at micromolar concentrations glufosinate treatment induces a shift from a M2-like to an M1-like phenotype in murine and human macrophages similarly to the effect displayed by MSO. This rewiring is associated with a decreased immunosuppression, angiogenesis, and metastatic burden in murine models of lung, breast cancer, and melanoma treated with glufosinate, similarly to what observed in the macrophage-specific GS knockout mice (Palmieri et al, 2017b), without any sign of specific liver or brain toxicity nor hematopoietic abnormalities at all tested doses. In the clinic, cancer death is mostly due to the appearance of diffused metastatic lesions that can be treated together with the primary tumor when the latter is unresectable or already in advanced stages (synchronous metastasis). Alternatively, metastasis treatment can start few or several years after primary tumor resection following the relapse of the disease far from the original site (metachronous metastasis) (Mekenkamp et al, 2010). The data hereby presented show that the antimetastatic effect of glufosinate is evident not only in a synchronous setting but also in a metachronous setting, increasing the clinical potential of our findings. This study provides a proof-of-concept of the role of in vivo pharmacological GS inhibition as immunometabolic strategy to reduce metastasis and highlights the significance of in vivo targeting of macrophagic metabolic checkpoints as a promising alternative to tackle tumor progression and metastasis. Results Glufosinate inhibits human recombinant GS The enzyme activity of the human recombinant GS (hGS), obtained by its expression in E. coli (Fig 1A), was evaluated in the presence of saturating concentrations of substrates for almost 90 min at 25°C and was found to be linear at least for 10 min at 25°C (Fig 1B). The Michaelis–Menten (half-saturation) constant (Km) of the human recombinant GS was determined by measuring the initial rate by varying l-glutamate or ATP concentrations in presence of a fixed saturating concentration of the other two substrates. By Lineweaver–Burk plots, the Km and Vmax values were 1.42 ± 0.17 mM and 0.82 ± 0.05 μmol/min/mg protein for l-glutamate, and 2.56 ± 0.15 mM and 0.91 ± 0.03 μmol/min/mg protein for ATP (Fig 1C and D). Figure 1. Glufosinate specifically inhibits human recombinant GS A. Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS–PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Bio-Rad). From right Lanes 1–4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropyl-β-D-1-thiogalattopiranoside (IPTG) 0.7 mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adjacent boxed lane: Western blotting analysis of GS. B–E. Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B). Lineweaver–Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence ( ), or in the presence of glufosinate (C) or MSO (D). Symbols ( ): 2 mM MSO or 0.025 glufosinate; () 3 mM of MSO or 0.050 mM of glufosinate; () 4 mM of MSO or 0.065 mM of glufosinate; () 5 mM of MSO or 0.075 mM of glufosinate. The insets represent the secondary plot of the slopes of Lineweaver–Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ± 0.08 μmol/(min x mg protein). F–K. Comparative analysis of GS structures from several organisms. Lateral (F) and top view (G) of the entire GS decameric (a dimer of pentamers) structure are reported in pink cartoon representation. (H) Cartoon representation showing the top view of the monomer–monomer interface hosting cofactors, substrates, or inhibitors (Dataset EV1) participating to or inhibiting the conversion of glutamate to glutamine. In particular, ADP (cyan), phosphate ions (brown), MSO phosphate (P3S, yellow), phosphoaminophosphonic acid-adenylate ester (ANP, white), glutamate (magenta), citrate (black), and the imidazopyridine inhibitor ((4-(6-bromo-3-(butylamino)imidazo(1,2-a)pyridin-2-yl)phenoxy) acetic acid, green) are reported in sticks representation, whereas Mn2+ ions are reported in pale blue spheres and Mg2+ in pale green spheres (involved in the coordination of the imidazopyridine inhibitor). (I) Superimposition of all the sampled 17 crystallized structures (Dataset EV1). (J) 2D representation of glutamate, P3S, glufosinate, and glyphosate. (K) Zoomed view of the crystallized P3S binding region and the docked glufosinate and glutamate binding regions within the superimposed human (white cartoon) and Z.mays (pink cartoon) GS structures. Source data are available online for this figure. Source Data for Figure 1 [emmm201911210-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint The effect of glufosinate on GS activity was investigated and was compared to that displayed by MSO on the human recombinant protein. Glufosinate and MSO inhibited human recombinant GS in a concentration-dependent manner (Fig 1E). Interestingly, glufosinate inhibition was much more effective compared to that of MSO as showed by the IC50 values that are of 0.051 and 2.4 mM, respectively (Fig 1E). To shed light into the inhibition mechanism, double-reciprocal plots were obtained from the reciprocal of initial rates versus the reciprocal of l-glutamate with or without different concentrations of MSO or glufosinate. The initial binding of glufosinate (Fig 1C) or MSO (Fig 1D) to human GS was competitive versus glutamate. The Ki value for MSO, estimated by secondary plot, was 0.88 mM in good agreement with the value previously obtained (Jeitner & Cooper, 2014) (inset of Fig 1D). Furthermore, the Ki value of glufosinate was estimated about 0.0195 mM (inset of Fig 1C) confirming a greater selectivity of this inhibitor when compared to that of MSO. To rule out an additional off-target role of glufosinate, a sequence database screening was performed by using alternatively H. sapiens or Z. mays GS sequences, searching for highly similar paralogous sequences in mammalia and plants (Dataset EV1). This search revealed no significant similarity between the query GS sequences and other mammalia or plant paralogous sequences, different from GS, nor identified structurally related proteins, beyond the available GS proteins, in the PDB until today (Dataset EV2). For gaining new insights about glufosinate competitive binding mechanism to GS binding region, in presence of glutamate, the available crystallized structures were manually inspected by using 3D visualizer. Thus, it was observed that several ligands participating in the reaction catalyzed