Spatially resolved metabolomics to discover tumor- associated metabolic alterations Chenglong Suna, Tiegang Lia, Xiaowei Songa, Luojiao Huanga, Qingce Zanga, Jing Xua, Nan Bib, Guanggen Jiaoc, Yanzeng Haoc, Yanhua Chena, Ruiping Zhanga, Zhigang Luoa, Xin Lia, Luhua Wangb, Zhonghua Wangd, Yongmei Songe, Jiuming Hea,1, and Zeper Abliza,d,1 aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 100050 Beijing, China; bDepartment of Radiation Oncology, Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 100021 Beijing, China; cDepartment of Pathology and Thoracic Surgery, Linzhou Esophageal Cancer Hospital, 456500 Linzhou, China; dCenter for Imaging and Systems Biology, Minzu University of China, 100081 Beijing, China; and eState Key Laboratory of Molecular Oncology, Cancer Institute, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 100021 Beijing, China Edited by Matthew G. Vander Heiden, Koch Institute at Massachusetts Institute of Technology, and accepted by Editorial Board Member Rakesh K. Jain November 27, 2018 (received for review May 24, 2018) Characterization of tumor metabolism with spatial information tissue samples is lost over the course of elaborate sample pre- contributes to our understanding of complex cancer metabolic treatment (7). Matrix-assisted laser desorption ionization mass reprogramming, facilitating the discovery of potential metabolic spectrometry imaging (MALDI-MSI) is an effective tool to vi- vulnerabilities that might be targeted for tumor therapy. However, sualize the spatial location of proteins and metabolites with high given the metabolic variability and flexibility of tumors, it is still spatial resolution, but high-vacuum environments and matrix challenging to characterize global metabolic alterations in heteroge- assistance are usually needed for ionization (8–11). Immuno- neous cancer. Here, we propose a spatially resolved metabolomics histochemistry (IHC) is a traditional tool to characterize the approach to discover tumor-associated metabolites and metabolic distribution and intensity of specific enzymes in tissue based on enzymes directly in their native state. A variety of metabolites antibody–antigen interactions. However, its dependency on tar- localized in different metabolic pathways were mapped by airflow- geted antibodies and its time-consuming procedure make it dif- assisted desorption electrospray ionization mass spectrometry imag- ficult to investigate multiple unknown features in tissues for CHEMISTRY ing (AFADESI-MSI) in tissues from 256 esophageal cancer patients. In tumor-associated enzyme discovery (12). combination with in situ metabolomics analysis, this method provided Metabolites are the direct products or substrates of metabolic clues into tumor-associated metabolic pathways, including proline enzymes, and the level of metabolites in tissue may reflect en- biosynthesis, glutamine metabolism, uridine metabolism, histidine zyme capacities (13). Characterization of the metabolic pathway- metabolism, fatty acid biosynthesis, and polyamine biosynthesis. Six related metabolites in heterogeneous cancer tissue provides abnormally expressed metabolic enzymes that are closely associated important insights into tumor-associated metabolic enzyme dis- with the altered metabolic pathways were further discovered in covery. Ambient MSI (AMSI) technology, represented by de- esophageal squamous cell carcinoma (ESCC). Notably, pyrroline-5- sorption electrospray ionization (DESI)-MSI can directly extract carboxylate reductase 2 (PYCR2) and uridine phosphorylase 1 (UPase1) were found to be altered in ESCC. The spatially resolved metabolomics Significance reveal what occurs in cancer at the molecular level, from metabolites to enzymes, and thus provide insights into the understanding of Tumor cells reprogram their metabolism to support cell cancer metabolic reprogramming. growth, proliferation, and differentiation, thus driving cancer progression. Profiling of the metabolic signatures in hetero- mass spectrometry imaging | metabolomics | esophageal cancer | metabolic geneous tumors facilitates the understanding of tumor me- alterations | airflow-assisted ionization tabolism and introduces potential metabolic vulnerabilities that might be targeted therapeutically. We proposed a spa- etabolic reprogramming represents cancer-associated tially resolved metabolomics method for high-throughput Mmetabolic changes during tumorigenesis and has been discovery of tumor-associated metabolite and enzyme al- recognized as a new hallmark of cancer (1). Profiling the dif- terations using ambient mass spectrometry imaging. Meta- ferential metabolic dependencies