Study On The Phenomenon And Mechanism Of Tumor-Derived GABA Promoting Lung Cancer Progression By Influencing TAMs Polarization

Background:Tumor-associated macrophages(TAMs)are a major component of the tumor microenvironment(TME).Initially,TAMs were considered a cell type differentiated from monocytes in the bone marrow,playing a critical role in tumor growth and metastasis.Chemotactic factors such as CSF1 and CCL2 secreted by tumor cells attract circulating monocytes to the TME,where they differentiate into macrophages.With the deepening of research,a general consensus has been reached regarding the role of TAMs in tumor progression.TAMs initially tend toward an M1 phenotype,which can phagocytose and present tumor antigens,activate T-cell-mediated tumor cell killing,and stimulate immune cells through the secretion of cytokines,enhancing the immune response and suppressing tumor progression.However,as the tumor progresses,tumor cells secrete specific signaling molecules that adjust the polarization of macrophages towards an M2 phenotype,promoting tumor growth and metastasis through various mechanisms.In addition,M2-type TAMs can suppress immune responses and facilitate tumor immune evasion.Gamma-aminobutyric acid(GABA)is a major neurotransmitter that plays an inhibitory regulatory role in the central nervous system.In recent years,some studies have suggested that GABA can promote macrophage secretion of IL-10 through GABAa-type receptors,hindering T-cell recruitment to tumors and attenuating the host’s anti-tumor activity.Other research has found that GABA itself can promote tumor growth and immune suppression through GABAb-type receptors,mediating β-catenin-dependent metabolism and enhancing tumor progression.GABA has been confirmed to be present in the murine tumor immune microenvironment and can participate in promoting tumor progression.This study employed GABAa-type receptor inhibitors in a mouse subcutaneous tumor model to investigate the role of GABA in tumor progression.Furthermore,exogenous GABA was added during the cultivation of bone marrow-derived macrophages(BMDMs)to explore GABA’s regulatory effect on TAM polarization.The study also confirmed the impact of TAMs on tumor progression under GABA conditions and demonstrated GABA’s pro-angiogenic role in tumor microvasculature through clinical samples and experiments.Based on these findings,we verified the phenomenon and mechanism by which tumor-derived GABA promotes lung cancer progression by affecting TAM polarization,providing a new therapeutic target for lung cancer treatment and confirming a novel mechanism of GABA in tumor progression.Research Objective:This study primarily investigates the role of tumor cell-derived GABA in lung cancer progression and verifies the regulatory mechanism of GABA on TAM polarization.It also explores the reasons behind GABA promoting tumor progression through its impact on TAMs,paving the way for new drug targets in cancer therapy.Research Methods:First,we analyzed the expression abundance of the GAD1 gene in lung cancer tissues using publicly available data and examined the survival curves of patients categorized based on GAD1 gene expression levels,verifying the crucial role of the GAD1 gene in tumors.Next,we established a mouse subcutaneous tumor model and treated it with GABAa receptor inhibitors to validate the impact of GABA on tumor cell growth in vivo.After depleting macrophages in mice,we further confirmed the role of macrophages in GABA-regulated tumor progression using mouse tumor-bearing experiments.Subsequently,we treated tumor-bearing mice or in vitro cultured murine BMDMs with GABAa receptor inhibitors or GABA and detected the proportion and polarization direction of macrophages in tumors or BMDMs using q PCR,flow cytometry,and Western blot.We induced BMDMs to polarize in different directions and collected macrophage-conditioned media.We then cultured tumor cells using these media and employed CCK8 assays,colony formation assays,transwell assays,and wound healing assays to verify the phenomenon of GABA affecting tumor cells through macrophages.Furthermore,we investigated the influence of GABA under different induction conditions on the JAK1/STAT6,JAK2/STAT3,and NF-κB pathways in BMDMs using Western blot.Lastly,we employed immunohistochemistry,immunofluorescence,and tube formation assays to explore the pro-angiogenic effects of GABA in clinical tissue samples,in vivo,and in vitro experiments.Additionally,we used PCR and ELISA to detect the expression levels of factors related to angiogenesis.Results:Lung cancer cells exhibited higher GAD1 gene expression relative to normal tissues,and high GAD1 gene expression in lung adenocarcinoma negatively correlated with patients’ overall survival rates.Mouse tumor-bearing experiments showed that GABAa receptor inhibitors could inhibit tumor progression by affecting the polarization direction of TAMs.Moreover,GABAa receptor inhibitors reduced the proportion of macrophages in tumors and increased the proportion of M2 macrophages among TAMs.Our in vitro experiments with BMDMs and RAW.264.7 cells revealed that under different macrophage stimulation conditions,GABA could bind to GABAa receptors on macrophage surfaces,activate the JAK1/STAT6 signaling pathway,and inhibit the JAK2/STAT3 and NF-κB pathways.This promoted macrophage polarization towards the M2 phenotype and inhibited polarization towards the M1 phenotype.However,GABA alone did not possess macrophage polarization capabilities.In addition,we verified through CCK8 assays,colony formation assays,transwell assays,and wound healing assays that GABA could promote tumor cell proliferation and migration by participating in macrophage-mediated regulation of tumor cells.Furthermore,immunohistochemistry results indicated a positive correlation between GABA expression levels in patient tumor tissues and tumor microvessel density.We assessed the expression levels of microvessels in mouse subcutaneous tumors using immunofluorescence and demonstrated through in vitro tube formation assays that GABA could bind to GABAa receptors on macrophage surfaces,regulate the expression levels of factors such as FGF2 in macrophages,and modulate the process of tumor microvessel formation.Conclusion:Tumor-derived GABA activates the JAK1/STAT6 signaling pathway through GABAa receptors on the surface of macrophages,promoting macrophage polarization towards the M2 phenotype and inhibiting the JAK2/STAT3 and NF-κB signaling pathways,which suppresses macrophage polarization towards the M1 phenotype.By modulating the polarization states of macrophages,GABA further enhances the proliferative and migratory capabilities of tumor cells.Additionally,by regulating the secretion of FGF2 from macrophages,GABA promotes the formation of tumor microvessels,providing a favorable growth environment for the tumor.