Abstract Macrophages integrate microenvironmental cues to orchestrate complex transcriptional and metabolic programs that drive functional polarization. Here, we demonstrate that STK11 links interleukin-4 (IL-4) signaling with metabolic reprogramming to restrain alternatively activated (M2) macrophage polarization. Through integrative transcriptomic and metabolomic analyses, we identified STK11 as a key transcriptional and metabolic regulator during M2 polarization. STK11 deficiency enhanced the expression of M2-associated markers and promoted glutamine metabolism in IL-4–stimulated macrophages. Mechanistically, STK11 deficiency led to increased FOXO1 activation, thereby promoting M2 polarization. Pharmacological inhibition of FOXO1 or glutamine metabolism effectively reversed the enhanced M2 polarization. In an orthotopic model of pancreatic ductal adenocarcinoma, myeloid-specific deletion of STK11 resulted in increased accumulation of M2-like tumor-associated macrophages, impaired antitumor immunity, and accelerated tumor progression. These findings uncover a previously unrecognized role for STK11 in modulating M2 macrophage polarization, offering mechanistic insights that may inform the development of immunometabolic therapies for pancreatic cancer. __________________________________________________________________ STK11 acts as a key regulator in M2 polarization through coordinating IL-4 signaling and glutamine metabolism. INTRODUCTION Macrophages play a pivotal role in maintaining tissue homeostasis and modulating immune responses against pathogens and tumor progression ([62]1). They adapt to the cytokine milieu in both steady-state and immune-challenged conditions by activating various signaling pathways and transcriptional programs, resulting in a broad spectrum of phenotypes and functions ([63]2). Classically activated (M1) macrophages, stimulated by lipopolysaccharide and interferon-γ (IFN-γ), produce high levels of nitrogen oxides, thereby enhancing inflammatory responses to combat pathogenic threats. In contrast, alternatively activated (M2) macrophages, induced by T helper 2 (T[H]2) cytokines such as interleukin-4 (IL-4), secrete anti-inflammatory cytokines like IL-10 and transforming growth factor–β (TGF-β), which facilitate tissue repair and the resolution of inflammation ([64]3). Within the tumor microenvironment, tumor-associated macrophages (TAMs) often acquire M2-like characteristics in response to elevated IL-4 levels, particularly in cancers such as pancreatic cancer ([65]4), thereby promoting tumor growth and progression ([66]5). A deeper understanding of the mechanisms governing M2 macrophage polarization may uncover previously unappreciated therapeutic strategies to enhance the efficacy of cancer immunotherapy ([67]6). Beyond cytokine-induced transcriptional regulation of macrophage polarization, rewiring of cellular metabolism has emerged as a crucial mechanism that links bioenergetic and biosynthetic demands to the modulation of immune responses ([68]7, [69]8). M1-like macrophages up-regulate aerobic glycolysis to support their proinflammatory activities ([70]9), while M2-polarized macrophages predominantly rely on mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to suppress inflammation and promote tissue repair ([71]8, [72]10, [73]11). Recent studies have shown that reprogramming glutamine metabolism significantly enhances mitochondrial OXPHOS, thereby facilitating M2 macrophage polarization ([74]12, [75]13). These metabolic pathways offer promising targets for modulating macrophage functional polarization to improve antitumor immunity ([76]14). However, the precise mechanisms by which cytokine signaling interfaces with metabolic reprogramming during M2 macrophage polarization remain poorly understood. Serine/threonine kinase 11 (STK11), also known as LKB1, is primarily recognized as a tumor suppressor gene mutated in Peutz-Jeghers syndrome, a condition associated with increased cancer risk ([77]15). STK11 acts as a key metabolic sensor that regulates cell growth, energy metabolism, and differentiation ([78]16). In cancer cells, STK11 mutations lead to uncontrolled growth and increased energy consumption, primarily via overactivation of mechanistic target of rapamycin (mTOR) signaling pathways ([79]16). Recent research has expanded our understanding of STK11, highlighting its critical role in regulating the homeostasis and function of immune cells, including regulatory T cells (T[reg] cells) and dendritic cells (DCs), through both mTOR-dependent and mTOR-independent mechanisms. In T[reg] cells, STK11 promotes metabolic reprogramming to support their survival and immunosuppressive function ([80]17–[81]19), while, in DCs, it enforces metabolic quiescence to help restrain tumor immune evasion ([82]20, [83]21). These findings suggest that STK11 influences the tumor immune microenvironment through both tumor cell–intrinsic and tumor cell–extrinsic mechanisms. Given the pivotal role of TAMs in regulating cancer progression, how STK11 regulates TAM polarization and function in antitumor immunity remains a critical but understudied question. In this study, we uncover a previously unknown role for STK11 signaling in coordinating transcriptional and metabolic reprogramming during IL-4–induced M2 macrophage polarization. We demonstrated that STK11 deficiency enhanced M2-like macrophage polarization, characterized by increased expression of M2-associatd markers, elevated glutamine metabolism, and mitochondrial OXPHOS. STK11-deficient macrophages exhibited heightened forkhead box protein O1 (FOXO1) activity, which not only up-regulated M2-associated markers but also induced genes involved in mitochondrial function and glutamine metabolism. Pharmacological inhibition of FOXO1, glutamine metabolism, or mitochondrial OXPHOS attenuated the enhanced M2 polarization observed in STK11-deficient macrophages. In an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC), an aggressive form of pancreatic cancer, myeloid-specific deletion of STK11 led to increased accumulation of M2-like TAMs, impaired antitumor immunity, and accelerated tumor growth. These findings underscore a critical role for STK11 in integrating transcriptional regulation with metabolic adaptation to control IL-4–driven M2 macrophage polarization. Targeting the STK11-FOXO1 axis in macrophages may offer a promising strategy to enhance antitumor immunity and suppress pancreatic cancer progression. RESULTS Identification of STK11 as a key regulator of transcriptional and metabolic reprogramming in M2 macrophage polarization The interplay between transcriptional networks and metabolic programs is essential for orchestrating macrophage functional polarization ([84]22). In this study, we aimed to identify novel regulators of transcriptional and metabolic reprogramming associated with M2 macrophage polarization by integrating transcriptomic and metabolomic data with upstream regulatory analyses ([85]Fig. 1A). Bone marrow cells from C57BL/6 (B6) wild-type (WT) mice were treated with macrophage colony-stimulating factor (M-CSF) to induce differentiation into bone marrow–derived macrophages (BMDMs). On day 7 of culture, BMDMs were either exposed to a vehicle (mock treatment) to maintain an M0 state or stimulated with IL-4 to induce polarization into M2-like macrophages. Successful polarization was confirmed by the up-regulation of M2-associated markers, including CD206, CD163, and CD301. Fig. 1. Identification of STK11 as a previously unknown regulator of M2-like macrophage polarization. [86]Fig. 1. [87]Open in a new tab (A) Schematic diagram of the integrative approach used to identify regulators of M2-like macrophage polarization. (B) Ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified key upstream regulators of M2-like macrophage polarization. (C) Heatmap showing expression levels of downstream genes of STK11 in M2-like macrophages. (D) Circos plot showing enriched pathways from both transcriptomic and metabolomic data. Red dots represent genes, while