Metabonomics of clinical cancer biofluids with distinct immuno-metabotypes

Abstract

Malignant pleural effusion (MPE) has pro-growth biological properties. However, this mechanism remains unfulfilled. Both diagnostic and prognostic biomarkers are urgently needed to improve the outcomes of patients with MPE. MPE is an in vivo human tumor immune microenvironment (TIME) model. Exometabolism analysis of MPE was conducted to find novel prognostic metabolic biomarkers and immuno-oncometabolites, and to explore potential therapeutic metabolic targets and corresponding immunotherapy. GG-MS and LC-MS/MS-based lipidomics found the high FASN expression in MPE promoted cancer progression by reprogramming lipid metabolism associated with poor overall survival. NMR and MS-based discovery metabolism revealed beta-hydroxybutyrate (BOHB) was a novel immuno-oncometabolite of MPE associated with shorter overall survival and reduced cytotoxicity. An inverse correlation between BOHB and an inflammatory marker, lactate dehydrogenase (LDH) ratio suggests BOHB is a marker of low immune cell cytotoxicity and indicated an altered immunometabolic tumor microenvironment (TME). The unsupervised clustering analysis of metabolomes suggested different metabolic signatures underlying BOHB-high and BOHB-low MPE immuno-metabotypes. BOHB immuno-metabotype may be enriched with other immune escape metabolic mediators enriched in MPE. To find immune escape mediators enriched in BOHB-high MPE, multi-omics exometabolism of proteomics, metabolomics, lipidomics, inflammation-related cytokine/chemokines, and integrated network analysis were conducted for BOHB-high and BOHB-low MPEs. Functional analysis indicated fatty acid activation, de novo fatty acid biosynthesis, and leukotriene metabolism. Targeted eicosanoid lipidomics analysis identified the active lipid mediators, LTB-4, 5-HETE, and 15-HETE. Tumor-associated macrophages (TAMs) with high 15-HETE expression aided the production of CCL2 involved in the recruitment of monocytes. 5-HETE and LTB4 promoted TAMs invasion. LC-MS/MS-based proteome showed apoproteins, corticosteroid-binding globulin (CBG), and zinc-α2-glycoprotein (ZAG) were upregulated. The overexpression of complement 3 (C3), coagulation factor XII (FXII), and antithrombin-III (ATIII) in high BOHB MPE further favor the polarization of M2-TAM. BOHB and C3 inhibited activation of the NLRP3 inflammasome. The increased expression of alpha-1-antichymotrypsin (ACT), alpha-1-antitrypsin (A1AT), and ceruloplasmin (Cp) indicated a cancer phenotype with tumor metastasis. The protein-metabolic interaction network analysis results highlighted the dysfunctional steroid hormone biosynthesis, sphingolipid, and lipoproteins metabolism. Increased sphingomyelin phosphodiesterase (SMPD1) activity was associated with suppressed Th1 and cytotoxic T cells. The cytokines and chemokines profiles imply that high BOHB MPE demonstrates a pro-angiogenesis and anti-inflammatory phenotype. The multi-omics and inflammation-related cytokine/chemokines pinpoint that BOHB modulated a novel TME subtype, high vs. low BOHB immuno-metabotype in MPE. The proof-of-concept study suggests BDH can be exploited for cancer immunotherapy for MPE.