Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non‐virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T. The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross‐reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140–1200 GE for G. parasuis and vtaA. The cut‐off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae. Glaesserella parasuis and its virulence marker vtaA distinguishing between highly virulent and non‐virulent strains. Furthermore, Mycoplasma hyorhinis and Mycoplasma hyosynoviae can be identified targeting 16S rRNA. The developed multiplex PCR provides a simple one‐tube assay, which can be performed rapidly and cost‐efficiently allowing an efficient throughput of samples in veterinary diagnostic laboratories.The aim of the study was to develop a multiplex PCR for identification of  Infected pigs can develop polyserositis, polyarthritis, meningitis, pneumonia, or septicemia. G. parasuis comprises strains with heterogeneous virulence capacities ranging from non‐virulent to highly virulent  genes, allowing for the differentiation between putative virulent and non‐virulent G. parasuis strains  system  from alignments of available sequences for HPS_219690793, vtaA, and 16S rRNA genes retrieved from the NCBI databank. Primers for G. parasuis were designed on gene HPS_219690793 with an amplicon length of 94 base pairs (bp), whereas the developed reverse primer (Primer_glaesserella_R) partly corresponded to the reverse primer published by Howell et al.  partly corresponded to the published forward primer AV1‐F  included a minor groove binding molecule at the 3ʹ‐end, allowing for a short 17 bp‐probe thereby increasing specificity. Probes detecting vtaA, M. hyorhinis, and M. hyosynoviae comprised Black Hole Quenchers (Eurogentec S.A.). All primer and probe sequences were tested for specificity by BLAST searches. For monitoring potential qPCR inhibition, eGFP was used as an internal amplification control as previously described  eGFP plasmid DNA and ultrapure distilled water to complete the total reaction volume. The thermocycling conditions of the PCR were as follows: initial denaturation at 95°C for 2 min, 40 cycles with denaturation at 95°C for 5 s, and annealing/extension at 62.5°C for 30 s. The three reference strains G. parasuis serovar 1, M. hyorhinis ATCC 17981T, and M. hyosynoviae NCTC 10167T were included in each qPCR run as positive controls. To exclude contaminations in the reaction mix, DNase‐ and RNase‐free water was tested as a negative control in each experiment. The multiplex qPCR was optimized using different concentrations of primer, probes, and annealing temperatures considering PCR efficiencies. Samples with a threshold cycle (Ct) value of <35 were considered positive.Multiplex qPCR experiments were executed using a Rotor‐Gene Q (Qiagen) and analyzed with the help of Rotor‐Gene Q Software 2.3.1 (Qiagen). The setup of the Rotor‐Gene instrument included an auto‐gain optimization step for each channel before starting with the first fluorescence acquisition at the beginning of the qPCR. The total reaction volume of the multiplex qPCR was 15 µL and consisted of 1 µL of genomic DNA (20 ng/µL), 1X QuantiNova Multiplex PCR Kit (Qiagen), 300 nM of primers targeting 2.3In a pilot study, different organs and swabs originating from 42 pigs from 31 farms were tested using the novel multiplex qPCR. Obtained samples  were first enriched in liquid tryptic soy broth at 37°C for 24 h. Afterward, sample disruption was achieved using Pathogen Lysis Tubes S (Qiagen) and the TissueLyser (Qiagen), according to the manufacturer's instructions. Subsequently, DNA was extracted using IndiSpin Pathogen Kit  following the instructions in the kit handbook.2 for up to 48 h and inspected for growth. To potentially cultivate Mycoplasma spp., swabs taken from infected tissue sites were inoculated in mycoplasma liquid media (Mycoplasma Experience Ltd.).In parallel, obtained clinical samples were grown on Chocolate agar with Vitoyx and Columbia Blood agar (Thermo Fisher Diagnostics AG) at 37°C in 5% COG. parasuis‐positive samples , Erysipelothrix rhusiopathiae (n = 1), Escherichia coli (n = 2), Mycoplasma hyopneumoniae (n = 1); nasal commensal microorganisms similar to G. parasuis: Moraxella spp. (n = 1) and Neisseria animaloris (n = 1); Gram‐negative coccobacilli, namely, Actinobacillus suis (n = 1), Actinobacillus pleuropneumoniae (n = 4), Actinobacillus minor (n = 1), Pasteurella multocida (n = 3), and Bordetella bronchiseptica (n = 2); and Gram‐positive rod Listeria monocytogenes (n = 1). Each strain was tested with a concentration of 20 ng/µL.The specificity of the multiplex qPCR was determined by testing an exclusivity panel consisting of 23 pathogenic bacteria comprising 12 additional species not including 2.5G. parasuis serovar 1, M. hyorhinis ATCC 17981T, and M. hyosynoviae NCTC 10167T were examined. With an estimated average genome size of 1.8 Mbp for G. parasuis , the following approximate DNA quantities corresponded to 1 genome equivalent (GE): 2 fg for G. parasuis, 0.95 fg for M. hyorhinis, and 0.93 fg for M. hyosynoviae. To obtain an accurate limit of detection (LoD) for each target and to identify a reasonable cut‐off Ct value, 20 replicates of each reference strain were analyzed with concentrations close to the LoD . The LoD at a confidence interval of 95% was defined as the concentration of DNA, where at least 95% positive replicates can be detected. The LoD was computed using the program GenEx version 7  by plotting the fraction of positive samples against the concentration of the DNA template, and was illustrated at a logarithmic scale.To determine the analytical sensitivities of the multiplex qPCR, the three reference strains G. parasuis serovar 1, M. hyorhinis ATCC 17981T, and M. hyosynoviae NCTC 10167T were analyzed by using tenfold dilution series in a range between 100 and 107 GE. DNA samples for each reference strain were measured in triplicates in three separate experiments.To examine the repeatability of the multiplex qPCR assay, the coefficients of intra‐ and inter‐assay variability were determined. For this purpose, reference strains 2.6Ct values of dilution series measured in triplicates were plotted against input DNA concentrations, resulting in a standard curve for reference strains of G. parasuis serovar 1 and its virulence marker vtaA, M. hyorhinis ATCC 17981T, and M. hyosynoviae NCTC 10167T. The slope (S) of the standard curve, obtained by dilution series in the linear range between 10 and 107 GE, was used to calculate PCR efficiency (E) as expressed in the following equation: E = (101/−S − 1) × 100.To calculate efficiencies of the multiplex qPCR for each target, 2.7G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates were tested.For further evaluating the multiplex qPCR, 20 ng of genomic DNA extracted from 21 33.1G. parasuis, vtaA, M. hyosynoviae, and M. hyorhinis can be recognized by channel green, crimson, orange, and yellow, respectively. The fifth channel red measures the internal control eGPF, which acts as an exogenous internal control monitoring the PCR for correct performance. For all samples measured, eGPF could be detected accurately.Amplification plots of the multiplex qPCR using different detection channels are shown in Figure 3.2S. suis) and not in every case clinical signs could be unambiguously attributed to either G. parasuis, M. hyorhinis, or M. hyosynoviae as a causative pathogen. Additionally, up to three different G. parasuis serovars of both virulent and non‐virulent kinds could be detected in five animals. Due to the fastidious cultivation of the strains, in many cases, the successful isolation of involved strains was hampered. From the obtained 25 G. parasuis‐positive samples in the novel multiplex qPCR, six samples were identified to be non‐virulent  of CV% < 5% for the inter‐assay variability and CV% < 4% for the intra‐assay variability demonstrating the multiplex qPCR to be a reliable and highly reproducible assay ; formal analysis ; investigation ; writing—original draft (lead). Sarah Schmitt: Conceptualization ; formal analysis . Fenja Rademacher: Investigation . Peter Kuhnert: Resources (lead); writing—review and editing . Giovanni Ghielmetti: Conceptualization . Sophie Peterhans: Conceptualization ; formal analysis ; investigation . Roger Stephan: Conceptualization ; formal analysis ; writing—review and editing .The authors declare no conflict of interest.The authors have nothing to report.