Cancer and stromal cell metabolism is important for understanding tumor development, which highly depends on the tumor microenvironment (TME). Cell or animal models cannot recapitulate the human TME. We have developed an ex vivo paired cancerous (CA) and noncancerous (NC) human lung tissue approach to explore cancer and stromal cell metabolism in the native human TME. This approach enabled full control of experimental parameters and acquisition of individual patient's target tissue response to therapeutic agents while eliminating interferences from genetic and physiological variations. In this two-case study of non-small-cell lung cancer, we performed stable isotope-resolved metabolomic (SIRM) experiments on paired CA and NC lung tissues treated with a macrophage activator β-glucan and 13C6-glucose, followed by ion chromatography–Fourier transform mass spectrometry (IC-FTMS) and nuclear magnetic resonance (NMR) analyses of 13C-labeling patterns of metabolites. We demonstrated that CA lung tissue slices were metabolically more active than their NC counterparts, which recapitulated the metabolic reprogramming in CA lung tissues observed in vivo. We showed β-glucan-enhanced glycolysis, Krebs cycle, pentose phosphate pathway, antioxidant production, and itaconate buildup in patient UK021 with chronic obstructive pulmonary disease (COPD) and an abundance of tumor-associated macrophages (TAMs) but not in UK049 with no COPD and much less macrophage infiltration. This metabolic response of UK021 tissues was accompanied by reduced mitotic index, increased necrosis, and enhaced inducible nitric oxide synthase (iNOS) expression. We surmise that the reprogrammed networks could reflect β-glucan M1 polarization of human macrophages. This case study presents a unique opportunity for investigating metabolic responses of human macrophages to immune modulators in their native microenvironment on an individual patient basis.

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Edited by Qi-Qun Tang Nucleotide synthesis is essential to proliferating cells, but the preferred precursors for de novo biosynthesis are not defined in human cancer tissues. We have employed multiplexed stable isotope-resolved metabolomics to track the metabolism of [ 13 C 6 ]glucose, D 2-glycine, [ 13 C 2 ]glycine, and D 3-serine into purine nucleotides in freshly resected cancerous and matched noncancerous lung tissues from nonsmall cell lung cancer (NSCLC) patients, and we compared the metabolism with established NSCLC PC9 and A549 cell lines in vitro. Surprisingly, [ 13 C 6 ]glucose was the best carbon source for purine synthesis in human NSCLC tissues, in contrast to the noncancerous lung tissues from the same patient, which showed lower mitotic indices and MYC expression. We also observed that D 3-Ser was preferentially incorporated into purine rings over D 2-glycine in both tissues and cell lines. MYC suppression attenuated [ 13 C 6 ]glucose, D 3-serine, and [ 13 C 2 ]glycine incorporation into purines and reduced proliferation in PC9 but not in A549 cells. Using detailed kinetic modeling, we showed that the preferred use of glucose as a carbon source for purine ring synthesis in NSCLC tissues involves cytoplasmic activation/compartmentation of the glucose-to-serine pathway and enhanced reversed one-carbon fluxes that attenuate exogenous serine incorporation into purines. Our findings also indicate that the substrate for de novo nucleotide synthesis differs profoundly between cancer cell lines and fresh human lung cancer tissues; the latter preferred glucose to exogenous serine or glycine but not the former. This distinction in substrate utilization in purine synthesis in human cancer tissues should be considered when targeting one-carbon metabolism for cancer therapy. De novo nucleotide biosynthesis is required to meet the demand for maintaining energy, nucleotide levels, and new nucleic acids in dividing cells (1, 2). Synthesis of pyrimidine nucleotides in cancer cells utilizes glucose and glutamine for the carbon of the uracil and cytosine rings, which then condense with the ribose subunit primarily derived from glucose via the pentose-phosphate pathway (1, 3). The purine nucleotides are synthesized by building the purine ring directly onto phosphoribosyl pyrophosphate (PRPP) 6 using glycine (direct route), CO 2 , and glycine-derived N 10-formyl tetrahydrofolate (CHO-THF, indirect route) as the carbon sources for C4,5, C6, and C2,8, respectively (cf. Fig. 1). Cellular glycine may derive from glucose via de novo synthesis from serine (1, 4), via protein degradation, and/or via uptake from external sources, i.e. common cell culture media (1, 2) or the circulating blood (5). Serine derived from glucose or external sources is converted (through one-carbon metabolism) into Gly and 5,10-methylene THF (CH 2-THF) via the serine hydroxymethyltransferase (SHMT) activity (Fig. 1). CH 2-THF is oxidized to CHO-THF via the methylene tetrahydrofolate cyclohydrolase activity, prior to incorporation into purine rings.