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Metabolomics Guided Pathway Analysis Reveals Link Between Cancer Metastasis, Cholesterol Sulfate, and Phospholipids Caroline H

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Metabolomics Guided Pathway Analysis Reveals Link Between Cancer Metastasis, Cholesterol Sulfate, and Phospholipids Caroline H Johnson et al. Cancer & Metabolism (2017) 5:9 DOI 10.1186/s40170-017-0171-2 RESEARCH Open Access Metabolomics guided pathway analysis reveals link between cancer metastasis, cholesterol sulfate, and phospholipids Caroline H. Johnson1,2,3*† , Antonio F. Santidrian4,5†, Sarah E. LeBoeuf4,6, Michael E. Kurczy1,7, Nicholas J. W. Rattray2, Zahra Rattray8, Benedikt Warth1, Melissa Ritland4,9, Linh T. Hoang1, Celine Loriot4, Jason Higa4, James E. Hansen3,8, Brunhilde H. Felding4* and Gary Siuzdak1* Abstract Background: Cancer cells that enter the metastatic cascade require traits that allow them to survive within the circulation and colonize distant organ sites. As disseminating cancer cells adapt to their changing microenvironments, they also modify their metabolism and metabolite production. Methods: A mouse xenograft model of spontaneous tumor metastasis was used to determine the metabolic rewiring that occurs between primary cancers and their metastases. An “autonomous” mass spectrometry-based untargeted metabolomic workflow with integrative metabolic pathway analysis revealed a number of differentially regulated metabolites in primary mammary fat pad (MFP) tumors compared to microdissected paired lung metastases. The study was further extended to analyze metabolites in paired normal tissues which determined the potential influence of metabolites from the microenvironment. Results: Metabolomic analysis revealed that multiple metabolites were increased in metastases, including cholesterol sulfate and phospholipids (phosphatidylglycerols and phosphatidylethanolamine). Metabolite analysis of normal lung tissue in the mouse model also revealed increased levels of these metabolites compared to tissues from normal MFP and primary MFP tumors, indicating potential extracellular uptake by cancer cells in lung metastases. These results indicate a potential functional importance of cholesterol sulfate and phospholipids in propagating metastasis. In addition, metabolites involved in DNA/RNA synthesis and the TCA cycle were decreased in lung metastases compared to primary MFP tumors. Conclusions: Using an integrated metabolomic workflow, this study identified a link between cholesterol sulfate and phospholipids, metabolic characteristics of the metastatic niche, and the capacity of tumor cells to colonize distant sites. Keywords: Cancer, Cholesterol sulfate, Phospholipids, Autonomous metabolomics, Mummichog, Metastasis, XCMS * Correspondence: [email protected]; [email protected]; [email protected] †Equal contributors 1Scripps Center for Metabolomics, The Scripps Research Institute, La Jolla, CA, USA 4Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Johnson et al. Cancer & Metabolism (2017) 5:9 Page 2 of 9 Background metabolites in tissues, we used an autonomous mass The biology of metastatic cancer is complex, and cells that spectrometry-based metabolomic workflow on primary enter the metastatic cascade encounter a multitude of tumor and metastasis tissue extracts [8]. challenges. The fraction of malignant cells that are shed from the primary tumor require traits that allow them to Cell culture survive in circulation and colonize distant organs [1]. MDA-MB-435 cells were grown in EMEM supplemented Once seeded, the cells must adapt to the new pool of with nonessential amino acids, vitamins, 2 mM L-glutam- extracellular metabolites available and survive immune ine, 1 mM pyruvate, and 10% fetal bovine serum. surveillance. To do this, cancer cells reprogram their microenvironment to assist tumor growth. Thus, the me- Animal treatment and sample collection tabolism, growth, and survival of the metastatic cells are Six- to eight-week-old female C.B-17/SCID mice were likely dependent on the attributes of the new site. injected with F-luc-tagged cancer cells: 2.5 × 105 (30 μl) As cancer cells proliferate, they require energy to MDA-MB-435 cells into the fourth mammary fat pad. synthesize macromolecules, support cell growth, and main- Primary tumors were removed surgically when they tain redox homeostasis [2, 3]. Sources of energy can be con- reached 300 mm3 in size (approximately 4 weeks post- trolled by the expression of metabolic enzymes such as injection), the endpoint. Mice were then examined hexokinase which converts