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Macrophages play diverse roles in development, homeostasis, and immunity. Accordingly, the dysfunction of macrophages is involved in the occurrence and progression of various diseases, such as coronavirus disease 2019 and atherosclerosis. The protective or pathogenic effect that macrophages exert in different conditions largely depends on their functional plasticity, which is regulated via signal transduction such as Janus kinase–signal transducer and activator of transcription, Wnt and Notch pathways, stimulated by environmental cues. Over the past few decades, the molecular mechanisms of signaling pathways in macrophages have been gradually elucidated, providing more alternative therapeutic targets for diseases treatment. Here, we provide an overview of the basic physiology of macrophages and expound the regulatory pathways within them. We also address the crucial role macrophages play in the pathogenesis of diseases, including autoimmune, neurodegenerative, metabolic, infectious diseases, and cancer, with a focus on advances in macrophage‐targeted strategies exploring modulation of components and regulators of signaling pathways. Last, we discuss the challenges and possible solutions of macrophage‐targeted therapy in clinical applications. We hope that this comprehensive review will provide directions for further research on therapeutic strategies targeting macrophage signaling pathways, which are promising to improve the efficacy of disease treatment. In this review, the authors introduce the research advances related to macrophage signaling pathways by first providing an overview of the basic physiology of macrophages and expound on the regulatory pathways within them. Then, the pathogenic effect of macrophages and the therapeutic strategies targeting macrophage signaling pathways are addressed in various diseases. Then, we focus on important signaling pathways regulating the functions of macrophages in a receptor‐centered way. On this basis, we discuss the current therapeutic approaches in clinical use or under clinical trials that target key components of these signaling pathways, including ligands, receptors and effectors, in different diseases. Finally, we discuss the challenges and future research directions of macrophage‐targeted therapy.2Macrophages, originally identified because of their phagocytic nature, are a heterogeneous group of immune cells that are widely distributed throughout the organism and play important roles in development and homeostasis maintenance. Furthermore, it is expected that the origins and polarization of macrophages will determine their phenotypes and functions in specific microenvironments. In this section, we will provide an overview of the basic biology of macrophages, including their origins, replenishment, polarization, and physiological functions.2.1Although macrophages were initially thought to originate primarily from hematopoietic stem cells (HSCs) and circulating monocytes,2.2Macrophages must maintain or increase their population to better perform functions, and two strategies are at stake.2.2.1Under tissue stress conditions, including inflammation and cancer, circulating monocytes can be recruited to specific sites and differentiate into macrophages with specific functions. This process relies on the participation of chemokines or cytokines.2.2.2Recent evidence points to the self‐renewal and numerical maintenance of macrophages in adult tissues under homeostatic conditions by in situ proliferation of TRMs rather than an influx of hematopoietic progenitors.2.3Polarization of macrophages is a process of altering phenotypes and function in response to microenvironmental stimuli,30, such as interleukin (IL)‐1, IL‐6, TNF‐α, nitric oxide (NO), reactive oxygen species (ROS), and downregulate the expression of IL‐12, IL‐23, and IL‐10.M1 and M2 macrophages represent the two main subpopulations of macrophages.To avoid tissue damage and maintain homeostasis, anti‐inflammatory M2 macrophage‐driven regulatory mechanisms suppress chronic inflammatory responses.As the study progressed, it was established that macrophage polarization is on a continuum, and in addition to M1 and M2 macrophages, there exist other phenotypes of macrophages, such as CD169+ and T cell receptor positive (TCR+) macrophages.In conclusion, macrophages are heterogeneous populations composed of multiple subpopulations with different phenotypes and functions, each with their own characteristics and functions. In vivo, macrophages can flexibly adopt specific functional phenotypes in response to subtle and continuous variations in the tissue microenvironment and then play an important role in development and homeostasis.2.4Macrophages play essential roles in all stages of development.Macrophages utilize their powerful biological functions to play diverse roles in tissue homeostasis maintenance as well.3The diversity of macrophage functions depends on their plasticity in response to environmental stimuli. In response to diverse microenvironmental signals or under distinct pathophysiological conditions, macrophages can obtain different functional phenotypes through polarization. This process is regulated by various ligand–receptor recognitions through receptor‐mediated signaling transduction and downstream signaling pathways. The integration of different stimulus signals and the crosstalk and balance among signaling pathways ultimately determine the phenotype and function of macrophages. And these ligands, receptors, and signaling pathways have been shown to serve as targets for disease treatment. Thus, we then introduce these important signaling pathways mainly in a receptor‐centered way.3.13.1.1TLRs are important PRRs expressed in macrophagesIL‐1, IL‐6, TNF‐α, and other inflammatory factor genes.Upon binding to ligands, some TLRs, such as TLR2, TLR4, and TLR6, recruit MyD88, which binds to the IL‐1R‐associated kinase (IRAK) 4 complex, leading to the autophosphorylation of IRAK1.TLR3 and TLR4 can also activate the TRIF‐dependent pathway.3.1.2The NLR family includes 22 members of three subfamilies, NLR family C‐terminal caspase activation and recruitment domain (CARD) containing protein (NLRC), NLR thermal protein domain associated protein (NLRP), and ICE‐protease activating factor (IPAF), among which NLRP3Nterm by removing the carboxyl terminus of gasdermin D (GSDMD).Nterm binds to the cell membrane to form membrane pores, leading to cell death and the release of large amounts of proinflammatory factors.Upon stimulation by multiple pathogen‐associated molecular patterns or damage‐associated molecular patterns, NLRP3 oligomerizes and recruits apoptosis‐associated speck‐like protein containing a C‐terCARD (ASC) to form ASC speck through its PYD domain.In contrast to NLRP3, NLRC3 inhibits macrophage activation and prevents inflammatory factor production by negatively regulating multiple inflammatory pathways.3.1.3The cGAS‐STING signaling pathway regulates macrophage polarization and the production of various cytokines, which play a role in pathogen clearance, the inflammatory response, and antitumor immunity.As an important PRR, cGAS is a direct sensor of DNA in host cells. cGAS can recognize and bind to cytoplasmic dsDNA to undergo conformational changes and activation and then catalyze the synthesis of cyclic GMP‐AMP (cGAMP) from intracellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP). cGAMP acts as a second messenger to bind to STING, a linker protein anchored in the endoplasmic reticulum (ER), causing conformational changes and activation.3.2TNF‐α plays an important role in regulating macrophage proliferation, apoptosis, and inflammation by activating signaling pathways, including NF‐κB and MAPK, through binding to its receptor TNFR1/2 , inducing stronger macrophage antigen‐presenting ability and the production of proinflammatory cytokines and chemokines.3.33.3.1Mrc1, Arg1, and IL‐10.IL‐4 is an important activator of macrophage M2 polarization and initiates the JAK‐STAT  triggers M1 macrophage polarization and proinflammatory mediator production mainly through the JAK2‐STAT5 signaling