Metabolomic profiling of rare cell populations isolated by flow cytometry from tissues
- University of Texas Southwestern Medical Center, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Abstract
Little is known about the metabolic regulation of rare cell populations because most metabolites are hard to detect in small numbers of cells. We previously described a method for metabolomic profiling of flow cytometrically-isolated hematopoietic stem cells (HSCs) that detects 60 metabolites in 10,000 cells (Agathocleous et al., 2017). Here we describe a new method involving hydrophilic liquid interaction chromatography and high-sensitivity orbitrap mass spectrometry that detected 160 metabolites in 10,000 HSCs, including many more glycolytic and lipid intermediates. We improved chromatographic separation, increased mass resolution, minimized ion suppression, and eliminated sample drying. Most metabolite levels did not significantly change during cell isolation. Mouse HSCs exhibited increased glycerophospholipids relative to bone marrow cells and methotrexate treatment altered purine biosynthesis. Circulating human melanoma cells were depleted for purine intermediates relative to subcutaneous tumors, suggesting decreased purine synthesis during metastasis. These methods facilitate the routine metabolomic analysis of rare cells from tissues.
Data availability
All data generated or analyzed during this study are included in the manuscript and the source data files.
Article and author information
Author details
Andrew W DeVilbiss
Funding
Howard Hughes Medical Institute
- Thomas P Mathews
- Sean J Morrison
National Institutes of Health
- Sean J Morrison
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Matthew G Vander Heiden, Massachusetts Institute of Technology, United States
Ethics
Animal experimentation: All mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center (protocols 2016-101360 and 2019-102632).
Version history
- Received: August 11, 2020
- Accepted: January 19, 2021
- Accepted Manuscript published: January 20, 2021 (version 1)
- Version of Record published: January 29, 2021 (version 2)
Copyright
© 2021, DeVilbiss et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,166
- views
-
- 843
- downloads
-
- 58
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Metabolomic profiling of rare cell populations isolated by flow cytometry from tissues
eLife 10:e61980.
https://doi.org/10.7554/eLife.61980
Further reading
-
- Developmental Biology
- Stem Cells and Regenerative Medicine
Endothelial and skeletal muscle lineages arise from common embryonic progenitors. Despite their shared developmental origin, adult endothelial cells (ECs) and muscle stem cells (MuSCs; satellite cells) have been thought to possess distinct gene signatures and signaling pathways. Here, we shift this paradigm by uncovering how adult MuSC behavior is affected by the expression of a subset of EC transcripts. We used several computational analyses including single-cell RNA-seq (scRNA-seq) to show that MuSCs express low levels of canonical EC markers in mice. We demonstrate that MuSC survival is regulated by one such prototypic endothelial signaling pathway (VEGFA-FLT1). Using pharmacological and genetic gain- and loss-of-function studies, we identify the FLT1-AKT1 axis as the key effector underlying VEGFA-mediated regulation of MuSC survival. All together, our data support that the VEGFA-FLT1-AKT1 pathway promotes MuSC survival during muscle regeneration, and highlights how the minor expression of select transcripts is sufficient for affecting cell behavior.
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody–RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.
