Children's Research Institute and Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Department of Dermatology, University Hospital Essen & German Cancer Consortium, Partner Site, Essen, Germany.
Nat Protoc. 2021 Nov;16(11):5123-5145. doi: 10.1038/s41596-021-00605-2. Epub 2021 Sep 17.
Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.
癌细胞经历多种代谢适应，以满足失调生长和增殖所带来的能量需求。评估完整肿瘤中的代谢情况，可以让研究者观察到众多内在和外在癌细胞因素的综合代谢效应，而这些因素在培养模型中是无法完全捕捉到的。我们已经开发了一些方法，使用稳定同位素标记的营养物质（例如 [C]葡萄糖）来在体内、在小鼠和人类中探测完整肿瘤内的代谢活性。在这些方法中，标记的营养物质通过静脉导管引入循环系统，然后再进行肿瘤和相邻非恶性组织的手术切除。在输注期间，这些组织内的代谢将同位素标记转移到由输注营养物质供应的代谢中间产物中。从手术标本中提取代谢物并分析其同位素标记模式，可以提供组织内代谢的信息。我们提供了有关该技术的详细信息，从引入标记示踪剂到数据分析和解释，包括从组织样本中提取的有意义代谢物中定量同位素标记的简化方法。我们重点介绍了 [C]葡萄糖的输注和质谱法在评估包括糖酵解、三羧酸循环和非必需氨基酸合成在内的中心代谢途径中的中间产物的同位素标记的应用。我们概述了将这些方法应用于接受实体瘤手术切除的人类受试者的实际考虑因素。我们还讨论了该方法的多功能性，并考虑了引入示踪剂、采集组织和分析数据的替代方法的相对优势和局限性。