of cancer at the highest bolic pathway-related metabolites and metabolic enzymes that achievable coverage is significant for understanding the complex are associated with tumor metabolism were efficiently dis- molecular processes taking place in tumors and will yield im- covered and visualized in heterogeneous esophageal cancer portant insights into how to target tumor metabolism (2). Me- tissues. Spatially resolved metabolic alterations hold the key to tabolites and metabolic enzymes are the two most important defining the dependencies of metabolism that are most limit- constituents of biological metabolic networks. Metabolic en- ing for cancer growth and exploring metabolic targeted strat- zymes, as important nodes in biological metabolic networks, can egies for better cancer treatment. regulate and control the flux of metabolites to maintain meta- Author contributions: J.H. and Z.A. designed research; C.S., L.H., Q.Z., N.B., G.J., Y.H., R.Z., bolic homeostasis (3). Emerging work suggests that the dysregula- Z.L., and L.W. performed research; C.S., T.L., X.S., J.X., Y.C., X.L., Z.W., Y.S., J.H., and Z.A. tion of metabolic pathways contribute to the pathogenesis of cancer, analyzed data; and C.S., J.H., and Z.A. wrote the paper. and this altered metabolism introduces metabolic liabilities that can The authors declare no conflict of interest. – be exploited for cancer therapy (4 6). However, high-throughput This article is a PNAS Direct Submission. M.G.V.H. is a guest editor invited by the discovery of tumor-associated metabolic alterations at both the Editorial Board. metabolite and metabolic enzyme levels is still a great challenge. This open access article is distributed under Creative Commons Attribution-NonCommercial- A technique that enables a direct view of altered enzyme NoDerivatives License 4.0 (CC BY-NC-ND). features from cancer tissues will facilitate the exploitation of 1To whom correspondence may be addressed. Email: [email protected] or zeper@ metabolic enzyme driven tumor-targeting therapy. Liquid chro- imm.ac.cn. matography–mass spectrometry (LC-MS)-based proteomic pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. filing of cancer tissue reveals differentially expressed proteins; 1073/pnas.1808950116/-/DCSupplemental. however, the spatial distribution discrepancy of heterogeneous www.pnas.org/cgi/doi/10.1073/pnas.1808950116 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 and visualize numerous molecular features in tissue sections (9, are directly associated with the altered metabolites in pathways, 14–20). Untargeted AMSI has been regarded as a rational ap- were further discovered. Notably, the expression levels of PYCR2 proach for monitoring lipid dysregulation in cancer tissues for and UPase1 were found to be altered in ESCC. The integration of biological and clinical studies (21–25). Airflow-assisted desorption spatially resolved enzyme information and the corresponding electrospray ionization (AFADESI)-MSI is a high-coverage am- downstream metabolite information will expand our understanding bient molecular imaging technique that was developed by our of tumor metabolism and could further facilitate the discovery of group, and it can map numerous functional metabolites located in altered metabolic pathways. Furthermore, extensively altered tumor different metabolic pathways (26). However, the chemical noise metabolism provides insights for developing novel drugs to target arising from tissue biomatrix makes the directly mapping of pro- multiple metabolic abnormalities. teins using AMSI techniques challenging, especially for the low- content functional metabolic enzymes. Results and Discussion In this study, we propose a strategy for high-throughput dis- Region-Specific Molecule Profiling. Postoperative ESCC tissue covery of cancer-associated metabolites and metabolic enzymes sections were divided into three histologic types based on the cell in their native state. The design of this strategy is shown in SI type and components: cancer tissue, epithelial tissue, and mus- Appendix, Fig. S1. High-coverage airflow-assisted desorption cular tissue. After tissue MSI, microscopy was integrated with electrospray ionization (AFADESI)-MSI was first applied to the MS image to form a microscopy-MSI overlay image, which acquire region-specific tissue metabolite profiles in 256 esopha- delivers both the spatial resolution of microscopy and the geal squamous cell carcinoma (ESCC) patients. Then, MSI- chemical signatures of MSI in one integrated whole (Fig. 1A). based metabolomics combined with multivariate statistical However, it is worth noting that the combined