Spaeth Erika L, Booth Christopher M, Marini Frank C
Department of Leukemia, MD Anderson Cancer Center.
J Vis Exp. 2013 Sep 22(79):e50385. doi: 10.3791/50385.
With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we've added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.
出于理解多种细胞成分对复杂组织发育的贡献的愿望；例如参与组织再生、肿瘤形成或血管生成的众多细胞类型，我们设计了一种肿瘤发育的多色细胞移植模型，其中细胞群体源自不同荧光标记基因小鼠，并被移植、植入或注射到发育中的肿瘤及其周围。这些有色细胞随后被募集并整合到肿瘤基质中。为了定量评估骨髓来源的肿瘤基质细胞，我们按照实验动物护理与使用委员会（IACUC）的批准，将表达绿色荧光蛋白（GFP）的转基因全骨髓移植到接受致死剂量照射且表达红色荧光蛋白（RFP）的小鼠体内。在移植后的6 - 8周，切除原位注射到移植小鼠体内的卵巢肿瘤，并使用多光谱成像技术分析肿瘤起源的骨髓标志物（GFP）以及检测肿瘤相关基质的抗体标志物。然后，我们采用了一种名为MIMicc - 多重细胞成分的多光谱分析方法，利用荧光探针的多光谱解混来定量评估哪些标记细胞来自哪些起始群体（基于原始报告基因标签），并且随着我们在每张载玻片上解混4、5、6种或更多光谱的能力提高，我们增加了与细胞谱系或分化相关的额外免疫组织化学方法以提高精度。利用软件检测共定位的多重荧光信号，可以根据标记染色对肿瘤基质群体进行追踪、计数和表征。