pathway. GM‐CSF binds to its receptors GM‐CSFRα and GM‐CSFRβ to form a dodecamer ligand‒receptor complex that promotes STAT5 phosphorylation and homodimerization via JAK2.3.63.6.1TGF‐β regulates macrophage polarization and function through the TβR in Sma‐ and Mad‐related protein (SMAD)‐dependent or SMAD‐independent signal transduction pathways Figure .112 Wh3.6.2EGF plays an important role in macrophage activation through MAPK signaling pathways Figure .119, 3.7GPCRs are a large family of receptors that can bind to various ligands, including chemokines, hormones, ions, neurotransmitters, and odorant molecules.2+, which activates various protein kinases, including protein kinase C (PKC) and calmodulin‐dependent protein kinase type II (CaMKII), to regulate macrophage activation and function.The binding of CCL2/CCL5 to its receptor CCR2/CCR5 promotes macrophage M2 polarization through the GPCR signaling pathway. When bound to its ligand, the GPCR undergoes conformational changes that cause the dissociation of G protein into Gα and Gβγ. Then, Gα and Gβγ activate their downstream signaling molecules and generate different intracellular signal transduction pathways  δ, and cyclin D1.Unlike CCR2/CCR5, Fzd1 is a member of another family of GPCRs. When combined with its ligand wingless homolog (Wnt), Fzd1 activates the Wnt/β‐catenin, Wnt/planar cell polarity (PCP), and Wnt/Ca2+ pathways , a myeloid inhibitory receptor containing immunoreceptor tyrosine‐based inhibitory motifs (ITIMs), inhibits macrophage phagocytosis and proinflammatory cytokine production by interacting with its cell surface ligand CD47.Similar to SIRPα, LILRB,3.11Arg 1 and FIZZ1.ROS are composed of free radicals and non‐free radicals formed by partial reduction of oxygen, including superoxide anion, hydrogen peroxide, and hydroxyl radical.3.12ATP6V0d2 expression by activating mTORC1. ATP6V0d2 is involved in the formation of vacuolar proton pumps that promote lysosomal acidification and protein degradation. Reduced expression of ATP6V0D2 leads to a reduction in the degradation of HIF2α by lysosomes, which induces high expression of VEGF and M2 homeostatic genes.Lactate drives macrophage polarization to anti‐inflammatory, pro‐repair phenotypes through regulation of multiple signaling pathways.3.13Glycine can shape macrophage polarization and function by regulating signaling pathways such as NF‐κB and PI3K/Akt.4Since macrophages are important contributors to development, tissue homeostasis, immunity, and repair, macrophage dysfunction is involved in the initiation and development of many diseases, such as autoimmune, neurodegenerative, metabolic, and infectious diseases and cancer. It is becoming increasingly clear that the flexible and precise transformation of the functional phenotypes of macrophages plays important roles in the progression or remission of diseases. Therapies targeting components and regulators of signaling pathways in macrophages hold great potential and are a prospective direction for the treatment of these diseases Table .4.1Autoimmune diseases refer to tissue trauma caused by excessive and persistent inflammation owing to autoantibodies or self‐reactive lymphocytes when immune tolerance is broken.4.1.1RA is characterized by the massive infiltration of inflammatory cells and proinflammatory mediators in the synovial membrane of multiple joints. Notably, macrophages are one of the most numerous cells in the RA synovium, the number of which is positively correlated with the severity of clinical signs.In view of the critical role of macrophages in the pathogenesis of RA, efforts are being made to develop macrophage‐targeted therapeutics. Several types of agents have succeeded in clinical trials for the treatment of RA. Inflammatory cytokine inhibitors targeting TNF‐α, IL‐6, IL‐1, and IL‐12/23 have been developed, among which the most widely used is anti‐TNF agents. These agents promote macrophage polarization toward the M2 phenotype and increase macrophage apoptosis, leading to the resolution of inflammation in RA.4.1.2IBD is a chronic inflammatory disease in the gastrointestinal tract, primarily encompassing Crohn's disease and ulcerative colitis. Patients with IBD have incomplete immune tolerance and thus initiate innate and adaptive immunity in the intestine with the participation of gut microbiota, leading to inflammation that is hyperactive and difficult to self‐limit. Intestinal macrophages play a major role in the establishment and maintenance of gut homeostasis, and dysfunction of intestinal macrophages is proposed to be the basis of chronic inflammation in IBD.Some of the anti‐TNF agents and anti‐IL‐12/23 agents used in RA are also widely used in IBD, and JAK inhibitors have entered late‐stage clinical trials. The well‐known mechanism for the effect of TNF‐α blockers on IBD is the induced differentiation of macrophages to the M2 phenotype and the secretion of the anti‐inflammatory cytokine IL‐10, which depends on FcR signaling.iNOS expression.In addition to the above agents, some of the first‐line treatments for IBD can have an impact on signaling pathways to alter the proinflammatory profiles of macrophages. For instance, corticosteroids direct monocytes to differentiate toward M2 macrophages by inactivating NF‐κB and AP‐1.4.1.3MS is an idiopathic inflammatory demyelinating disease of the CNS that is caused by immune responses to self‐antigens. The role of microglia/macrophages in MS pathogenesis is similar to that of other autoimmune diseases mentioned above, such as oxidative damage and proinflammatory cytokine and chemokine production, resulting in inflammation and destruction in the brain.Drugs targeting chemokines, especially the CCL2/CCR2 axis, achieve satisfactory efficacy in preclinical studies and animal models; however, they encounter challenges in clinical research.NF‐κB and reducing the activity of the JNK and Akt pathways, respectively.Fingolimod (FTY720) is an agonist of the S1P receptor, which skews microglial polarization to the M2 phenotype, decreases the expression of inflammatory cytokines and induces apoptosis by modulating several signaling pathways involving STAT1/3, IRF8, p38, PI3K/Akt, and NF‐κB.4.2Neurodegenerative diseases feature progressive and irreversible loss of selective populations of neurons, mainly AD, PD, and ALS, according to primary clinical features.4.2.1AD is the most prevalent degenerative disease of the CNS and is characterized by progressive memory loss, behavioral change, and cognitive dysfunction. One of the most characteristic pathological features of AD is the deposition of Aβ and consequently the formation of neurotic plaques, which is considered by many scholars to be the initial factor of neuronal degeneration.TNF is a common target to suppress inflammation in various diseases, and the same is true in AD. However, whether to use TNF‐α inhibitors for the treatment of AD is a matter of debate both in clinical trials and preclinical research. Pilot studies reported improvement in efficacy variables with treatment with etanercept, a TNF‐alpha inhibitor in patients with AD,Nuclear receptor PPARγ is another promising target. Activated PPARγ in microglia can stimulate NF‐κB  to reduce the production of inflammatory mediators.Moreover, attempts have been made to modulate other targets in microglia, such as p38, TLRs, NF‐κB, NLRP3, TREM‐2, and the CD200‐CD200R axis,4.2.2PD is a class of movement disorders characterized by bradykinesia, muscular rigidity, rest tremor, and postural and gait impairment. Its key pathological features include loss of dopaminergic neurons within the substantia nigra of the midbrain, Lewy pathology with α‐synuclein aggregation, and neuroinflammation.NLRP3, pro‐IL‐1β, and pro‐IL‐18. In α‐synuclein high expression mouse models, inhibiting the component of the TLR‐NF‐κB‐NLRP3 axis demonstrated a reduction in neuroinflammation, nigral dopaminergic degeneration and loss, and behavioral deficits.The induction effect of α‐synuclein on neuroinflammation depends in part on the activation of the TLR/NF‐κB pathway in activated microglia, which causes the increased transcription of 4.2.3ALS is a motor neuron disease that is characterized by the progressive loss of both upper and lower motor neurons. It can affect any voluntary muscle, resulting in heterogeneous