The Role And Mechanism Of Targeting Monocarboxylate Transporter 1 In The Treatment Of Malignant Pleural Effusion

Objectives:Malignant pleural effusion(MPE)is an immunosuppressive tumor microenvironment(TME).In this‘cold’TME,the antitumor effects of CD8~+T cells and natural killer T(NKT)cells are inhibited,while the immunomodulatory effects of regulatory T(Treg)cells are enhanced.Various cell populations,including tumor cells,cancer-associated fibroblasts(CAFs)and tumor-associated macrophages(TAMs),as well as elevated metabolites and cytokines,such as lactate and interleukin-1β(IL-1β),all contribute to the immunosuppressive MPE.However,few studies have focused on the association between metabolic reprogramming and MPE progression.In this study,we aimed to explore the therapeutic value of targeting lactate metabolism in controlling the progression of MPE.Methods:The proportions of different immune cell subpopulations in MPE and corresponding peripheral blood(PB)were detected by flow cytometry analysis.Differentially expressed genes and metabolic differences between FOXP3~+natural killer T-like(NKT-like)cells and FOXP3~-NKT-like cells were analyzed by single-cell RNA sequencing(sc RNA-seq).The lactate concentrations in the supernatant of MPE and corresponding PB were detected using a commercial L-lactate concentration assay kit.The expression of FOXP3 in cultured NKT-like cells was detected by flow cytometry,and the histone lactylation(H3K18la)level in cultured NKT-like cells was analyzed by western blotting.Possible lactylation sites were detected by chromatin immunoprecipitation quantitative real-time PCR(Ch IP-q PCR).The mouse MPE model was constructed by intrapleural injection of LLC or LLC-Luc cells.In vivo bioluminescence imaging was used to investigate the progression of intrapleural tumor burden.Pan-cancer analysis was used to explore the expression of monocarboxylate transporter 1(MCT1)in non-small cell lung cancer(NSCLC)tissues.CRISPR-Cas9 technology was applied to investigate the effect of MCT1 knockout on the progression of MPE.Results:(1)The proportions of FOXP3~+NKT-like cells were significantly higher in MPE than in corresponding PB.A higher proportion of FOXP3~+NKT-like cells in human MPE was positively associated with a higher tumor proportion,higher performance status score,and poorer prognosis of MPE patients.(2)Compared with FOXP3~-NKT-like cells,FOXP3~+NKT-like cells expressed higher levels of ICOS,CTLA-4,TIGIT,Helios,GITR,PD-1,LAG-3,PD-L1,IL-4,IL-10,and TGF-β,indicating that the immunomodulatory function of FOXP3~+NKT-like cells was enhanced.(3)Compared with FOXP3~-NKT-like cells,FOXP3~+NKT-like cells expressed higher levels of MCT1 and lactate dehydrogenase B(LDHB).The sc RNA-seq analysis also showed that lactate metabolism was highly activated in FOXP3~+NKT-like cells.In addition,the proportion of FOXP3~+NKT-like cells in MPE was positively correlated with lactate concentration in MPE supernatant.(4)In vitro culture experiments demonstrated that chronic high lactate significantly increased FOXP3 expression and H3K18la levels in NKT-like cells,while MCT1 small molecule inhibitor 7ACC2 had the opposite effect.Furthermore,Ch IP-q PCR analysis showed that there were H3K18la binding sites in the promoter region of the FOXP3 gene in NKT-like cells.(5)The construction of the mouse MPE model showed that the proportions of mouse invariant NKT(i NKT)cells and FOXP3~+NKT cells slowly increased with the progression of MPE.(6)In vivo bioluminescence imaging and flow cytometry analysis showed that combined therapy of 7ACC2 and NKT cell agonistα-Gal Cer significantly inhibited MPE progression,decreased intrapleural tumor burden,prolonged overall survival,and increased the proportion of i NKT cells in MPE and intrathoracic tumor.(7)Pan-cancer analysis demonstrated that MCT1 expression in NSCLC tissues was significantly increased,which was negatively associated with the prognosis of NSCLC patients.(8)MCT1 knockout significantly inhibited the progression of MPE and increased the proportion of NKT cells in MPE.Conclusions:Overall,this study demonstrates a promising strategy to target lactate metabolism to control the progression of MPE.