glucose to glucose-6-phosphate weekly (IVIS 200, Xenogen) by non-invasive biolumines- in the glycolysis pathway, as seen in many cancers, including cence imaging, 10 min after i.p. injection of D-luciferin glioblastoma multiforme [4]. However, cancer cells can also (100 mg/kg) to assess the presence of lung metastasis. utilize transporters to help produce and import metabolites Animals were sacrificed 24 h after bioluminescence sig- from the microenvironment. A recent study revealed that nal on the chest reached 1 × 107 photons/s/cm2.In metastatic cells release an enzyme into the extracellular order to eliminate any artefacts that could occur with space catalyzing the phosphorylation of creatine. Phospho- the enzymatic procedure, i.p. injection of D-luciferin was creatine is then imported into disseminated cancer cells to not used for the final dissection. Animal work complied generate adenosine triphosphate (ATP), fueling metastatic with the National Institutes of Health and institutional survival [5]. Moreover, stromal-epithelial metabolic coupling guidelines (TSRI is AAALAC accredited). has been described for symbiotic nutrient sharing in cancer. A recent report revealed that adipocytes in the omentum Tissue sample extraction provide fatty acids for primary ovarian cancers promoting Three sections were taken from different regions of rapidtumorgrowthandmetastasis [6]. Metabolites can also mammary fat pad tumors (core, middle, edges) with a be directly involved in increasing cancer cell growth, for ex- combined approximate weight of 15 mg. The tissues ample, succinate and fumarate are known to inhibit prolyl were added to high recovery glass vials (Agilent Tech- hydroxylase enzymes producing a pseudo-hypoxic state, nologies, Santa Clara, CA) on dry ice. Lung metastases driving glycolysis, and tumor proliferation [7]. Therefore, were first assessed by bioluminescence imaging, as de- understanding the metabolism of cancer cells in a primary scribed above, to guide the microdissection under the tumor versus those that have metastasized to a secondary microscope. The microdissected metastasis was then organ site can reveal metabolic adaptions of a disseminating pooled for each mouse, then similarly placed in vials on cancer cell. dry ice. Each sample was homogenized in 400 μl metha- A recently developed autonomous metabolomic workflow nol/water (4:1) and 1 mm glass beads (BioSpec Products, was implemented with integrative metabolic pathway ana- Bartlesville, OK) for 30 s at 3500 rpm. The homogenate lysis to identify metabolites and guide further biological in- was added to glass vials, sonicated for 10 min at room vestigation [8]. A MDA-MB-435 xenograft model was used temperature, and stored at − 20 °C overnight. Samples to generate mammary fat pad (MFP) tumors in the mouse were then centrifuged at 13,000 rpm for 15 min and the and spontaneous metastases to the lung, to test the hypoth- supernatant transferred to a new 1.5-ml centrifuge tube. esis that metastatic cells undergo a metabolic adaption to The pellet was resuspended in 600 μl ice cold acetone, survive the new microenvironment [9]. These cancer tissues vortexed for 10 s, and sonicated for 10 min at room were subjected to comprehensive metabolomics and pathway temp. The pellet samples were stored at − 20 °C for 1 h analyses, which revealed a number of metabolic dependen- followed by centrifugation (13,000 rpm for 15 min). The cies for cancer cells that resided in these tissues. supernatant was pooled with the supernatant collected earlier and dried down in a Speedvac for 4 h. The pellet Methods was used for protein quantification by microBCA The aims of this study were to assess the metabolic dif- (Thermo Fisher Scientific, Waltham, MA). ferences between primary tumors and spontaneous me- Samples were resuspended in acetonitrile/water/iso- tastases. To carry out a comprehensive analysis of propanol (50/40/10 v/v) according to the protein Johnson et al. Cancer & Metabolism (2017) 5:9 Page 3 of 9 concentration; the lowest concentration was resus- spectra, released after 0.15 min; and a threshold of 200 pended in 40 μl and the other samples adjusted accord- counts. Two contaminant ions with m/z’s 172.9297 and ingly. Each sample was then sonicated for 10 min, 248.9762 were excluded throughout the whole run. Me- centrifuged at 13,000 rpm for 15 min, and the superna- tabolite identifications were further confirmed by com- tants transferred to glass HPLC vials for LC/MS analysis. paring retention time and tandem MS to standard compounds. For targeted
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