presentations of motor deficits. The degree of microglial activation in the motor cortex is significantly correlated with the severity of clinical signs in ALS.Minocycline, a widely used broad‐spectrum antibiotic, selectively inhibits microglia by downregulating NF‐κB, that is, it suppresses M1 microglia during the progressive phase but does not affect the function of M2 microglia.Novel treatment approaches are under development for patients with ALS. NP001, a formulation of purified sodium chlorite, converts into taurine chloramine within monocytes/macrophages that induce proinflammatory macrophages to transform to a phagocytic state by inhibiting the NF‐κB pathway.4.3Metabolic homeostasis in the human body depends on the coregulation of metabolic organs such as the pancreas, liver, and adipose tissue. Meanwhile, the immune response and metabolic regulation are highly integrated, and their proper function depends on each other.4.3.1Obesity is the major cause of insulin resistance. However, obese patients do not always develop T2DM. Hyperglycemia occurs only when islet inflammation impairs the function of β cells, preventing them from producing enough insulin to compensate for insulin resistance. Macrophages that present the proinflammatory classically activated M1‐like phenotype dominate in T2DM islet inflammation,Considering the injurious effect of TNF‐α and IL‐1 produced by macrophages on the insulin signaling pathway, insulin resistance and glycemic control can theoretically be improved by therapies targeting these cytokines. In fact, the treatment of neutralizing cytokines is indeed efficacious in diabetic rodent models.PPARγ agonists, metformin, and glucagon‐like peptide‐1 receptor agonists (GLP‐1RAs) are three widely used and effective classes of antidiabetic agents. Their inhibitory effect on macrophages likely helps to explain their glucose‐lowering role. PPARγ agonists used in T2DM include thiazolidinedione, pioglitazone, and rosiglitazone, the modulation mechanism of which has been discussed in the AD section. Metformin has anti‐inflammatory actions by suppressing the activity of the NLRP3 inflammasome and the maturation of IL‐1β in macrophages by activating the AMPK pathway.4.3.2FFAs released from adipocytes, damaged hepatocytes, endotoxins, and translocated bacteria resulting from increased intestinal permeability and changes in gut microbiota are factors that activate Kupffer cells in NAFLD.At present, there are a variety of macrophage signaling‐targeted therapeutic approaches for NAFLD treatment that have shown efficacy in disease models.Cenicriviroc, a dual CCR2/CCR5 inhibitor, can inhibit monocyte‐derived macrophage recruitment mediated by CCL2 and significantly relieve hepatic steatosis and liver fibrosis in experimental models.4.3.3AS is the pathological basis of many cardiovascular diseases, such as myocardial infarction (MI), stroke, and cardiovascular death. Macrophages are considered to be the central cellular effectors of AS progression and run throughout all stages of disease development.Although measures to prevent AS have mainly focused on lipid‐lowering treatment until now, progress has been made in targeting inflammatory pathways. The well‐known CANTOS trial is the first large‐scale, randomized, double‐blind trial in human AS. This trial tested the efficacy of canakinumab, an IL‐1β monoclonal antibody, in 10,061 patients with previous MI on the basis of standard therapy.Colchicine, which is used for gouty arthritis and familial Mediterranean fever, has been extended to AS. The mechanism of action of colchicine is to disrupt tubulin and subsequently downregulate multiple inflammatory pathways and modulate innate immunity. Its impact on macrophages is mainly reflected in suppressing the release of various proinflammatory substances, such as ROS, NO, and TNF‐α, and inhibiting the maturation of IL‐1β via the NALP3/caspase‐1 or ROCK/caspase‐1 pathway.4.4When host defense is challenged by invading infectious microbes, macrophages are recruited and differentiate into a proinflammatory phenotype after engagement and recognition of pathogens. These macrophages are activated through inflammatory signaling pathways and can secrete inflammatory mediators to promote pathogen killing in the early stages of the disease; however, they can cause tissue damage and even organ failure in diseases such as tuberculosis (TB) and coronavirus disease 2019 (COVID‐19).4.4.1Mycobacterium tuberculosis (Mtb), an intracellular pathogenic bacterium.Mtb infection, in which macrophages can present dual characteristics of eliminating pathogenic Mtb and establishing niches for the persistence and dissemination of Mtb.TB, a worldwide infectious disease that has led to major health issues, is caused by Mtb chemotherapy. Imatinib, a TKI, can promote acidification and maturation of macrophage phagosomes, thus reducing intracellular Mtb survival.Mtb growth and replication in macrophage cytoplasm.Mtb infections.Mtb infection or exacerbate active Mtb infection if not administered concurrently with antimycobacterial chemotherapy. Therefore, further research on drug dose and clinical value is needed.Currently, there are two approaches targeting macrophages in the treatment of TB.4.4.2COVID‐19 is a contagious viral disease caused by SARS‐CoV‐2 and has become a global health concern due to its worldwide pandemic. Innate immunity functions as the first line of host defense, in which macrophages play a crucial role.Macrophages are first activated through the TLR4/TLR7‐TRAF6‐NF‐κB pathway by identifying the SARS‐CoV‐2 structure. Meanwhile, the virus enters the macrophage cytoplasm through the ACE2 receptor, activating the NLRP3 inflammasome in macrophages. Chemokines released by infected epithelial cells and cytokines released by activated NK cells and T cells further promote the recruitment and activation of macrophages through the JAK‐STAT pathway.It has been shown that blocking the entry and replication of virus in macrophages, as well as inhibiting the production of inflammatory factors, inflammasome activation and the type I IFN response can reduce the overactive immune inflammatory response. On the one hand, antiviral drugs remdesivir, molnupiravir, and nirmatrelvir‐ritonavir are proven to be effective in preventing hospitalization and death.4.5The approach for cancer treatment in recent years has focused on the tumor microenvironment (TME), which includes malignant, endothelial, stromal, and immune cells. Tumor‐associated macrophages (TAMs) are a plastic and heterogeneous cell population that account for the largest fraction of the myeloid infiltrate in most of human solid malignancies.4.5.1In tumor‐bearing mice, blockade of CCL2/CCR2 signaling inhibits the recruitment of inflammatory monocytes, delays cancer growth and metastasis, reduces postsurgical recurrence, and enhances survival.Several clinical trials targeting CCR2/CCL2 are ongoing. For example, a phase 1b trial in patients with borderline resectable and locally advanced pancreatic cancer showed decreased infiltration of TAMs in tumors and increased responsiveness to chemotherapy in patients treated with the small‐molecule CCR2 inhibitor PF‐04136309 combined with folinic acid, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), compared with those receiving FOLFIRINOX alone.In mouse models of various cancer types, including colorectal cancer (CRC), gastric cancer, and breast cancer, CCR5 inhibitors, including maraviroc, vicriviroc, and TAK‐779, have demonstrated antitumor effects.Preclinical studies with intracranial gliomas in both mice and rats clearly demonstrate that the marked increase in macrophage entry into irradiated tumors is mediated by the CXC motif ligand (CXCL) 12‐CXC chemokine receptor (CXCR) 4 pathway and that inhibition of this pathway blocks macrophage entry, thereby improving local control and survival after radiation therapy.4.5.2Several small molecules and antibodies of the CSF‐1/CSF‐1R axis have been developed and tested in a substantial array of animal models. For example, in one study, RG7155, a monoclonal antibody that inhibits CSF‐1R activation, caused macrophage cell death in vitro and reduced TAM densities accompanied by an increase in the CD8(+)/CD4(+) T‐cell ratio in vivo.In addition, various drugs targeting CSF‐1(R), including