blue dots indicate metabolites. The y axis depicts the log[2] (fold change) in IL-4–stimulated bone marrow–derived macrophages (BMDMs) compared to vehicle-treated controls. (E) Alteration in expression levels of genes and metabolites in the tricarboxylic acid (TCA) cycle and glutaminolysis. Bar plots depict fold changes in metabolite levels. Up-regulated genes are represented with varying intensities of red. CoA, coenzyme A; succinate-CoA ligase GDP/ADP-forming subunit alpha (SUCLG1), succinate dehydrogenase complex subunits A (SDHA), SDHB, SDHD, citrate synthase (CS), aconitase 2 (ACO2), fumarate hydrase (FH), oxoglutarate dehydrogenase (OGDH), malate dehydrogenase 1 (MDH1), isocitrate dehydrogenase1 (IDH1), glutamate dehydrogenase 1 (GLUD1), GLUD2, glutaminase (GLS), and solute carrier family 1 member 5 (SLC1A5). (F) Western blot analysis of phosphorylation of STK11 at serine-428 (p-STK11^S428), phosphorylation of signal transducer and activator of transcription 6 (STAT6) at tyrosine-641 (p-STAT6^T641), and β-actin expression in BMDMs stimulated with vehicle or IL-4 for the indicated time points. Data are representative of one experiment [(B) to (E)] or at least three independent experiments (F) and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test (E). ***P < 0.001 and ****P < 0.0001. h, hours. We performed RNA sequencing (RNA-seq) on both M0 and M2 BMDMs and conducted ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) in M2-like BMDMs. This analysis identified several upstream regulators implicated in M2 macrophage polarization, with the top four being STK11, tripartite motif-containing 24 (TRIM24), estrogen-related receptor alpha (ESRRA), and signal transducer and activator of transcription 6 (STAT6) ([88]Fig. 1B). While TRIM24, ESRRA, and STAT6 are well-established regulators of M2 polarization ([89]23, [90]24), the involvement of STK11, which exhibited the highest activation z-score, has not been previously characterized in this context. Among the genes targeted by STK11, the transcription factors (TFs) Cebpb and Myc, both known to promote M2 polarization ([91]25, [92]26), were significantly up-regulated in IL-4–stimulated BMDMs ([93]Fig. 1C). Stat5a and Stat1, which are more commonly associated with M1 macrophage polarization ([94]27, [95]28), were also up-regulated ([96]Fig. 1C). These findings suggest that STK11 may orchestrate the polarization of M2-like macrophages by modulating signaling and transcriptional networks in response to IL-4 stimulation. To investigate the metabolic programs associated with the transcriptional regulation of M2 macrophage polarization, we performed metabolomic profiling of M0 and M2 BMDMs (fig. S1A). Metabolites were extracted and analyzed using liquid chromatography–tandem mass spectrometry in both negative (mode N) and positive (mode P) ionization modes ([97]29). Principal component analysis (PCA) revealed distinct metabolic features between M0- and M2-polarized BMDMs (fig. S1, B and C), with numerous differential metabolites uniquely enriched in M2-polarized BMDMs (fig. S1, D and E). Integration of transcriptomic and metabolomic data with pathway enrichment analysis showed that M2 BMDMs displayed elevated metabolic activity in pathways including OXPHOS, the tricarboxylic acid (TCA) cycle, glutathione metabolism, and the metabolism of alanine, aspartate, and glutamate ([98]Fig. 1D), all of which are known to support M2-macrophage polarization ([99]22). Notably, the phosphatidylinositol 3-kinase (PI3K)–Akt, FOXO, and AMP-activated protein kinase (AMPK) signaling pathways were also associated with these metabolic changes in M2-like macrophages ([100]Fig. 1D), with AMPK being a well-established downstream effector of STK11 signaling ([101]16). This integrated analysis further revealed links between these signaling and metabolic pathways with various cancer types, including pancreatic, colorectal, and small cell lung cancers ([102]Fig. 1D), suggesting a broader relevance of these networks in shaping M2-like TAM polarization within the tumor microenvironment. Given the role of glutamine as a major carbon source for the TCA cycle, we analyzed TCA cycle intermediates and glutaminolysis-related metabolites in M0- and M2-like BMDMs. In M2-polarized BMDMs, we observed a marked increase in TCA cycle metabolites, including succinate (Suc), fumarate, malate, and cis-aconitate, alongside elevated levels of glutamine and glutamate ([103]Fig. 1E and fig. S1D). This metabolic reprogramming was accompanied by the up-regulation of key enzymes and transporters involved in the TCA cycle and glutaminolysis, including Suclg1, Sdha, Sdhb, Sdhd, Cs, Aco2, Fh, Ogdh, Mdh1, and Slc1a5, in M2-polarized BMDMs ([104]Fig. 1E). To investigate whether IL-4 influences STK11 activation, we examined STK11 phosphorylation in IL-4–stimulated BMDMs over a time course. IL-4 treatment induced phosphorylation of STK11 at serine-428 (p-STK11^S428) as early as 1 hour posttreatment, and this phosphorylation was sustained for at least 24 hours ([105]Fig. 1F). In contrast, phosphorylation of STAT6 at tyrosine-641 (p-STAT6^T641), a key event in M2-like macrophage polarization ([106]30, [107]31), was rapidly induced at 1 hour but gradually declined over time ([108]Fig. 1F). These results suggest that STK11 may serve as a critical regulator that links IL-4 signaling to both transcriptional and metabolic reprogramming, thereby modulating M2-like macrophage polarization. STK11 restrains polarization of M2-like macrophages To investigate the role of STK11 in M2-like macrophage polarization, we generated mice with myeloid-specific deletion of STK11 by crossing mice harboring loxP-flanked Stk11 alleles (Stk11^fl/fl) with LysM^Cre mice, which express Cre recombinase in myeloid cells. These mice were designated as LysM^CreStk11^fl/fl. Efficient deletion of STK11 in BMDMs and peritoneal macrophages (PMs) from LysM^CreStk11^fl/fl mice was confirmed (fig. S2A). To determine whether STK11 deficiency affects IL-4–induced macrophage polarization, we examined the expression of M2-associated genes. STK11-deficient BMDMs exhibited significantly increased expression of Mrc1 and Arg1 ([109]Fig. 2A), as well as Il10 ([110]Fig. 2B), while Tgfb expression remained unchanged ([111]Fig. 2B). Consistently, these cells showed elevated levels of M2 surface markers, including CD206 (encoded by Mrc1), CD163, and CD301 ([112]Fig. 2, C to E). Similarly, IL-4–stimulated STK11-deficient PMs displayed increased expression of Mrc1 and Arg1 ([113]Fig. 2F) and Il10 ([114]Fig. 2G), with no notable change in Tgfb expression ([115]Fig. 2G). Collectively, these results indicate that STK11 deficiency promotes macrophage polarization toward an M2-like phenotype. Fig. 2. STK11 deficiency promotes M2-like macrophage polarization. [116]Fig. 2. [117]Open in a new tab (A and B) Relative expression of Mrc1 and Arg1 mRNA (A), and Il10 and Tgfb mRNA (B) in WT and LysM^CreStk11^fl/fl BMDMs stimulated with IL-4 (20 ng/ml) for 24 hours. (C to E) Comparisons of CD206 (C), CD163 (D), and CD301 (E) expression on IL-4–stimulated WT and LysM^CreStk11^fl/fl BMDMs. Numbers in graphs indicate the mean fluorescence intensity (MFI). (F and G) Relative expression of Mrc1 and Arg1 mRNA (G), and Il10 and Tgfb mRNA (H) in WT and LysM^CreStk11^fl/fl PMs stimulated with IL-4 (20 ng/ml) for 24 hours. (H to J) Percentages of CD4^+ T cells producing IFN-γ (H), or IL-17 (I), or IL-4 (J) in the spleen from WT and LysM^CreStk11^fl/fl mice (n = 5 per group). (K) Percentages of IFN-γ–producing CD8 T cells in the spleen from WT and LysM^CreStk11^fl/fl mice (n = 5 per group). (L) Representative images of hematoxylin and eosin staining of the lung, small intestine, and colon from WT and LysM^CreStk11^fl/fl mice (scale bar, 100 μm). Data are representative of at least three independent experiments [(A) to (L)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A) to (K)]. *P < 0.05 and **P < 0.01; n.s., not significant. We next examined whether myeloid-specific deletion of STK11 affects macrophage and other