低氧和酸中毒是被广泛认可的肿瘤微环境（TME）生物标志物，与癌症进展相关。癌症氧化还原状态和代谢的改变与细胞内谷胱甘肽（GSH）和间质磷酸盐（Pi）水平升高也有关。本研究旨在评估这些生物标志物的能力，区分不同癌症分期并提示向恶性转变的转变趋势。这些研究使用自发发展乳腺癌并模拟人类肿瘤分期的MMTV-PyMT（ +）雌性转基因小鼠进行。利用L频段电子顺磁共振光谱和多功能三苯基和GSH敏感的亚硝酰胺探针，对体内氧浓度（pO2）、细胞外酸度（pHe）、Pi和GSH进行了评估。TME的分析显示，从前恶性病变（pre-S4）到恶性阶段（S4），测得的生物标志物明显偏离。对于联合标记物HOP：（pHe × pO2）/Pi，数值>186表明在被分析的乳腺腺体中，肿瘤在85%的情况下为前恶性。数值<186时，肿瘤在42%的情况下为恶性。对于GSH来说，数值<3 mM表明在74%的情况下肿瘤为前恶性，而数值>3 mM时，肿瘤在80%的情况下为恶性。唯一一个在S1阶段就明显偏离其前S1的数值的标志物是升高的Pi，随后是pHe和pO2的下降以及细胞内GSH的增加。分子TME分析揭示了肿瘤氧化还原和代谢在肿瘤分期过程中的变化。S1早期间质Pi的升高可能反映了肿瘤代谢的变化，这与高速生长假说所需的磷供应增加一致。这些代谢变化得到了体外pHe的下降的支持，因为肿瘤高度依赖于糖酵解，并且细胞内GSH增加，这是一个主要的细胞内氧化还原缓冲系统。只有在恶性S4阶段观察到明显的TME pO2下降，这显然是由于肿瘤质量增长，对灌注效果下降和随着肿瘤细胞增殖氧气消耗增加所致。© 2023. 作者（们），在世界分子成像学会独家许可下。

Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy.These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO2), extracellular acidity (pHe), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes.Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pHe × pO2)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3 mM indicated that the tumors were pre-malignant 74% of the time, and when > 3 mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pHe and pO2 and increase in GSH at later stages.Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pHe due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO2 was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.© 2023. The Author(s), under exclusive licence to World Molecular Imaging Society.