small‐molecule inhibitors such as sotuletinib, ARRY‐382, and pexidartinib and antagonistic monoclonal antibodies such as emactuzumab, cabiralizumab, LY3022855, and axatilimab, have been or are being tested in clinical trials. For patients with tenosynovial giant cell tumor, a rare, locally aggressive neoplasm that overexpresses CSF‐1, objective and complete responses were observed during a phase 1 study of emactuzumab,4.5.3CD47 in many cancer types, disguising them as healthy cells and avoiding phagocytosis. Therefore, strategies targeting the CD47/SIRPα axis to enable tumor cell killing through cellular phagocytosis have emerged as promising cancer immunotherapies.Tumor cells overexpress In a variety of immunocompetent mouse tumor models, including ovarian cancer,Clinical trials are underway for both solid and hematological malignancies using anti‐CD47 antibodies. To date, anti‐CD47 antibodies such as Hu5F9‐G4,As mentioned above, Siglec‐10 is an inhibitory I‐type lectin that can be expressed by TAMs and binds the potent anti‐phagocytic signal CD24 that is overexpressed in several solid tumor types. The binding of CD24 to Siglec10 elicits inhibitory signaling that blocks the cytoskeletal rearrangement required for cellular engulfment by macrophages. Clinical data have shown that CD24 expression is significantly higher in triple‐negative breast cancer cells than in normal breast cells.4.5.4The NF‐κB signaling pathway is important in cancer‐related inflammation and malignant progression. Studies have shown that when NF‐κB signaling is inhibited specifically in macrophages in the TME, they switch to M1 and become cytotoxic to tumor cells.PI3K, the upstream regulator of Akt, has been shown to mediate M2 macrophage phenotypes. Activation of PI3Kγ signaling in macrophages has been reported to drive TAM immunosuppressive activities in models of lung cancer,5Macrophages are widely present in almost all tissues of adult and play important roles in physiological and pathological processes. Although extraordinary efforts have been made to understand the functions and regulating mechanisms of macrophages, our evolving knowledge about the intricate signaling network within and between macrophage and its microenvironment is still not satisfactory yet.Streptococcus uberis infection.CCL2 expression through TLR4/MyD88 signaling, which activates JNK and promotes M1 polarization;The crosstalk of different signaling pathways and the bidirectional effect of the same signaling pathway leads to the complexity of macrophage regulation. First, the perception and integration of various external stimuli by macrophages lead to the activation of multiple signaling pathways and significant crosstalk of different signaling pathways, while the complicated crosstalk of signaling pathways can cause synergistic or antagonistic effects in macrophage regulation. An example of the synergistic regulation of macrophage inflammatory responses is the crosstalk between PI3K/Akt/mTOR and TLRs/NF‐κB signaling to promote inflammatory responses in It is the complexity of the associated regulatory signaling pathways that allows macrophages to exhibit a remarkable degree of functional and phenotypic plasticity and heterogeneity in response to environmental stimuli. Many of these macrophage phenotypes are tissue‐ and environment‐specific and play different or even opposing roles in human diseases. We have outlined various types of antibody‐based drugs and small molecule drugs targeting environmental signals or macrophage receptors and intracellular signaling pathways. These drugs exert therapeutic effects by blocking or activating macrophage‐related signaling pathways and gene transcription to alter the functional or physiological properties of macrophages.However, with the development of drugs aiming at different molecular targets of macrophages, the next challenge is to effectively and selectively deliver these drugs to specific disease‐related macrophage subpopulations to improve efficacy and reduce off‐target effects. Studies have found that targeting of macrophages by phagocytosis of nanoparticles or liposomes or by antibodies binding to macrophage‐specific receptors can improve the therapeutic efficacy of drugs.Furthermore, due to the complexity of macrophage regulatory pathways, the efficacy of a single drug is limited, and combination therapy is a promising direction. For instance, CD40 could enhance the phagocytic activity of macrophages recovered by CD47‐SIRPα blockade, and a fusion protein with high affinity to bind CD40 and CD47 performed better than either CD47 blockade or CD40 agonist alone in a mouse CT26 tumor model.To sum up, macrophage‐targeted therapy has been proven to be a promising therapeutic strategy. However, due to the wide distribution of macrophages throughout the organism and the diversity and complexity of regulatory factors, further understanding of the functional and phenotypic heterogeneity of macrophages in specific microenvironment or disease is needed to improve the efficacy of targeted macrophage therapy and reduce off‐targeting. We comprehensively introduce the signal pathways that regulate macrophages and some key regulators in these pathways that can be used as therapeutic targets. The in‐depth understanding of these signal pathways and molecular targets is important for guiding the development of new therapeutic approaches.M.L., M.J.W., Y.J.W., and H.F.Z. conceived and drafted the manuscript. G.N.Z. edited and revised the manuscript. Q.L.G. supervised and revised the manuscript. All authors read and approved the final manuscript.The authors declare no conflicts of interest.No ethical approval was required for this study.


GPCR) family stands as the largest group of membrane proteins. Alterations in GPCR gene expression and dysregulation of signal transduction have been recognized as the markers of malignancy. As a member of the GPCR family, G protein‐coupled receptor 37 (GPR37) exhibits unknown functions in tumors, particularly in non‐small‐cell lung cancer (NSCLC)Lung cancer is a major public health concern and represents the most common cause of cancer‐related death worldwide. Among eukaryotes, the G protein‐coupled receptor  In NSCLC, the expression of GPR37 is markedly higher than that in corresponding normal tissues. We found that elevated GPR37 expression predicts an unfavorable prognosis. (2) It was demonstrated that GPR37 positively regulates NSCLC cell invasion, migration, and proliferation, suppresses cell apoptosis, heightens resistance to cisplatin, and promotes tumor formation and growth. Conversely, we observed that GPR37 knockdown suppresses NSCLC cell invasion, migration, and proliferation, promotes cell apoptosis, increases sensitivity to cisplatin, and affects tumor formation and growth. (3) GPR37 activates PI3K/Akt/mTOR signal transduction pathways to mediate epithelial‐mesenchymal transition (EMT), thereby promoting the progression of NSCLC.It was suggested that GPR37 acts a crucial role in promoting the occurrence and development of NSCLC. Knockdown of GPR37 significantly inhibits the occurrence and development of NSCLC. Therefore, our findings demonstrated that GPR37 may represent a viable therapeutic target for NSCLC. In 2021, World Health Organization (WHO) data show that lung cancer is the most common cause of cancer‐related deaths in 60 countries and the second largest malignant tumor worldwide. Despite significant advancements in medical technology and therapeutic strategies, the 5‐year survival rate of patients afflicted with non‐small cell lung cancer (NSCLC) stands at an approximate 20%.G protein‐coupled receptors (GPCRs) constitute the most extensive family of membrane proteins within eukaryotes. Altered GPCR gene expression and dysregulated signal transduction are recognized hallmarks of malignancy.In the early 1980s, a correlation between cancer and epithelial‐mesenchymal transition (EMT) was reported. EMT is a ubiquitous process that characterizes the vast majority of tumors during their progressive stage; thus, cancers of epithelial origin are determined by EMT processes.Moreover, the PI3K/Akt/mTOR pathway acts a crucial role in modulating cell growth and metabolism. The relevance of PI3K in oncogenesis was initially postulated in 1985 and has since been well‐documented.Through the