myeloid cells populations under steady-state conditions. Both WT and LysM^CreStk11^fl/fl mice displayed comparable proportions of macrophages in the spleen (fig. S2B) and peritoneal cavity (fig. S2C). PMs can be divided into two major subsets: large PMs (CD11b^+F4/80^hiMHCII^lo) and small PMs (CD11b^+F4/80^loMHCII^hi) ([118]32). The frequencies of these subsets were comparable between WT and LysM^CreStk11^fl/fl mice (fig. S2D). We also assessed other myeloid populations, including neutrophils and eosinophils, and found no substantial differences between the two groups (fig. S2, E and F). Furthermore, the percentages and absolute numbers of splenic DCs were similar in LysM^CreStk11^fl/fl mice and WT mice (fig. S2G). Expression levels of activation markers MHCII and CD86 on DCs were also comparable (fig. S2, H and I), indicating that DCs in LysM^CreStk11^fl/fl mice maintain a quiescent state under homeostatic conditions. Given the critical role of macrophages in regulating T cell homeostasis ([119]33), we next examined cytokine production in CD4^+ and CD8^+ T cells from WT and LysM^CreStk11^fl/fl mice. The frequencies of CD4^+ T cells producing IFN-γ ([120]Fig. 2H), IL-17 ([121]Fig. 2I), and IL-4 ([122]Fig. 2J) were comparable between the two groups. Similarly, the proportion of IFN-γ–producing CD8^+ T cells did not show notable differences between WT and LysM^CreStk11^fl/fl mice ([123]Fig. 2K). Consistent with these findings, histological analysis using hematoxylin and eosin staining revealed normal tissue architecture and immune homeostasis in the lung, small intestine, and colon of LysM^CreStk11^fl/fl mice ([124]Fig. 2L). Collectively, these finding suggest that, while STK11 plays a crucial role in regulating M2-like macrophage polarization, its deletion in myeloid cells does not substantially disrupt immune homeostasis under steady-state conditions. STK11 in macrophages regulates T[reg] cell homeostasis and differentiation Given that M2-like macrophages can promote T[reg] cell homeostasis and differentiation ([125]33, [126]34), we investigated whether STK11 deficiency in macrophages affects T[reg] cell populations across various lymphoid and nonlymphoid tissues. In the spleen, peripheral lymph nodes, and mesenteric lymph nodes, LysM^CreStk11^fl/fl mice exhibited significantly increased percentages and numbers of T[reg] cells compared to WT mice ([127]Fig. 3A). In contrast, T[reg] cell percentages and numbers in the thymus were unchanged ([128]Fig. 3A). In nonlymphoid tissues, including the lung and liver, LysM^CreStk11^fl/fl mice also displayed elevated percentages and numbers of T[reg] cells ([129]Fig. 3, B and C). Similar increases in T[reg] cell proportions were observed in the blood (fig. S3A) and peritoneal cavity (fig. S3B). Moreover, T[reg] cells from LysM^CreStk11^fl/fl mice expressed higher levels of Ki67 ([130]Fig. 3D), a marker of cell proliferation, suggesting that STK11 deficiency in macrophages promotes T[reg] cell homeostatic expansion. Fig. 3. LysM^CreStk11^fl/fl mice display increased proportions of T[reg] cells. [131]Fig. 3. [132]Open in a new tab (A to C) Percentages and numbers of T[reg] cells (Foxp3^+CD4^+) in lymphoid tissues (A), lung (B) and liver (C) from WT and LysM^CreStk11^fl/fl mice (n = 5 per group). (D) Comparison of Ki67 expression in splenic T[reg] cells from WT and LysM^CreStk11^fl/fl mice (n = 5 per group). Numbers in graphs indicate the MFI of Ki67. (E) Percentages of CD103^+ T[reg] cells in the spleen from WT and LysM^CreStk11^fl/fl mice (n = 5 per group). (F and G) Flow cytometry analysis of Foxp3^+ (F) and CD103^+Foxp3^+ (G) populations in OT-II CD4^+ T cells cocultured with WT or LysM^CreStk11^fl/fl BMDMs in the presence of OVA and TGF-β for 4 days. (H and I) Flow cytometry analysis of Foxp3^+ (H) and CD103^+Foxp3^+ (I) populations in OT-II CD4^+ T cells cocultured with WT or STK11-deficient BMDMs in the presence of OVA and TGF-β, along with IgG or IL-10 neutralizing antibodies for 4 days. Data are representative of at least three independent experiments [(A) to (I)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A) to (E)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n.s., not significant. T[regs], T[reg] cells. We further characterized T[reg] cell subsets, specifically central T[reg] cells (cT[reg] cells; CD62L^hiCD44^lo) and effector T[reg] cells (eT[reg] cells; CD62L^loCD44^hi), which play distinct roles in maintaining immune homeostasis ([133]35). The proportions of cT[reg] cells and eT[reg] cells were similar between WT and LysM^CreStk11^fl/fl mice (fig. S3C). In addition, T[reg] cells from LysM^CreStk11^fl/fl mice exhibited comparable expression of key T[reg] cell–associated markers, including Foxp3, CD25, CD73, glucocorticoid-induced TNFR-related protein (GITR), and tumor necrosis factor receptor superfamily member 4 (OX40), relative to WT controls (fig. S3, D to H). The expression of Helios and NRP1, markers indicative of the thymus-derived natural T[reg] cells ([134]36), was also preserved (fig. S3I). Notably, T[reg] cells from LysM^CreStk11^fl/fl mice displayed significantly increased expression of CD103 ([135]Fig. 3E), a marker associated with T[reg] cell migration to nonlymphoid tissues and crucial for the suppression of inflammatory responses ([136]37–[137]39). Because IL-10 is a key cytokine that promotes T[reg] cell homeostasis and function ([138]40), we hypothesized that elevated IL-10 production by STK11-deficient macrophages may promote T[reg] cell differentiation. To test this, we cocultured WT or STK11-deficient BMDMs with naive CD4^+ T cells isolated from ovalbumin (OVA)–specific OT-II transgenic mice in the presence of TGF-β and OVA for 4 days. STK11-deficient BMDMs significantly increased the generation of OT-II Foxp3^+ T[reg] cells ([139]Fig. 3F) and up-regulated CD103 expression on these cells ([140]Fig. 3G) compared to WT BMDMs. To assess the role of IL-10 in this process, we added either a control IgG or an IL-10–neutralizing antibody to the cocultures. The IL-10–neutralizing antibody, but not the control IgG, abrogated the enhanced T[reg] cell induction ([141]Fig. 3H) and CD103 expression ([142]Fig. 3I) observed in cultures with STK11-deficient BMDMs. Collectively, these findings indicate that STK11 in macrophages restrains T[reg] cell differentiation and CD103 expression in an IL-10–dependent manner. STK11 orchestrates transcriptional control of M2 macrophage polarization To determine the molecular and metabolic pathways regulated by STK11 signaling, we performed RNA-seq to compare the transcriptomes of WT and STK11-deficient BMDMs under both mock- and IL-4–stimulated conditions. PCA revealed distinct transcriptional profiles between WT and STK11-deficient BMDMs in both conditions (fig. S4A). We identified 3772 DEGs in mock-stimulated STK11-deficient BMDMs and 1890 DEGs in IL-4–stimulated STK11-deficient BMDMs, relative to their WT counterparts ([143]Fig. 4A). Gene ontology analysis showed that STK11-deficient BMDMs up-regulated genes associated with macrophage activation and function. These included cell surface molecules (Ccr1, Cxc3cr1, Cxcr4, Nt5e, S1pr4, and Tlr5), signaling molecules (Dusp1, Dusp5, Mapk13, Plcb1, and Prkca), TFs (Cebpb, Fosb, Irf7, Nr4a1, and Spic), and chemokines and effector molecules (Ccl6, Cxcl1, Cxcl2, Mmp9, Pf4, and S100a8) ([144]Fig. 4B). Additionally, STK11 deficiency led to increased expression of M2 macrophage-associated genes, including Ahr, Arg1, Ccl6, Cd274 (encoding PD-L1), and Nrg1 ([145]Fig. 4C). These findings indicate that STK11 plays a crucial role in shaping the transcriptional landscape that controls macrophage activation and polarization. Fig. 4. STK11 orchestrates transcriptional regulation of M2-like macrophage polarization. [146]Fig. 4. [147]Open in a new tab (A) Heatmap with unsupervised hierarchical clustering of the DEGs in WT and LysM^CreStk11^fl/fl BMDMs stimulated with vehicle or IL-4 (n = 3 per group). (B) Heatmap showing the DEGs of the conditioned WT and STK11-deficient BMDMs. (C) Heatmap showing