Regulatory Effect Of Kynurenine On Macrophage Recruitment And Differentiation In Tumor Microenvironment Of Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma(OSCC)is one of the most familiar malignant tumors of the head and neck.OSCC has the characteristics of high local infiltration rate,strong metastatic ability,and is easy to relapse in the later stage.tumor-associated macrophages(TAMs)plays an important role in the regulation of tumor microenvironment(TME).The occurrence,progression and local metastasis of OSCC have relation to TME,but the specific mechanism is not clear.TAMs can produce a variety of cytokines and pro-inflammatory factors and affect the M2 polarization of TAMs to regulate tumor growth,invasion and metastasis.Indoleamine 2,3-dioxygenase 1(IDO1)as a key enzyme can metabolize tryptophan into L-Kynurenine(KYN).In recent years,it has been confirmed that KYN,a metabolite produced by tumor cells decomposing tryptophan through IDO,can promote tumor growth and migration,participate in the immune editing process,and inhibit tumor clearance by the immune system.However,few study reported the correlation function and molecular mechanism of KYN and other metabolites in OSCC,and the specific mechanism of KYN in OSCC tumor microenvironment is not clear.Therefore,the main purpose of this study is to explore the role of differential metabolites between cancer tissue and paracancerous tissue of oral squamous cell carcinoma in growth and metastasis of OSCC,and to further clarify the interaction between tumor cells and TAMs in the TAM of OSCC and its possible mechanism,so as to supply potential therapeutic strategies for the intervention and treatment of OSCC.Aim and MethodsPart Ⅰ: We collected 10 pairs of cancer tissue samples from patients with OSCC,and conducted non-targeted amino acid metabonomics study of these tissues based on a liquid chromatograph triple quadrupole mass spectrometer(LC-MS/MS)method.The differences of metabolites between cancerous tissues and paracancerous tissues were studied by multi-dimensional statistical analysis,and the metabolites with significant differences were detected out.16 pairs of preoperative and postoperative plasma samples from patients with OSCC,and 18 healthy plasma samples were verified in ELISA to determine its diagnostic efficacy for head and neck tumors,and to determine the role of its evaluation index in the diagnosis of oral squamous cell carcinoma.Part Ⅱ: CAL27 cells were treated with 0.06 μmol/L,0.6 μmol/L and 6 μmol/L L-Kynurenine or N-Acetylphenylalanine(KYN or ACE)respectively.CCK-8(Cell counting kit-8),colony formition assay and Transwell assay were used to discover the effect of KYN or ACE on tumor cell proliferation and metastatic ability of CAL27 cells.CAL27 cells were transfected by IDO1 siRNA.The suppressed expression of IDO1 gene in CAL27 cells was detected by Real-time PCR assay.CCK-8 was used to test the effect of inhibition of IDO1 target gene expression on tumor cell proliferation and Transwell chamber assay to detect the effect on the metastatic ability.Part Ⅲ: M0,M1 and M2 macrophages were obtained by sequence stimulation to differentiate THP-1 cells.After observing different morphologies under microscope and identifying their phenotypes by flow cytometry,a co-culture system was established with CAL27 cells suspension containing 1 μmol/L KYN or ACE.Transwell assay and Real-time PCR were used to discover the influence of KYN or ACE on M0 macrophage migration and differentiation.ResultsPart Ⅰ: Based on LC-MS/MS method,we found that KYN,ACE and other metabolites were discovered in Tryptophan metabolism pathway.The expression of KYN and ACE in cancer tissues was higher than that in para-cancerous tissues.What’s more,they have high sensitivity and specificity.But there is no difference in the concentration of KYN in plasma between patients and healthy subjects in ELISA.Part Ⅱ: CCK-8 showed that the OD value of the experimental group with KYN conditioned medium was not different from that of the control one,but the number of clones in the group with KYN was higher than that of the control one,and in a certain concentration-dependent manner.However,there was no difference in CCK-8 and colony formation assay between ACE and control group.The result of Transwell assay was that the number of CAL27 cells migrated to KYN and ACE in the lower chamber was increased,and the best concentration was 0.6 μmol/L.CCK-8 and Transwell assay showed that the OD value and the number of migrating cells of CAL27 cells transfected with siRNA of IDO1 was lower than that of the NC group.Part Ⅲ: The phenotypes of M0,M1 and M2 macrophages were successfully identified according to microscopic observation of different morphology and Real-time PCR.The results of Transwell chamber assay showed that KYN could promote the migration of M0 type macrophages to CAL27 cells,and then Real-time PCR identified the differentiation phenotype of M0 type macrophages.It was found that the addition of KYN could promote the differentiation of M0 type macrophages to M2 cells.ACE did not have the founction of promoting the recruitment and differentiation of M0 macrophages.ConclusionsPart Ⅰ: The metabolic levels of KYN and ACE which were in cancer tissues were higher than those in paracancerous tissues,and the level of the two metabolites had high sensitivity and specificity,but the same results were not detected in plasma.Part Ⅱ: The KYN metabolic pathway regulated by IDO1 can promote the proliferation and migration of CAL27 cells.Part Ⅲ: KYN can promote the migration of M0-type macrophages and make them differentiate into M2-type macrophages.