screening of the Cancer Genome Atlas (TCGA) database, we found that the expression of GPR37 shows a marked increase in patients afflicted with NSCLC and appears to exert a discernible influence upon disease prognosis. Based on the analysis of clinical data at our center, we also obtained similar findings, that is, the expression of GPR37 in para‐cancerous tissues of NSCLC was lower than that in tumor. Moreover, the study demonstrated that the expression of GPR37 was lower in normal alveolar epithelial cells than that in different tumor cell lines. GPR37 can drive the invasion, migration and proliferation of tumor cells, and has a certain anti‐apoptotic effect. GPR37 is able to elevate the resistance of platinum‐based chemotherapy drugs and promote EMT in NSCLC and activate PI3K/Akt/mTOR signal transduction pathways. GPR37 knockdown can inhibit EMT in NSCLC and affect the activation of PI3K/Akt/mTOR signal transduction pathways. Consequently, further in‐depth research on GPR37 will not only help reveal the development mechanism and tumor drug resistance mechanism of NSCLC but also use GPCR as a drug target to provide a theoretical basis for individualized treatment options for NSCLC.22.1We used the database information in TCGA, applied the online analysis tool TIMER2.0, and entered GPR37 in the Cancer Exploration module to compare the expression of GPR37 between normal tissues and tumors. Information obtained from TCGA encompassed 501 samples of lung squamous cell carcinoma (LUSC) with 51 samples of normal control tissues, 515 samples of lung adenocarcinoma (LUAD) with 59 samples of normal control tissues. Moreover, the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) was progressed to select the patient information from TCGA and GTEx databases, and the differential analysis of GPR37 expression was performed by statistically analyzing the data in LUSC and LUAD tumor, and normal control tissues. We also analyzed the NSCLC dataset of NCBI‐GEO database to investigate the differential expression of GPR37 in NSCLC samples and normal samples. We obtained gene expression profiles of GSE18842,The Survival Analysis module of GEPIA2 was applied to evaluate the overall survival (OS) of GPR37. The GEPIA platform implements the log‐rank test for hypothesis evaluation, with the inclusion of 95% confidence interval findings in its outcomes.2.2In this study, 20 patients with a histologically confirmed diagnosis of NSCLC who underwent surgery were recruited. The lung cancer and adjacent tissues of these participants were collected. The admission criteria were as follows: (1) age ≥18 years, (2) definite diagnosis of NSCLC by postoperative histopathology, and (3) no history of other tumors. The exclusion criteria were as follows: (1) chemotherapy and radiotherapy for other diseases, (2) history of other malignant tumors, and (3) diseases with immunodeficiency. Specimens were collected and then cryopreserved in liquid nitrogen for storage at −80°C until RNA extraction was performed.All participants provided informed consent before enrolling in this study, which was endorsed by the Ethics Committee of the First Hospital of Jilin University (2021‐170).2.3Sampling and fixing, dehydration, immersion in wax, embedding, sectioning, and dewaxing were performed. For hematoxylin and eosin (HE) staining of tissue sections, HE staining, eosin staining, dehydrating sections, transparent sectioning, and mounting were performed.2.4To prepare human lung tissue slices, the sodium citrate buffer was added, boiled, fixed, and washed, and then, an endogenous peroxidase‐blocking agent was added. After washing, a nonspecific staining blocker was added. After drying, the GPR37 polyclonal antibody was added dropwise as the primary antibody . The primary antibody was removed, and a secondary antibody of goat anti‐rabbit polymer  was subsequently introduced. Streptavidin‐peroxidase was utilized, and the specimen was then incubated at room temperature. Thereafter, DAB chromogenic solution was added and then soaked in hematoxylin for counterstaining, and the slides were dehydrated in gradient alcohol and then soaked in xylene. Mount the slides for observation under a microscope.2.52. According to different requirements, 10% fetal bovine serum (Biosharp), penicillin–streptomycin liquid (HyClone), and Hepes (Solarbio) were added to the culture medium.Frozen Mrc‐5, BEAS‐2B, CCD‐19Lu, A549, H1299, Calu‐1, Calu‐3, H460, and HCC827 cells were taken from −80°C liquid nitrogen storage and immediately placed in a 37°C water bath. BEAS‐2B, CCD‐19Lu, A549, H1299, and H460 cells were cultured in Dulbecco's modified eagle medium (Gibco), Mrc‐5, Calu‐3, and Calu‐1 cells were cultured in minimum essential medium (Invitrogen), while HCC827 cells were cultured in RPMI 1640 (Gibco). All cells were cultured under standard conditions, which involved incubating them at 37°C in a 5% CO2.6For the extraction of total tissue protein, human lung tumor tissue and matched para‐cancerous tissue were taken out from −80°C liquid nitrogen storage. The tissues were shredded, thoroughly grounded, and added with radioimmunoprecipitation assay  to fully lyse it. Then, the sample was supplemented with precooled phosphate‐buffered saline , following which it was resuspended and subjected to centrifugation. The resulting supernatant was then collected via aspiration.Extraction of total cell protein: The original culture medium was removed from the cell culture dish, washed with PBS, and then added with an appropriate amount of lysate (the amount of lysate depends on the size of the culture dish) until the lysate completely covered the cells and fully lysed. The lysate and cell debris were transferred to an Eppendorf tube and centrifuged, and the supernatant was aspirated.The bicinchoninic acid protein concentration assay kit (Biosharp) was utilized to calculate the protein content. The protein content was transferred to polyvinylidene difluoride membranes and subsequently obstructed with 5% nonfat dry milk for 60 minutes. Once obstructed, the membrane was incubated with primary antibodies. The primary antibodies include GPR37 , GAPDH , vimentin , N‐cadherin , E‐cadherin , P‐AKT1 , P‐EIF4EBP1 , p70S6K , P‐p70S6K , mTOR , P‐mTOR , PI3K , P‐PI3K , EIF4EBP1 , and AKT1 . The secondary antibodies were HRP goat anti‐rabbit IgG and HRP goat anti‐mouse IgG .2.7Total RNA was extracted from lung cancer tissues, para‐cancerous tissues, and cells via Trizol reagent (Invitrogen). Using a TransStart Top Green qPCR SuperMix Kit (TransGen), the extracted RNA was reverse‐transcribed into cDNA. Then, Real‐time PCR System (Applied Biosystems) was carried out for qRT‐PCR. The expressions of GPR37 mRNA and GAPDH mRNA were determined through qRT‐PCR. qPCR data were normalized according to 2−ΔΔCt method, which facilitates the analysis of relative gene expression in qRT‐PCR experiments.2.8The GPR37‐siRNA sequences were as follows: GPR37‐shRNA‐1F, CCGGCGAGGGAATAAACGGCAGATTCTCGAGAATCTGCCGTTTATTCCCTCGTTTTT; GPR37‐shRNA‐1R, AATTAAAAACGAGGGAATAAACGGCAGATTCTCGAGAATCTGCCGTTTATTCCCTCG; GPR37‐shRNA‐2F, CCGGCCTTAATATCATCAGCCAGTTCTCGAGAACTGGCTGATGATATTAAGGTTTTT; GPR37‐shRNA‐2R, AATTAAAAACCTTAATATCATCAGCCAGTTCTCGAGAACTGGCTGATGATATTAAGG; Mock‐shRNA‐F, CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG; Mock‐shRNA‐R, AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG. Plasmids were extracted based on the instructions of the plasmid extraction kit . Transfection was performed using Lipofectamine 3000 (Invitrogen). In this study, the lentivirus packaging plasmid with GPR37 overexpression and knockdown were constructed and transfected into H1299, H460, and A549 cells to obtain stable expressions of GPR37 overexpression (GPR37 group), empty vector group (vector group), GPR37 knockdown group (shGPR37 group), and GPR37 knockdown control group (sh‐mock group).2.9To evaluate cell proliferation, three distinct assays were employed: the colony formation assay, the 5‐ethynyl‐2′‐deoxyuridine (EdU) Cell Proliferation Kit (Invitrogen), and the Cell Counting Kit‐8  assay. For conducting the CCK‐8 assay, 4000 cells per well were seeded onto 96‐well plates and thereafter cultivated for a period of 24 h. Subsequently, 10 μL of CCK‐8 reagent was added to each well at 24, 48, 72, and 96 h, and the plate was left to incubate for 2 h in