the expression of M2-associated markers in BMDMs. (D) IPA results indicating the top canonical pathways enriched in the DEGs of IL-4–stimulated STK11-deficient BMDMs. GM-CSF, granulocyte-macrophage colony-stimulating factor; GTPases, guanosine triphosphatases; MAPK, mitogen-activated protein kinase; NFAT, nuclear factor of activated T cells. (E and F) Comparisons of PD-L1 (E) and PD-L2 (F) expression on IL-4–stimulated WT and STK11-deficient BMDMs. (G) Gene set enrichment analysis results showing up-regulation of OXPHOS in STK11-deficient BMDMs. NES, normalized enrichment score. FDR, false discovery rate. (H) Measurement of OCR in IL-4–stimulated WT and LysM^CreStk11^fl/fl BMDMs. R/A, rotenone plus antimycin. (I) Spare respiratory capacity (SRC) in WT and LysM^CreStk11^fl/fl BMDMs. (J) Measurement of OCR in IL-4–stimulated BMDMs in response to the indicated mitochondrial inhibitors. (K) Eto-sensitive OCR in IL-4–stimulated BMDMs. Data are representative of one [(A) to (D) and (G)] or at least three [(E), (F), and (H) to (K)] independent experiments and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(E), (F), (I), and (K)] or two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test [(H) and (J)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. We next performed IPA on the DEGs in IL-4–stimulated STK11-deficient BMDMs to identify STK11-dependent signaling and metabolic pathways. This analysis revealed increased activity in several canonical signaling pathways, including Toll-like receptor signaling, Rho GDP-dissociation inhibitor (RHGDI) signaling, phosphatase and tensin homolog (PTEN) signaling, vitamin D receptor (VDR)/retinoid X receptor (RXR) activation, and the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) cancer immunotherapy pathway ([148]Fig. 4D). In contrast, pathways such as the P2Y purinergic receptor pathway, PI3K signaling in B lymphocytes, and cell cycle control of chromosomal replication were significantly down-regulated ([149]Fig. 4D). Given that PD-1 signaling is known to promote M2 macrophage generation and function ([150]41–[151]44), we further investigated the impact of STK11 deficiency on the expression of the PD-1 ligands. STK11-deficient BMDMs exhibited significantly elevated expression of both PD-L1 and PD-L2 following IL-4 stimulation ([152]Fig. 4, E and F). Additionally, gene set enrichment analysis revealed that, even under mock-treated conditions, STK11-deficient BMDMs up-regulated gene associated with several metabolic pathways, including OXPHOS ([153]Fig. 4G), fatty acid metabolism, tryptophan metabolism, and glutathione metabolism (fig. S4, B to D). To validate the impact of STK11 deficiency on mitochondrial OXPHOS in macrophages, we used a Seahorse extracellular flux analyzer to measure oxygen consumption rates (OCRs). STK11-deficient BMDMs displayed elevated basal OCR under both vehicle- and IL-4–stimulated conditions ([154]Fig. 4H and fig. S4E). Upon sequential treatment with oligomycin (Oligo), carbonyl cyanide p-trifluoromethoxyphenylhydrazon (FCCP), and rotenone plus antimycin, STK11-deficient BMDMs showed a significantly higher maximal OCR in response to FCCP compared to WT counterparts ([155]Fig. 4H and fig. S4E). This was accompanied by an increased spare respiratory capacity ([156]Fig. 4I), calculated as the difference between maximal and basal OCR, indicating an enhanced mitochondrial energy production potential. To further assess the impact of STK11 loss on FAO, we measured OCR in IL-4–stimulated WT and STK11-deficient BMDMs, following sequential treatment with Oligo, FCCP, and etomoxir (Eto), a carnitine palmitoyltransferase 1 (CPT1) inhibitor that blocks the mitochondrial import of long-chain fatty acids and FAO. In STK11-deficient BMDMs, OCR significantly decreased upon Eto treatment ([157]Fig. 4J), resulting in a higher Eto-responsive OCR ([158]Fig. 4K), indicating enhanced FAO capacity due to STK11 loss. Together, these findings demonstrate that STK11 orchestrates transcriptional regulation, mitochondrial OXPHOS, and FAO, collectively shaping M2 macrophage polarization. STK11 inhibits FOXO1 activity during M2 macrophage polarization We next investigated the signaling pathways downstream of STK11 that regulate M2 polarization. Although STK11 is known to suppress the mTOR signaling pathway and inhibit cancer cell proliferation ([159]16, [160]45), our data showed that STK11 deficiency did not substantially alter the phosphorylation of 4E-BP1 at threonine-37 and threonine-46 (p-4E-BP1^T37/46) or Akt at serine-473 (p-Akt^S473) (fig. S5A), suggesting that mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 activation remains largely unaffected in STK11-deficient macrophages. We also assessed the levels of p-STAT6^T641 and interferon regulatory factor 4 (IRF4), two key TFs crucial for M2-like macrophage polarization ([161]31, [162]46). STK11 deficiency did not markedly alter p-STAT6^T641 (fig. S5B) or IRF4 expression (fig. S5C) in IL-4–stimulated BMDMs. Additionally, given the potential role of recombination signal binding protein for immunoglobulin kappa J region (RBP-J)–mediated Notch signaling pathway in promoting M2 polarization ([163]47, [164]48), we examined whether STK11 regulates RBP-J expression. Upon IL-4 stimulation, RBP-J levels were comparable between WT and STK11-deficient BMDMs (fig. S5D). By performing IPA, we identified FOXO1 and FOXO3 as potential upstream activators in IL-4–stimulated STK11-deficient BMDMs ([165]Fig. 5A). To determine whether STK11 influences the activity of FOXO1 and FOXO3 during M2 polarization, we examined their phosphorylation at sites associated with proteasomal degradation: FOXO1 at threonine-24 (p-FOXO1^T24) and FOXO3 at threonine-32 (p-FOXO3^T32) ([166]49). In WT BMDMs, IL-4 stimulation markedly increased p-FOXO1^T24 levels ([167]Fig. 5B), while this increase was significantly attenuated in STK11-deficient BMDMs ([168]Fig. 5B). In contrast, STK11 deficiency resulted in only a modest reduction in p-FOXO3^T32 levels ([169]Fig. 5B). Consistent with these findings, FOXO1 protein accumulated notably in STK11-deficient BMDMs ([170]Fig. 5C), while FOXO3 levels remained largely unchanged (fig. S5E). Notably, Foxo1 transcript levels were comparable between WT and STK11-deficient BMDMs (fig. S5F), suggesting that observed accumulation of FOXO1 was posttranslationally regulated. Further analysis of FOXO1- and FOXO3-regulated genes in IL-4–stimulated STK11-deficient BMDMs revealed that 302 genes were uniquely regulated by FOXO1, 20 by FOXO3, and 45 were coregulated by both ([171]Fig. 5D). FOXO1-specific target genes included several M2-associated markers, such as Ctsl, Fgf1, Hmox1, Nt5e, and Tgm2 ([172]Fig. 5E). Collectively, these findings indicate that FOXO1, rather than FOXO3, plays a dominant role in driving M2 polarization in STK11-deficient BMDMs. Fig. 5. STK11 restrains FOXO1 activation upon IL-4 stimulation. [173]Fig. 5. [174]Open in a new tab (A) IPA results showing enhanced activation of FOXO1 and FOXO3 in IL-4–stimulated STK11-deficient BMDMs. (B) Immunoblotting of p-FOXO1^T24, p-FOXO3^T32, and β-actin in BMDMs stimulated with vehicle or IL-4. (C) Immunoblotting of FOXO1 and β-actin expression in BMDMs stimulated with vehicle or IL-4. h, hours. (D) Venn diagram showing the DEGs uniquely regulated or coregulated by FOXO1 and/or FOXO3 in IL-4–stimulated BMDMs (n = 3). (E) Heatmap showing the expression of the DEGs uniquely regulated by FOXO1 in IL-4–stimulated BMDMs. (F and G) Comparison of PD-L1 and PD-L2 expression (F) or CD206 and CD163 expression (G) on IL-4–stimulated BMDMs in the presence of vehicle or FOXO1i. (H) Bar graph showing Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome Pathway Database (REACTOME) pathways significantly enriched in the DEGs uniquely regulated by FOXO1 in IL-4–stimulated STK11-deficient BMDMs. SRP, signal recognition particle. (I) Heatmap showing the expression of FOXO1-regulated metabolic genes in IL-4–stimulated WT and STK11-deficient BMDMs (n = 3). Data are representative of one [(A), (D), (E), (H), and (I)] or three [(B), (C), (F), and (G)] independent experiments and are presented as the means ± SEM. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test [(F) and (G)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. To investigate the role of FOXO1 in regulating M2 polarization in STK11-deficient BMDMs, we treated WT BMDMs with AS1842856, a selective and potent FOXO1 inhibitor (FOXO1i) ([175]50), in the presence of IL-4 and assessed the expression of M2-associated markers. FOXO1 inhibition significantly reduced the expression of PD-L1 and PD-L2 in IL-4–stimulated WT BMDMs (fig. S5G), with a similar reduction observed in WT PMs treated with FOXO1i (fig. S5H). Notably, FOXO1 inhibition also reversed the increased expression of PD-L1 and PD-L2 in STK11-deficient BMDMs ([176]Fig. 5F) and attenuated the enhanced up-regulation of CD206 and CD163 ([177]Fig. 5G). To further explore the FOXO1-dependent pathways altered in IL-4–stimulated STK11-deficient BMDMs, we performed Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway enrichment analysis on the genes uniquely regulated by FOXO1. This analysis revealed significant enrichment of metabolic pathways, including glutathione metabolism, central carbon metabolism in cancer, and the glucagon signaling pathway ([178]Fig. 5H). Of note, STK11-deficient BMDMs showed increased expression of key enzymes, including Gclc, Gclm, Ggt5, Glul, Gsr, Gsta3, and Idh1 ([179]Fig. 5I), which are critical for glutamine metabolism and the TCA cycle, both of which support M2 macrophage polarization ([180]8, [181]12, [182]13). Collectively, these results indicate that STK11 suppresses FOXO1 transcriptional activity to regulate glutamine metabolism and the TCA cycle, thereby restraining M2-like macrophage polarization. STK11 reprograms glutamine metabolism to modulate M2 polarization Given that glutamine metabolism supports mitochondrial OXPHOS and promotes M2 macrophage polarization ([183]12, [184]13, [185]51), we investigated whether STK11 deficiency alters glutamine-derived metabolites in M2-polarized macrophages. STK11-deficient BMDMs exhibited significantly elevated intracellular levels of glutamine ([186]Fig. 6A) and glutamate ([187]Fig. 6B) under both mock- and IL-4–stimulated conditions. This increase in glutamate is likely due to enhanced conversion of glutamine by glutaminase 1 (GLS1). To assess the contribution of glutamine metabolism to M2 polarization in STK11-deficient macrophages, we used a panel of metabolic inhibitors targeting distinct steps of the glutamine metabolic pathway ([188]Fig. 6C). These included V-9302, an inhibitor of the amino acid transporter ASCT2 that blocks glutamine uptake; bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BTPES), a selective GLS1 inhibitor that prevents the conversion of glutamine to glutamate; and R162, an inhibitor of glutamate dehydrogenase 1, which blocks the conversion of glutamate to α-ketoglutarate (α-KG). Treatment with V9302 reversed the elevated expression of CD206 and PD-L2 ([189]Fig. 6D) and CD163 (fig. S6A) in STK11-deficient BMDMs. Similarly, BPTES reduced the expression of CD206 and PD-L2 ([190]Fig. 6E). Notably, R162 treatment abolished the enhanced expression of CD206, PD-L2, and CD163 in STK11-deficient BMDMs ([191]Fig. 6F and fig. S6B). Together, these findings indicate that STK11 restricts glutamine metabolism to suppress M2 macrophage polarization. Fig. 6. STK11 connects glutamine metabolism to modulate M2 macrophage polarization. [192]Fig. 6. [193]Open in a new tab (A and B) Measurement of cellular glutamine (A) and glutamate (B) in WT and LysM^CreStk11^fl/fl BMDMs stimulated with vehicle or IL-4. (C) Schematic diagram of specific inhibitors targeting the glutamine transportation and catabolism pathway. (D to F) Comparison of CD206 and PD-L2 expression on IL-4–stimulated WT and STK11-deficient BMDMs treated with vehicle or V-9302 (D), or BPTES (E), or R162 (F) (WT with vehicle treatment (mock) is set to 1, n = 3). (G) Relative fold change of α-KG/Suc ratio in IL-4–stimulated WT and STK11-deficient BMDMs. (H) Measurement of OCR in WT and LysM^CreStk11^fl/fl BMDMs treated with mock or without DE-Suc in response to Oligo, FCCP, and R/A stimulation. (I and J) Comparison of CD206 and PD-L2 (I) and CD163 (J) expression on IL-4–stimulated WT and STK11-deficient BMDMs supplemented with mock or DE-Suc (WT with mock treatment is set to 1, n = 3). Data are representative of three independent experiments [(A), (B), and (D) to (J)] and are presented as the means ± SEM. P values were calculated using two-tailed Student’s t test [(A), (B), and (G)] or one-way ANOVA with Tukey’s multiple comparison test [(D) to (F), (I), and (J)]. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. To support mitochondrial OXPHOS, glutamate is converted into α-KG, which is subsequently transformed into Suc. A high α-KG–to-Suc (α-KG/Suc) ratio in macrophages has been shown to favor M2 polarization ([194]12). To determine whether STK11 regulates this metabolic balance, we assess the α-KG/Suc ratio in IL-4–stimulated macrophages. STK11-deficient BMDMs exhibited a significantly higher α-KG/Suc ratio compared to WT controls ([195]Fig. 6G). To further examine the role of this ratio, we supplemented IL-4–stimulated STK11-deficient BMDMs with diethyl disuccinate (DE-Suc), a cell-permeable form of Suc analog ([196]Fig. 6C). DE-Suc supplementation normalized the elevated OCR observed in STK11-deficient BMDMs ([197]Fig. 6H). Consistent with these metabolic shifts, DE-Suc supplementation reversed the up-regulation of M2 markers, including CD206, PD-L2, and CD163 ([198]Fig. 6, I and J). Together, these findings indicate that STK11 regulates the α-KG/Suc balance to restrain mitochondrial metabolism and limit M2 macrophage polarization. We further examined whether pharmacological inhibition of mitochondrial OXPHOS could suppress M2 polarization in STK11-deficient BMDMs. Treatment with Oligo, a known OXPHOS inhibitor, significantly reduced the elevated expression of CD206 and PD-L2 in STK11-deficient BMDMs (fig. S6C). Although 2-deoxy-d-glucose (2-DG) is traditionally known as a glycolysis inhibitor, recent studies have demonstrated its ability to impair M2 polarization by inhibiting mitochondrial OXPHOS ([199]10). Consistent with these findings, 2-DG treatment effectively suppressed mitochondrial OXPHOS in both WT and STK11-deficient BMDMs (fig. S6D). Furthermore, 2-DG also reduced the elevated expression of CD206 and PD-L2 in STK11-deficient BMDMs (fig. S6E). Collectively, these results support a model in which STK11 functions as a key metabolic regulator that suppresses glutamine metabolism and mitochondrial OXPHOS to restrain M2 macrophage polarization. LysM^CreStk11^fl/fl mice exhibit accelerated progression of orthotopic pancreatic cancer An increase in both the abundance and immunosuppressive function of M2-like TAMs is commonly associated with impaired antitumor immunity and accelerated tumor progression. Analysis of data from The Cancer Genome Atlas revealed that low STK11 expression significantly correlated with reduced overall survival across multiple cancer types, including pancreatic adenocarcinoma (PAAD), uterine corpus endometrial carcinoma, and head and neck squamous cell carcinoma ([200]Fig. 7, A to C). Given that both tumor cell–intrinsic and tumor cell–extrinsic mechanisms contribute to tumorigenesis, we next assessed the role of tumor cell–intrinsic STK11 in PAAD. Analysis of the PAAD dataset used in [201]Fig. 7A showed that only 2.7% of all patients harbored STK11 mutations (fig. S7A), suggesting that altered expression, rather than mutation, may be more relevant in this context. To evaluate whether STK11 expression levels influence immune cell composition within the tumor microenvironment, we performed TIMER deconvolution analysis on the PAAD dataset, stratifying tumors into “STK11-low” and “STK11-high” groups. As shown in fig. S7B, the STK11-high group exhibited reduced proportions of M2 macrophages and T[H]2 cells but increased