甲硫氨酸依赖是癌细胞对外源性甲硫氨酸在生长和存活中的升高需求，称之为霍夫曼效应。甲硫氨酸依赖的癌细胞合成正常或过量的甲硫氨酸，但仍需外源性甲硫氨酸。通过甲硫氨酸限制（MR），通过甲硫氨酸缺乏培养基，在体通过低甲硫氨酸饮食或甲硫氨酸酶，可选择性地使癌细胞阻滞在晚期S/G2细胞周期阶段，但对正常细胞没有影响。本研究主要比较了MR条件下和正常条件下癌细胞产生和分泌外泌体的情况。MDA-MB-231细胞（三阴性乳腺癌），含有标记为CD63-GFP的外泌体（CD63-GFP外泌体），被荧光显微镜可视化。在含有甲硫氨酸（MET+）或无甲硫氨酸（MET-）的DMEM条件下，通过超速离心从培养MDA-MB-231细胞的条件培养基中分离外泌体，并通过纳米颗粒跟踪分析（NTA）和免疫印迹鉴定这些外泌体。当MDA-MB-231-CD63-GFP细胞在MR条件下培养时，它们停滞了生长，并且CD63-GFP表达的外泌体在细胞中显著增加。MR导致每个细胞外泌体产生和分泌增加了约2倍，即使细胞生长被阻滞。甲硫氨酸限制因此导致了每个存活细胞的外泌体产生和分泌增加。癌细胞在MR条件下产生和分泌外泌体增加，暗示了MR与外泌体产生和分泌之间的关系。 版权所有©2023，国际抗癌研究学会（George J. Delinasios博士），保留所有权利。

Methionine addiction is the elevated requirement for exogenous methionine for growth and survival of cancer cells, termed the Hoffman effect. Methionine-addicted cancer cells synthesize normal or excess amounts of methionine but still need an external source of methionine. Methionine restriction (MR) by either a methionine-free medium or in vivo by a low-methionine diet or by methioninase, selectively arrests cancer cells in the late S/G2 cell cycle phase, but not normal cells. The present study focuses on the comparison of production and secretion of exosomes by cancer cells under MR and normal conditions.MDA-MB-231 cells (triple-negative breast cancer), containing exosomes labeled with CD63-GFP (CD63-GFP exosomes), were visualized by fluorescence microscopy. MDA-MB-231 cells expressing exosome-specific CD63-GFP were cultured in methionine-containing (MET+) or in methionine-free (MET-) DMEM conditions. Exosomes were isolated from conditioned medium of cultured MDA-MD-231 cells by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and Western blotting.When MDA-MB-231-CD63-GFP cells were cultured under MR conditions, they arrested their growth and CD63-GFP-expressing exosomes were strongly increased in the cells. MR resulted in approximately a 2-fold increase in exosome production and secretion per cell, even though cell growth was arrested. Methionine restriction thus resulted in elevated exosome production and secretion per surviving cell.Exosome production and secretion in the cancer cells increased under MR, suggesting a relation between MR and exosome production and secretion.Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.