a 37°C incubator. Then, the absorbance at 450 nm was quantified through a microplate reader. For the EdU assay, NSCLC cells were seeded into 6‐well culture plates and analyzed based on the kit instructions. For the colony formation assay, cells in different groups were taken for digestion, blown repeatedly to form a single cell, and prepared into a cell suspension. Then, 200 cells per well were added to a 6‐well plate and placed in an incubator. After culturing for 2 weeks, formaldehyde was added for fixation and then stained with 0.1% crystal violet.2.10Detection of apoptosis was executed by implementing the Cell Cycle and Apoptosis Analysis Kit (Beyotime). All procedures were progressed according to the manufacturer's instructions. Cell apoptosis was detected using the DxFLEX flow cytometer (Beckman Coulter). At the excitation wavelength of 488 nm, light scattering and red fluorescence were detected. Different cell lines adjusted different parameters.2.11Subsequent to cell digestion, a single‐cell suspension was established and deployed for seeding into 6‐well culture plates. Plates were scratched manually after a 24 h incubation. The scratched culture plate was gently washed with PBS to remove scratched cells, serum‐free medium was utilized, and pictures were taken at 0 h. Afterward, the culture plate was put into the incubator again for 24 h. Using a microscope, the peripheral cells were checked whether they have migrated to the central scratch area, and pictures were taken.2.125/mL. Following this, 200 μL of the refined cell suspension was added to the top chamber while taking keen precautions to avoid air bubbles as much as possible. Subsequent to the addition of serum‐free medium containing the tumor cells into the upper chamber, 500 μL of complete medium augmented with fetal bovine serum was supplemented to the lower well. The resultant plate was thereafter placed inside the incubator for 48 h. As soon as the incubation was completed, the cells situated in the lower chamber were duly fixed in precooled 10% formaldehyde and stained using 0.1% crystal violet. Finally, cell enumeration was conducted under a microscope.Tumor cells from different groups of distinct cell lines were extracted and resuspended in serum‐free medium. Thereafter, the cells were counted, and the resultant cell density was calibrated to 1–10 × 102.136 cells) were mixed with Matrigel and injected subcutaneously into the middle and outer sides of the right axilla of nude mice. The skin of the nude mice was disinfected before injection. The body weight of nude mice and subcutaneous tumor size were detected every 7 days. On Day 28, the nude mice were sacrificed. The tumor volume was measured as follows: volume = length × width2 × Π/6, and the tumor growth curve was drawn.Twenty‐four healthy Balb/c nude female mice of specific pathogen‐free level  were selected for the experiment. The Animal Experiment Center of Jilin University provided nude mice. Throughout the experiment, all nude mice were consistently maintained under standard conditions, with the ambient temperature being 22 ± 2°C and the relative humidity remaining around 50% ± 5%, artificially controlled diurnal cycle lighting for 12 h, kept in separate cages, and given full‐price nutritional feed. No differences in other exposure factors, such as age, and body weight, were found among the groups. The operators involved in the animal experimentation conducted at the First Hospital of Jilin University had all duly cleared the qualification examination for experimental animals and performed the procedures within a strictly controlled environment. The experimental methodology for all animal studies incorporated in this study had been granted ethical clearance by the Animal Experiment Ethics Committee of the First Hospital of Jilin University (20210270). Different groups of A549 tumor cells .All data analyses in this experiment were progressed via IBM SPSS Statistics version 19.0 (IBM Corp.). The outcomes of continuous variables have been delineated herein as mean ± standard deviation. Subsequently, comparative analysis between two groups was performed via 33.1p < 0.001). The outcomes of this study have been graphically depicted in Figure To seek the expression of GPR37 between normal tissues and different tumors, TCGA database was used, and the online analysis tool of TIMER 2.0 was applied to input GPR37 in the Cancer Exploration module. Notably, the expressions of GPR37 in LUAD and LUSC were markedly elevated compared to the control group (p > 0.05). The outcomes of this analysis have been graphically presented in Figure The GEPIA2 online tool was used to select lung cancer patient information from Genotype‐Tissue Expression (GTEx) and TCGA databases, and differences in GPR37 expression were analyzed in LUAD and LUSC tissues and normal tissues. In 483 LUAD tissues and 347 normal tissues analyzed, it was observed that the expression of GPR37 was indeed amplified in the LUAD tissues. Among the analyzed 338 normal tissues and 486 LUSC tissues, it was discovered that the expression of GPR37 was notably enhanced in the LUSC tissues. In contrast, the analysis showed that the expressions of GPR37 in the two types of lung cancers were not remarkably divergent from those detected in the control group  from the GEO database to study the differential expression of GPR37 in NSCLC samples and normal samples. The three data sets obtained 85 NSCLC samples and 84 control samples. Data analysis revealed that the expression level of GPR37 was significantly increased in NSCLC samples compared with controls Figure . KEGG paThe Survival Analysis module of GEPIA2 was used to evaluate the OS and disease‐free survival (DFS) of GPR37. Participants were divided into the high and low GPR37 expression groups. Patients with LUAD and LUSC in TCGA database were selected for statistical analysis.p < 0.05). In addition, no remarkable difference was detected in DFS between the two groups (p > 0.05). These results are exhibited in Figure Evaluating the OS, the findings showed that the high GPR37 expression group exhibited a notably inferior prognosis in contrast to the low GPR37 expression group, displaying significant statistical significance (3.2p < 0.001) . Both tumor and para‐cancerous tissue samples were additionally subjected to IHC analyses, which confirmed a significant difference in GPR37 expression levels between NSCLC and para‐cancerous tissues (p < 0.05). A detailed outline of the examination results is available in Table The demographic characteristics of the patients are shown in Table ) Figure . Moreovep > 0.05). The results of the BEAS2B cell line, which was utilized as the control group in the statistical analysis, were conducted to evaluate the expression levels. However, the expressions of GPR37 in the six NSCLC cell lines were considerably greater than those observed in the BEAS2B cell line, with highly statistical differences (p < 0.001). We chose H1299, H460, and A549 cell lines with higher expression for subsequent experiments. By WB detection, the expression levels of Mrc5, BEAS2B, and CCD‐19Lu were very low in normal human lung tissue cells; however, the expression of GPR37 was notably amplified in NSCLC cell lines (p < 0.001).Three normal human lung tissue cell lines  were selected as the control group, and different NSCLC cell lines  were used to verify GPR37 expression. Through RT‐PCR and WB detection, the expressions of GPR37 in normal human lung cells and tumor cells were observed, as shown in Figure 3.3GPR37 gene had a knockdown effect. In addition, the expression level in the shGPR37‐1 cell line is more inhibited than that in the shGPR37‐2 cell line, considering that the knockdown efficiency of shGPR37‐1 is better.Two A549 cell lines with stable knockdown of GPR37 were successfully constructed. After 10 passages, the GPR37 knockdown efficiency was detected by WB Figure . WB detep < 0.001). In H1299, H460, and A549 cell lines, the cell viability rate was lower in the shGPR37‐1 group than in the shGPR37‐2 group. Statistically remarkable differences were detected amid the three groups . The findings corresponded to the results in Figure To further verify the expression efficiency of shGPR37‐1 and shGPR37‐2, three cell lines including H1299, H460, and A549 were constructed, and the