populations of tumor-killing immune cells, including natural killer (NK) cells and T[H]1 cells, compared to the STK11-low group. These findings suggest that tumor cell–intrinsic STK11 expression may shape the immune landscape and promote antitumor immunity in PAAD. However, whether myeloid-specific STK11 directly regulates TAM accumulation and function to modulate antitumor immunity in PAAD remains to be determined. Fig. 7. LysM^CreStk11^fl/fl mice display increased growth of orthotopic pancreatic tumors. [202]Fig. 7. [203]Open in a new tab (A to C) Kaplan-Meier survival curves of patients with high (red) or low (blue) STK11 expression in pancreatic adenocarcinoma (PAAD) (A), uterine corpus endometrial carcinoma (UCEC) (B), and head and neck squamous cell carcinoma (HNSC) (C). (D) Experimental design for in vivo imaging system (IVIS) imaging of mice orthotopically inoculated Luc^+ KPC tumor cells. (E) Bioluminescent images of WT and LysM^CreStk11^fl/fl mice bearing orthotopically inoculated Luc^+ KPC tumors (n = 5 per group). (F) Bioluminescent analysis of KPC tumor growth in WT and LysM^CreStk11^fl/fl mice. (G) Growth of KPC tumors in WT and LysM^CreStk11^fl/fl individuals based on bioluminescent analysis (n = 5 per group). (H) Survival curves of the KPC tumor-bearing WT and LysM^CreStk11^fl/fl mice (n = 5 per group). (I) Representative images of KPC tumors from WT and LysM^CreStk11^fl/fl mice (n = 4 per group). (J and K) KPC tumor volume (J) and weight (K) from WT and LysM^CreStk11^fl/fl mice in (I). Data are from one experiment [(A) to (C) and (E) to (H)] or representative of two-independent experiments [(I) and (K)] and are presented as means ± SEM. P values were calculated using Log-rank test [(A) to (C) and (H)], two-way ANOVA with Bonferroni’s multiple comparison test (F), or two-tailed Student’s t test [(J) and (K)]. *P < 0.05, **P < 0.01, and ****P < 0.0001. To investigate whether STK11 regulates TAM accumulation and function during pancreatic tumor progression, we used an orthotopic model of PDAC ([204]52), an aggressive subtype of PAAD. Specifically, we used KPC cells, which are pancreatic cancer cells derived from genetically engineered mice harboring Kras^G12D and Trp53^R172H mutations driven by Pdx1-Cre (commonly referred to as KPC mice). A total of 2 × 10^5 of luciferase expressing KPC cells were injected into the pancreas of WT and LysM^CreStk11^fl/fl mice. Tumor progression was monitored using in vivo imaging system (IVIS) on days 15, 22, 29, 36, and 50 postinoculation ([205]Fig. 7D). Bioluminescence imaging revealed significantly higher signals in LysM^CreStk11^fl/fl mice compared to those in WT controls, indicating accelerated tumor growth ([206]Fig. 7E), which was further supported by increased total flux values ([207]Fig. 7, F and G). Consistent with these results, LysM^CreStk11^fl/fl mice exhibited significantly reduced survival compared to WT mice ([208]Fig. 7H). To exclude potential confounding effects of luciferase expression, we repeated the experiment using non–luciferase-expressing KPC cells (5 × 10^4 cells) injected orthotopically into the pancreas. As observed previously, LysM^CreStk11^fl/fl mice developed significantly larger tumors ([209]Fig. 7, I and J) and exhibited greater tumor weights compared to WT mice ([210]Fig. 7K). Together, these results underscore the critical role for STK11 in TAMs in restraining PDAC progression. STK11 suppresses M2-like TAM polarization to enhance antitumor immunity in PDAC The shift of TAMs toward a M2-like phenotype is known to suppress antitumor immunity and promote tumor progression. To further investigate how STK11 deficiency affects TAM polarization and the antitumor immune response in KPC tumors, we orthotopically inoculated WT and LysM^CreStk11^fl/fl mice with KPC cells and analyzed tumors 21 days postinoculation. Using a Cytek Immunoprofiling assay, we examined the immune cell composition within the tumor microenvironment. Visualization by t-distributed stochastic neighbor embedding (t-SNE) revealed the distribution of myeloid cell subsets, including DCs, eosinophils, macrophages, and neutrophils, within the CD11b^+ compartment ([211]Fig. 8A). Notably, the tumors from LysM^CreStk11^fl/fl mice displayed a significant increase in eosinophil frequency compared to WT mice ([212]Fig. 8, A and B). Although the overall proportion of TAMs was comparable between groups ([213]Fig. 8A), LysM^CreStk11^fl/fl mice exhibited a markedly increased population of M2-like TAMs, as evidenced by elevated expression of CD206 ([214]Fig. 8C) and PD-L2 ([215]Fig. 8D). Furthermore, the proportion of IL-10–producing TAMs was significantly higher in LysM^CreStk11^fl/fl mice ([216]Fig. 8E). These findings indicate that STK11 constrains the acquisition of M2-like features in TAMs to suppress PDAC progression. Fig. 8. STK11-deficient TAMs acquire a M2-like phenotype in pancreatic cancer. [217]Fig. 8. [218]Open in a new tab (A) t-SNE analysis (left) and stacked bar graph (right) of CD11B^+ cell populations in KPC tumors from WT and LysM^CreStk11^fl/fl mice (n = 4 per group). (B) Percentages of eosinophils (Siglec-F^+CD11b^+) in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (C and D) Percentages of F4/80^+CD206^+ (C) and F4/80^+PD-L2^+ (D) M2-like TAMs in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (E) t-SNE analysis of IL-10–producing CD11b^+ myeloid cells (left) and percentages of IL-10–producing macrophages (right) in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (F) t-SNE analysis (left) and stacked bar graph (right) of CD11b^− cell populations in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (G and H) Percentages of IOCS^+T[reg] cells (G) and KLRG1^+T[reg] cells (H) in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (I) Flow cytometry analysis (left) and percentages (right) of PD-1–expressing CD8^+ T cells in KPC tumors from WT and LysM^CreStk11^fl/fl mice. (J) Percentages of IFN-γ–expressing CD8 T cells in KPC tumors from WT and LysM^CreStk11^fl/fl mice. Data are representative of two independent experiments [(A) to (J)] and are presented as means ± SEM. P values were calculated using two-tailed Student’s t test [(B) to (D) and (G) to (J)]. *P < 0.05, **P < 0.01, and ***P < 0.001. We further analyzed the composition of CD11b^− immune cells within KPC tumors from WT and LysM^CreStk11^fl/fl mice, focusing on CD4^+ T cells, CD8^+ T cells, T[reg] cells, B cells, and NK cells ([219]Fig. 8F). t-SNE analysis revealed a significant reduction in the proportion of NK cells, a critical population for controlling pancreatic cancer progression ([220]53), in tumors from LysM^CreStk11^fl/fl mice compared to those from WT mice ([221]Fig. 8F and fig. S8A). While the overall frequency of tumor-infiltrating T[reg] cells was comparable between groups ([222]Fig. 8F), LysM^CreStk11^fl/fl mice exhibited a marked increase in T[reg] cells expressing effector markers such as inducible costimulator protein (ICOS) ([223]Fig. 8G) and killer cell lectin-like receptor G1 (KLRG1) ([224]Fig. 8H), which are associated with enhanced suppressive function in the tumor microenvironment ([225]54, [226]55). To investigate whether STK11-deficient TAMs influence T cell phenotypes and cytokine production, we performed additional immune profiling. Although t-SNE analysis showed comparable proportions of tumor-infiltrating CD4^+ and CD8^+ T cells between WT and LysM^CreStk11^fl/fl mice ([227]Fig. 8I), the frequency of PD-1^+CD8^+ T cells, a hallmark of T cell exhaustion, was significantly higher in KPC tumors from LysM^CreStk11^fl/fl mice ([228]Fig. 8J). Consistent with this observation, tumor-infiltrating CD8^+ T cells from LysM^CreStk11^fl/fl mice produced significantly less IFN-γ compared to those from WT mice ([229]Fig. 8J). Moreover, the proportion of CD8^+ T cells producing both IFN-γ and TNFα was also reduced (fig. S8B). Similarly, IFN-γ production by tumor-infiltrating CD4^+ T cells was diminished in LysM^CreStk11^fl/fl mice (fig. S8C). These findings indicate that STK11 signaling in TAMs plays a