Metabolomics revealed the influence of breast cancer on lymphatic endothelial cell metabolism, metabolic crosstalk, and lymphangiogenic signaling in co-culture

Suehelay Acevedo-Acevedo,Paulo F Cobra,Melissa Skala,Sean P Palecek,Peter Favreau,Douglas C Millar,Aaron D Simmons

doi:10.1038/s41598-020-76394-7

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 ^1hbox {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}-labeled glucose experiments did not reveal lactate shuttling into LECs from breast cancer cells, yet showed other ^{13}hbox {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.

Highlights

Lymphatic endothelial cells (LECs) to support tumor vascularization and growth
To determine what changes occurred in lymphatic endothelial cells (LECs) metabolism in the presence of breast cancer cells, LECs were cultured for four days in a reduced medium composed of endothelial basal media 2 (EBM2) with 5% fetal bovine serum (FBS) using a Transwell co-culture system
The impact of breast cancer sub-types on the metabolism of LECs in vitro and their relation to lymphangiogenesis was investigated. These results provide an initial indicator of how LECs and breast cancer cells interact metabolically in the tumor microenvironment (TME)

Summary

Introduction

Lymphatic endothelial cells (LECs) to support tumor vascularization and growth. 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. Lymphatic vessels are a favorable route of metastasis since primary tumors can express lymphangiogenic growth factors, vascular endothelial growth factor-C (VEGF-C) and VEGF-D, which promote tumor-associated lymphangiogenesis leading to invasion into the lymphatic vasculature, lymph node metastasis, and tumor dissemination[6,7,12,13]. Expression of these lymphangiogenic growth factors correlated with poor patient prognosis in certain human cancers such as invasive breast carcinoma, lung, prostate, colorectal, and thyroid c ancer[12,14,15,16]. A recent report by Ayuso et al showed that breast cancer cells altered transcription of LECs which correlated with glucose permeability into the v asculature[18]

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Results

Conclusion