by GS were crystallized in complex at the monomer–monomer interface with GS (Fig 1F–K), namely ADP, phosphate ion, L-methionine-S-sulfoximine phosphate (P3S), phosphoaminophosphonic acid-adenylate ester (ANP), glutamate, citrate, and the imidazopyridine inhibitor ((4-(6-bromo-3-(butylamino)imidazo(1,2-a)pyridin-2-yl)phenoxy) acetic acid) (Dataset EV2). Given the structural similarity of glufosinate with glutamate ligand and P3S, after superimposition of the three molecules in the GS catalytic site, it is observed that glufosinate might bind most of residues involved in the binding of glutamate ligand (in 4hpp.pdb) and P3S (i.e., in 2qc8.pdb) (Fig 1F–K and Dataset EV3). Glufosinate skews macrophages away from an M2-like phenotype and promotes an M1-like phenotype in vitro Based on the inhibitory capacity of glufosinate on the human recombinant protein, together with our previous findings on the role of GS in macrophages (Palmieri et al, 2017b), we asked whether pharmacological GS targeting affects polarization of primary human macrophages. To this end, macrophages derived from human monocytes were skewed toward an M2-like phenotype with IL10 in presence or absence of glufosinate and the expression levels of M1 and M2 markers were measured. In IL10-stimulated macrophages (IL10-macrophages for simplicity), M1-like genes, such as CD80, CXCL9, and CXCL10 (Fig 2A–C), were upregulated by glufosinate in a concentration-dependent fashion, except for TNFA, which strongly increased only at 20 μM concentration (Fig 2D). Concomitantly, up-regulation of M2-specific markers upon IL10 stimulation, such as MSR1 (CD204) and MRC1 (CD206), CCL17 and CCL18, was reduced (Fig 2E–H). These data indicate that glufosinate effectively blocks GS activity in macrophages and this prevents the expression of M2 markers while promoting the acquisition of M1 features, in a more effective fashion compared to MSO (Palmieri et al, 2017b). From a metabolic point of view, glufosinate/IL10 macrophages displayed increased glutamate and succinate levels, with a decrease in glutamine compared to IL10 macrophages (Fig EV1A), phenocopying the metabolic reprogramming displayed by MSO-treated IL10 macrophages (Palmieri et al, 2017b). Figure 2. Glufosinate promotes a rewiring of IL10 macrophages toward a M1-like phenotype through HIF1α stabilization and abolishes immunosuppressive effect of hypoxia A–D. Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20 μM)-stimulated IL10 macrophages (n = 3). E–H. Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, CCL17, and CCL18 mRNA in IL10, MSO-, and glufosinate (10 and 20 μM)-stimulated IL10 macrophages (n = 3). I–M. Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86, CD80, and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20 μM)- and acriflavine/glufosinate (10 and 20 μM)-IL10 macrophages (n = 3). N–P. Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20 μM)-treated, and acriflavine/glufosinate (10 and 20 μM)-treated IL10 macrophages (n = 3). Q. Quantification of cancer cell motility through a matrigel-coated membrane in presence of IL10, MSO/IL10, and glufosinate (10 and 20 μM)-IL10-treated macrophages after 24 h of incubation (n = 6). R. Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20 μM)/IL10 after 4 h of incubation with HUVEC cells (n = 6). S. CD8+ T-cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20 μM)/IL10 for 24 h (n = 4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi/well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control. T. CD8+ T-cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20 μM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n = 4). U. Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6, and P70S6K (in their phosphorylated and unphosphorylated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20 μM), rapamycin (20 nM) and a combination of both (n = 3). V. CD8+ T-cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20 μM), rapamycin, and a combination of both for 24 h (n = 4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi/well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control. Data are reported as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001.Exact P values and statistical tests are reported for each experiment in Appendix Table S2. Source data are available online for this figure. Source Data for Figure 2 [emmm201911210-sup-0007-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Glufosinate triggers a metabolic and functional switch of macrophages toward an M2-like phenotype A. LC-MS/MS quantification of intracellular glutamate, glutamine, and succinate in IL10, MSO-, and glufosinate (10 and 20 μM)-treated IL10 (n = 4). B. Western blotting analysis of TNFα and CCL18 in IL10 alone or glufosinate (10 and 20 μM)- and acriflavine/glufosinate (10 and 20 μM)-IL10 macrophages (n = 3). C. Representative images of A549 cell migration through a matrigel-coated micropore filter in presence of IL10, MSO/IL10, and glufosinate (10 and 20 μM)-IL10 pre-stimulated macrophages after 24 h of incubation (n = 6). Five images per field were analyzed. Scale bar: 50 μm. D. Representative pictures of capillary network formation of HUVEC cells cocultured with macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20 μM)/IL10 after 4 h of incubation with HUVEC cells (n = 6). Five images per field were analyzed. Scale bar: 100 μm. Data are reported as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001.Exact P values and statistical tests are reported for each experiment in Appendix Table S2. Source data are available online for this figure. Download figure Download PowerPoint Glufosinate-treated macrophages display HIF1α activation and relevant anti-tumor functions in vitro Since glufosinate promotes succinate accumulation and skews IL-10 macrophages toward an M1-like phenotype, which is characterized by succinate accumulation (Fig EV1A), we tested whether HIF1α, known to be stabilized by succinate (Tannahill et al, 2013), is upstream to the expression of a pro-inflammatory M1 phenotype (Takeda et al, 2010). Treatment with the HIF1α inhibitor acriflavine prevented the M2 to M1 phenotypical rewiring in glufosinate-treated IL-10 macrophages, since the expression of typical markers of classically activated macrophages (such as TNFA, CXCL10, CD86, CD80, and CXCL9) was significantly decreased (Fig 2I–M). C
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