pivotal role in modulating the tumor immune landscape, limiting the development of an immunosuppressive microenvironment and promoting antitumor immunity in PDAC. To further confirm the intrinsic role of myeloid-specific STK11 in regulating antitumor immunity, we used the murine colon adenocarcinoma cell line MC38, which does not harbor mutations in Stk11 ([230]56, [231]57). WT and LysM^CreStk11^fl/fl mice were subcutaneously inoculated with MC38 cells, and tumor growth was monitored every other day. Compared to WT controls, LysM^CreStk11^fl/fl mice developed significantly larger tumors, as evidenced by increased tumor volumes (fig. S8D) and visibly greater tumor mass (fig. S8E). These results suggest that myeloid-specific deletion of STK11 promotes tumor growth by fostering an immunosuppressive tumor microenvironment. To further investigate this, we analyzed tumor-infiltrating immune cell populations and observed a significant increase in M2-like TAMs in MC38 tumors from LysM^CreStk11^fl/fl mice, while M1-like TAMs exhibited a decreasing trend that did not reach statistical significance (fig. S8F). Additionally, tumor-infiltrating CD8^+ T cells isolated from LysM^CreStk11^fl/fl mice produced significantly lower levels of IFN-γ (fig. S8G) and perforin (fig. S8H) compared to those from WT mice. Similarly, the proportion of CD4^+ T cells producing IFN-γ was markedly reduced in LysM^CreStk11^fl/fl tumors (fig. S8I). Collectively, these findings highlight the intrinsic role of myeloid-specific STK11 in constraining M2-like TAM polarization and preserving effective antitumor T cell responses. DISCUSSION Macrophages adapt to their microenvironment by integrating diverse cytokine signals and transcriptional networks, which collectively govern their phenotypic and functional heterogeneity in health and disease ([232]58, [233]59). Beyond cytokine-mediated regulation, metabolic reprogramming plays a crucial role in shaping macrophage function across various immune contexts ([234]7). Our study identifies STK11 as a key molecular and metabolic checkpoint that links IL-4 signaling to the metabolic reprogramming required for M2 macrophage polarization. We demonstrate that STK11 restrains M2-like macrophage polarization by limiting FOXO1 activity and modulating glutamine metabolism and mitochondrial OXPHOS. This regulatory axis enhances antitumor immune responses and mitigates pancreatic cancer progression (fig. S8J). These findings underscore the therapeutic potential of targeting the STK11-FOXO1 pathway and glutamine metabolism in TAMs to attenuate their immunosuppressive functions and improve outcomes in pancreas cancer. Cellular signaling pathways are essential for integrating microenvironmental cytokines and transcriptional networks that guide macrophage polarization. IL-4–induced activation of the mTORC2 signaling is critical for M2 polarization, as it facilitates the activation of STAT6 and IRF4 ([235]60, [236]61), which, in turn, promote the expression of M2-associated markers ([237]3, [238]46). In contrast, excessive activation of the mTORC1 signaling pathway impairs M2 macrophage polarization ([239]62). Our study demonstrates that STK11 deficiency enhances IL-4–induced polarization toward M2-like macrophages, but this occurs independently of mTOR signaling. In IL-4–stimulated macrophages, loss of STK11 does not markedly alter mTORC1 or mTORC2 activation, nor does it affect STAT6 phosphorylation or IRF4 induction. Instead, a key finding from our study is that STK11 deficiency reduces FOXO1 phosphorylation, leading to increased its accumulation and transcriptional activity. This enhances the expression of M2 macrophage markers, and pharmacological inhibition of FOXO1 reverses this effect. These findings reveal a previously unknown mechanism by which the STK11-FOXO1 axis regulates M2-like macrophage polarization and function, highlighting an alternative regulatory pathway in macrophage biology. Reprogramming of cellular metabolism is fundamental to macrophage polarization, meeting the biosynthetic and bioenergetic demands required for distinct immune responses ([240]63). A metabolic shift from glycolysis toward mitochondrial OXPHOS and FAO is essential for the sustained functionality of M2-like macrophages, which are known to suppress antitumor immunity and promote tumor growth. Recent research has identified enhanced glutamine metabolism and an elevated α-KG/Suc ratio as critical metabolic signals driving M2 macrophage polarization ([241]8, [242]13, [243]64), although the upstream regulatory mechanisms remain poorly defined. Our study demonstrates that STK11 signaling plays a pivotal role in regulating mitochondrial OXPHOS and glutamine metabolism during M2 macrophage polarization. In STK11-deficient macrophages, levels of glutamine and glutamate are elevated, accompanied by an increased α-KG/Suc ratio, thereby promoting M2 polarization. Additionally, STK11 loss induces the expression of genes involved in glutathione metabolism, which facilitates ROS detoxification and further supports M2 polarization ([244]65). Notably, supplementation with cell-permeable DE-Suc restores the balance of mitochondrial OXPHOS and suppresses M2 macrophage polarization in STK11-deficient macrophages. These findings position STK11 as a critical metabolic checkpoint that coordinates mitochondrial OXPHOS and glutamine metabolism to restrain M2 macrophage polarization. PDAC is an aggressive malignancy with poor prognosis and high mortality rate. Both tumor cell-intrinsic and tumor cell-extrinsic mechanisms contribute to the initiation and progression of PDAC ([245]66). While activating mutations of KRAS and TP53 are well-established drivers of pancreatic cancer, our analysis reveals that high STK11 expression in patients with PDAC is associated with improved overall survival and increased infiltration of cytotoxic immune cells. In contrast, low STK11 expression is associated with an enrichment of immunosuppressive cell populations, including M2-like TAMs and T[H]2 cells within the tumor microenvironment. These findings indicate that STK11 shapes the tumor immune microenvironment and antitumor immunity in PDAC through both tumor cell–intrinsic and myeloid cell–intrinsic mechanisms. Elevated levels of IL-4, frequently observed in patients with PDAC ([246]67), promote M2-like TAM polarization, thereby contributing to PDAC progression by reshaping the tumor immune microenvironment to support tumor growth and immune evasion ([247]68). M2-like TAMs secrete immunosuppressive cytokines such as IL-10 and TGF-β, produce chemokines that inhibit cytotoxic CD8^+ T cell function, and express immune checkpoint ligands like PD-L1, which induce T cell exhaustion and facilitate T[reg] cell accumulation, collectively suppressing antitumor immune responses. Despite this, the molecular mechanisms driving M2-like polarization in PDAC remain incompletely understood. Our findings reveal a pivotal role for myeloid-specific STK11 in regulating TAM polarization and function in PDAC. Loss of STK11 in myeloid cells leads to increased accumulation of M2-like TAMs, which is associated with elevated effector T[reg] cells, enhanced T cell exhaustion, and diminished cytokine production by tumor-infiltrating lymphocytes (TILs), ultimately promoting tumor progression. These results suggest that restoring STK11 signaling in TAMs may suppress their immunosuppressive phenotype and enhance the efficacy of immune checkpoint blockade therapies in PDAC. Collectively, our study demonstrates that STK11 regulates M2-like macrophage polarization and contributes to immune remodeling of the tumor microenvironment through both tumor cell– and myeloid cell–dependent mechanisms. While previous studies have shown that STK11 promotes T[reg] cell function, our current findings indicate that STK11 deficiency in myeloid cells indirectly fosters T[reg] cell expansion. This apparent dichotomy underscores the context-dependent and cell-type–specific roles of STK11, highlighting the need for future studies to dissect the