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Abstract Breast cancer metastasis occurs via blood and lymphatic vessels. Breast cancer cells ‘educate’ lymphatic endothelial cells (LECs) to support tumor vascularization and growth. However, despite known metabolic alterations in breast cancer, it remains unclear how lymphatic endothelial cell metabolism is altered in the tumor microenvironment and its effect in lymphangiogenic signaling in LECs. We analyzed metabolites inside LECs in co-culture with MCF-7, MDA-MB-231, and SK-BR-3 breast cancer cell lines using $$^1\hbox {H}$$ 1 H nuclear magnetic resonance (NMR) metabolomics, Seahorse, and the spatial distribution of metabolic co-enzymes using optical redox ratio imaging to describe breast cancer-LEC metabolic crosstalk. LECs co-cultured with breast cancer cells exhibited cell-line dependent altered metabolic profiles, including significant changes in lactate concentration in breast cancer co-culture. Cell metabolic phenotype analysis using Seahorse showed LECs in co-culture exhibited reduced mitochondrial respiration, increased reliance on glycolysis and reduced metabolic flexibility. Optical redox ratio measurements revealed reduced NAD(P)H levels in LECs potentially due to increased NAD(P)H utilization to maintain redox homeostasis. $$^{13}\hbox {C}$$ 13 C -labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other $$^{13}\hbox {C}$$ 13 C signals in LECs suggesting internalized metabolites and metabolic exchange between the two cell types. We also determined that breast cancer co-culture stimulated lymphangiogenic signaling in LECs, yet activation was not stimulated by lactate alone. Increased lymphangiogenic signaling suggests paracrine signaling between LECs and breast cancer cells which could have a pro-metastatic role.
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Metabolomics studies of cell–cell interactions using single cell mass spectrometry combined with fluorescence microscopy
Cell–cell interactions are critical for transmitting signals among cells and maintaining their normal functions from the single-cell level to tissues. In cancer studies, interactions between drug-resistant and drug-sensitive cells play an important role in the development of chemotherapy resistance of tumors. As metabolites directly reflect the cell status, metabolomics studies provide insight into cell–cell communication. Mass spectrometry (MS) is a powerful tool for metabolomics studies, and single cell MS (SCMS) analysis can provide unique information for understanding interactions among heterogeneous cells. In the current study, we utilized a direct co-culture system (with cell–cell contact) to study metabolomics of single cells affected by cell–cell interactions in their living status. A fluorescence microscope was utilized to distinguish these two types of cells for SCMS metabolomics studies using the Single-probe SCMS technique under ambient conditions. Our results show that through interactions with drug-resistant cells, drug-sensitive cancer cells acquired significantly increased drug resistance and exhibited drastically altered metabolites. Further investigation found that the increased drug resistance was associated with multiple metabolism regulations in drug-sensitive cells through co-culture such as the upregulation of sphingomyelins lipids and lactic acid and the downregulation of TCA cycle intermediates. The method allows for direct MS metabolomics studies of individual cells labeled with fluorescent proteins or dyes among heterogeneous populations.
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- Award ID(s):
- 1634630
- PAR ID:
- 10378334
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 13
- Issue:
- 22
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 6687 to 6695
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d2sc02298b