corresponding GPR37 knockdown negative control group (sh‐mock group) was created. Through CCK‐8 experiment, the absorbance with time under the 450 nm wavelength light of the microplate reader was compared Figure . After 73.4p < 0.001).Three stably transfected cell lines of A549, H1299, and H460 overexpressing GPR37 (GPR37 group) and a stably transfected cell line transfected with an empty vector packaging plasmid (vector group) were constructed. Through the CCK‐8 experiment, absorbance change times under the 450‐nm wavelength light of the microplate reader were compared Figure . After 7p < 0.05). Moreover, the cell colony formation potential of the shGPR37 group was notably diminished in contrast to that of the sh‐mock group. In A549, H1299, and H460 cell lines, the results were all statistically significant (p < 0.01).The colony formation ability was detected by a clone formation experiment. The ordinate on the right of the histogram is the number of clones formed, and the abscissa presents the groups analyzed Figure . It was p < 0.01, p < 0.05, and p < 0.05). Conversely, a notable decrease in DNA synthesis ability was observed in the shGPR37 group concerning the sh‐mock group .The DNA synthesis ability was detected by the EdU experiment. The vertical axis on the right of the histogram is the ratio of EdU‐positive cells to the total number of cells, and the horizontal axis presents the groups analyzed Figure . In H1293.5p > 0.05, p > 0.05, and p > 0.05). The apoptosis rate of the shGPR37 group was remarkably higher than that of the sh‐mock group .FACS was employed to detect cell apoptosis. H1299, H460, and A549 cell lines were evaluated Figure . In H1293.6p < 0.001, p < 0.001, and p < 0.001). The cell migration rate of the shGPR37 group was considerably weaker than that of the sh‐mock group, showing statistical differences .The migration ability of A549, H1299, and H460 cell lines in the GPR37, vector, shGPR37, and sh‐mock groups was observed by the cell scratch test Figure . In H129p < 0.001, p < 0.01, and p < 0.01). The cell migration rate of the shGPR37 group was weaker than that of the sh‐mock group, showing statistical differences .The cell invasion ability was evaluated via the Transwell experiment Figure . In H1293.7p < 0.05). As the concentration increased, the difference remained significant and persisted  until the concentration reached 3.0 μg/mL; no statistical difference was noted in the cell viability between the two groups (p > 0.05). Meanwhile, H1299 and H460 cell lines obtained similar results.Gradient concentrations of cisplatin  were added and cultivated for 24 h. The CCK‐8 experiment was conducted to explore the influence of GPR37 on tumor cell regulation in the context of cisplatin sensitivity, as displayed in Figure p < 0.01). As the concentration increased, the difference was still significant and persisted . No statistical difference in cell viability was detected between the two groups until the concentration reached 2.5 μg/mL (p > 0.05). The results for H460 and H1299 cell lines trended similarly to those of the A549 cell line.After adding different concentrations of cisplatin, changes in the cell viability in the sh‐mock and shGPR37 groups were analyzed Figure . In the p < 0.01), whereas the apoptosis rate of the shGPR37 group was higher than that of the sh‐mock group (p < 0.01). The results for the H460 and H1299 cell lines trended similarly to those for the A549 cell line. In the FACS detection, the H1299 and H460 cell apoptosis levels in different groups under the action of cisplatin were similar to that of the A549 cell line.H1299, H460, and A549 cell lines in the logarithmic growth phase were selected in different groups. Cisplatin at a concentration of 1.0 μg/mL was added for 24 h. The effect of GPR37 on the regulation of tumor cell sensitivity to cisplatin was observed by FACS Figure . In the 3.8p < 0.001). The tumor volume and weight of the shGPR37 group were both lower than those of the sh‐mock group, and the difference was statistically significant (p < 0.001). Figure To evaluate the effect of GPR37 on NSCLC growth in animals, 24 randomly selected female nude mice were separated into four groups, as described in the methodology. The A549 cells in the vector, GPR37, sh‐mock group, and shGPR37 groups were treated and injected subcutaneously into the middle and outer sides of the right axilla of nude mice. The volume of the subcutaneous tumor was calculated every 7 days after injection, and the body weight of the nude mice was measured every 7 days until Day 28 . In addition, the high expression of GPR37 was shown to predict poor prognosis in NSCLC patients (p < 0.05), suggesting that GPR37 could pose as an independent prognostic indicator for NSCLC. These findings highlight the potential clinical utility of GPR37 in guiding personalized cancer treatment and outcomes for NSCLC patients. Subsequently, through clinical verification, the expressions of GPR37 in different tumor tissues were remarkably higher than that in paired para‐cancerous tissues (p < 0.001). However, because the follow‐up period of the patients was <5 years, no further analysis of the OS was performed. Related work still needs to be further improved. Through cell experiments, we found that GPR37 was upregulated in NSCLC cell lines, which verified the important role of GPR37 in NSCLC. Studies have demonstrated that GPR37 is highly expressed in NSCLC and impacts the prognosis, suggesting its important role in NSCLC progression.Our study also involved a screening of a public database, where we discovered a significant raise in GPR37 expression in NSCLC patients compared to the healthy population  and transforming growth factor Beta (TGF‐β) lead to EMT, they can also induce drug resistance of NSCLC cells to cisplatin and paclitaxel.PI3KCA frequently mutates in human cancers,Dysregulated PI3K/Akt pathway is oncogenic in some cases. The crucial role of dysregulated PI3K activity in cancer onset and progression has been extensively documented. For instance, the PI3K p110 subunit gene This study also has shortcomings. The number of clinical verification samples was too small. The different expression levels and prognosis of GPR37 were not analyzed clinically. This study lacks in‐depth animal studies to verify the mechanism of GPR37 in promoting NSCLC development. The novelty of this study is related to its evaluation of the involvement of GPR37 in NSCLC. GPR37 knockdown was found to act a tumor‐suppressor role in NSCLC and could be used as a potential therapeutic target in NSCLC. This study also confirmed for the first time that GPR37 can promote the EMT of NSCLC and GPR37 can activate the PI3K/Akt/mTOR signal transduction pathway to promote NSCLC progression. This study elucidated the expression changes of a variety of downstream genes caused by the high GPR37 expression in NSCLC at the molecular level, pointing out the molecular mechanism of accelerating the proliferation of NSCLC cells, inhibiting cell apoptosis, increasing cell migration and invasion ability, and increasing the probability of drug resistance. This new idea of GPR37 as an important regulatory gene was proposed and might be a novel target in the therapy of NSCLC.Han Liu: Funding acquisition ; methodology ; project administration ; writing – original draft . Yingjie Zhu: Methodology . Huikun niu: Data curation ; formal analysis . Jing Jie: Data curation . Shucheng Hua: Conceptualization ; investigation . Xiaoxue Bai: Methodology . Shuai Wang: Conceptualization ; writing – review and editing . Lei Song: Conceptualization ; writing – review and editing .This work was supported by Science and Technology Development Program Project of Jilin Provincial Department of Science and Technology (YDZJ202201ZYTS108) and the Science and Technology Development Project of Changchun Science and Technology Bureau (21ZGM20).The authors confirm that the study was performed without any financial or commercial relationships that might be perceived as potential conflicts of interest.The research method of animal experiments was endorsed by the Animal Experiment Ethics Committee of the First Hospital of Jilin University (20210270). All patients signed the informed consent. The trial was endorsed by the Ethics Committee of the First Hospital of Jilin University (2021–170).