intercellular cross-talk among STK11-deficient immune populations within the tumor microenvironment. In summary, our research identifies STK11 as a critical regulator in M2 macrophage polarization, orchestrating the integration of IL-4 signaling with transcriptional and metabolic pathways that govern M2 polarization. By suppressing FOXO1 activity and modulating glutamine metabolism and mitochondrial OXPHOS, STK11 constrains the polarization toward M2-like phenotypes, thereby enhancing antitumor immunity in pancreatic cancer. Moreover, therapeutic strategies targeting STK11, either alone or in combination with interventions that inhibit glutamine metabolism, may offer a synergistic approach to improve treatment outcomes in pancreatic cancer. MATERIALS AND METHODS Experimental design This study aimed to identify previously unknown regulators that coordinate transcriptional and metabolic networks underlying IL-4–induced M2-like macrophage polarization. Unbiased integrative analyses of transcriptomic and metabolomic datasets from M0 and M2 BMDMs identified the tumor suppressor STK11 as a key regulator of M2-like macrophage polarization. To investigate its function, we generated a previously unknown mouse strain with myeloid-specific ablation of STK11 and assessed the impact of STK11 deletion on M2-like macrophage polarization, metabolic reprogramming, and immune homeostasis. We revealed that STK11 acts a negative regulator of M2-like macrophage polarization by limiting FOXO1 transcriptional activity, glutamine metabolism, and mitochondrial OXPHOS. Using both an orthotopic PDAC model and a subcutaneous MC38 tumor model, we further demonstrated that myeloid-specific STK11 suppresses the formation of M2-like TAMs and promotes antitumor immunity. Replicate numbers of each experiment and statistical analysis methods are indicated in the figure legends. All experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the Indiana University School of Medicine (protocol nos. 23147 and 23023). Mice Stk11^fl/fl mice and LysM^Cre mice were purchased from the Jackson Laboratory. C57BL/6, CD45.1^+, and OT-II transgenic mice were purchased from the Jackson Laboratory or a breeding core facility at Indiana University School of Medicine. All mice were kept in a specific pathogen–free facility in the Animal Resource Center at Indiana University School of Medicine, and all animal experiments involved in this study were evaluated and approved by the IACUC. BMDM differentiation and polarization Marrow was flushed from bones, dissociated by pipetting, and cultured in mouse M-CSF (40 ng/ml; R&D Systems) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin for 7 days. For macrophage polarization, BMDMs were stimulated with IL-4 (20 ng/ml; for M2 activation) for 24 hours. To examine the effects of different inhibitors on M2 polarization, WT and STK11-deficient BMDMs were stimulated with IL-4 (20 ng/ml) in the presence of vehicle or the indicated inhibitors for 24 hours, including FOXO1i (2 μM), Oligo (2 μM), 2-DG (1 mM), V-9302 (20 μM), BPTES (20 μM), R162 (25 μM), and DE-Suc (200 μM), followed by flow cytometry analysis of the expression of various M2-associated markers. Immunoblot and quantitative reverse transcription polymerase chain reaction Immunoblots were performed as described previously, using the following antibodies: p-LKB1^S428, p-AKT^S473, p-4E-BP1^T37/46, p-STAT6^Tyr641, p-FOXO1^Thr24/FOXO3^Thr32, FOXO1, FOXO3, RBP-J, IRF4, and β-actin (all antibodies are from Cell Signaling Technology). RNA was extracted using a microRNA isolation kit (QIAGEN), and RNA concentration was determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized with SuperScript III reverse transcriptase (Thermo Fisher Scientific). An ABI 7500 Real-time PCR system was used for quantitative polymerase chain reaction with the probe sets listed in the key resources table in the Supplementary Materials. Metabolic assays BMDMs (5 × 10^5 per well) were cultured in a 24-well plate for 2 days in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin. WT and STK11-deficient BMDMs were stimulated with IL-4 in the presence of vehicle or the indicated inhibitors, including DE-Suc (200 μM) and 2-DG (1 mM) for 24 hours. BMDMs (5 × 10^4 per well) were transferred to XF cell culture plate that had been coated with poly-d-lysine overnight. OCR was measured in XF medium (nonbuffered DMEM containing 5 mM glucose, 2 mM l-glutamine and 1 mM sodium pyruvate), under basal conditions and in response to Oligo (1 μM), FCCP (2 μM), Eto (200 μM), and rotenone/antimycin A (1 μM) using the XF-96 Extracellular Flux Analyzer (Seahorse Bioscience). Concentrations of cellular glutamine and glutamate in WT and STK11-deficient BMDMs stimulated with or without IL-4 were determined by a Glutamine/Glutamate-Glo assay kit (Promega). An α-Ketoglutarate Colorimetric assay kit (Abcam) and a Succinate Colorimetric Assay kit (Abcam) were used to respectively determine the concentrations of cellular α-KG and Suc in WT and STK11-deficient BMDMs according to the product manual. Orthotopic pancreatic cancer mouse model Orthotopic injections were conducted on 6-week-old C57BL/6 and LysM^CreStk11^fl/fl mice. A total of 2 × 10^5 Luc^+GFP^+ KPC cells suspended in 50 μl of PBS buffer were injected into the pancreas. Before injection, the mice were anesthetized, and a small incision was made in the skin and muscle layer to expose the pancreas. Following cell injection, the incision was closed using surgical sutures and wound clips. Tumor progression and survival were monitored weekly using the IVIS Spectrum In Vivo Imaging System, with imaging performed five times between days 15 and 50. For tumor microenvironment analysis 5 × 10^4 KPC cells suspended in 50 μl of PBS buffer were orthotopically injected into pancreas of WT and LysM^CreStk11^fl/fl mice. Isolation and analysis of tumor-infiltrating immune cells from KPC tumors Tumors were processed into single-cell suspensions using a tumor dissociation kit (Miltenyi) and a MACS dissociator in C-tubes. Cells were activated with a Cell Activation Cocktail (containing brefeldin A) for 4 hours at 37°C in a CO[2] incubator. After activation, cells were blocked with an Fc blocker (Invitrogen) for 10 min at room temperature. Intracellular staining was performed using the True-Nuclear Transcription Factor Buffer according to the manufacturer’s instructions. Samples were analyzed using a Cytek Aurora flow cytometer, and dead cells were identified using Zombie NIR dye (BioLegend). Antibodies used in the Cytek analysis are detailed in the key resources table in the Supplementary Materials. Data were analyzed using FlowJo software (version 10.10.0) and the Cytobank platform. MC38 tumor model Murine MC38 colon adenocarcinoma cells were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. Gender- and age-matched WT and LysM^CreStk11^fl/fl mice were injected subcutaneously with 2 × 10^5 MC38 adenocarcinoma cells at the right flank. Tumors were measured with digital calipers every other day. Tumor volumes were calculated by the formula: length × width × [(length × width)^0.5] × 𝜋𝜋/6. To isolate TILs, tumors were collected, minced, and digested in RPMI 1640 buffer containing 2% FBS and collagenase IV (1 mg/ml) (Sigma-Aldrich) at 37°C for 30 min. The digested tissues were filtered using nylon mesh. The cell suspension was centrifuged and separated by a 40 to 70% Percoll density gradient (GE Healthcare). The cells layered between 40 and 70% fraction were collected and used for staining. Referenced accessions The RNA-seq data have been deposited to the Gene Expression Omnibus (GEO) with accession number, GSE228251. Statistical analysis P values were calculated by one-way analysis of variance (ANOVA) for one-factor analysis, two-way ANOVA for two factor analysis, and two-sided two-sample Student’s t test for comparison between two independent groups using GraphPad Prism, unless otherwise noted. P values of less than 0.05 were considered as significant. All error bars represent the SEM. Acknowledgments