Hypoxia Studies with Pimonidazole in vivo

Therapy-induced hypoxia drives changes in the tumor microenvironment that contribute to the poor response to therapy. Hypoxia is capable of driving the expression and/or activation of specific signaling cascades (e.g., c-Met, Axl, CTGF), the recruitment of tumor promoting immune cells, and the induction of cell survival pathways including autophagy (Phan et al., 2013; Hu et al., 2012; Ye et al., 2010). We have recently shown that anti-VEGF therapy-induced hypoxia can result in changes in the extracellular matrix that contribute to the aggressiveness of tumors post therapy (Aguilera et al., 2014). Importantly, therapies that induce hypoxia do not always increase epithelial plasticity and tumor aggressiveness (Ostapoff et al., 2013; Cenik et al., 2013). We have used pimonidazole to evaluate hypoxia in tumors and herein provide a detailed protocol for this useful tool to interrogate the levels of hypoxia in vivo. The utility of the HypoxyprobeTM (pimonidazole hydrochloride) immunohistochemical analysis approach allows for the assessment of hypoxia in different tissues as well as cell types. Pimonidazole is a 2-nitroimidazole that is reductively activated specifically in hypoxic cells and forms stable adducts with thiol groups in proteins, peptides, and amino acids (Cenik et al., 2013; Arnold et al., 2010; Raleigh and Koch, 1990; Raleigh et al., 1998). Furthermore, the amount of pimonidazole that is detected is directly proportional to the level of hypoxia within tumors., 治疗诱导的缺氧驱动肿瘤微环境中的变化，其导致对治疗的差的反应。缺氧能够驱动特异性信号级联（例如，c-Met，Axl，CTGF）的表达和/或活化，肿瘤促进免疫细胞的募集和细胞存活途径的诱导，包括自噬（Phan等人，2013; Hu等人，2012; Ye等人，2010）。我们最近已经显示，抗VEGF治疗诱导的缺氧可以导致细胞外基质的变化，其有助于治疗后肿瘤的侵袭性（Aguilera等人，2014）。重要的是，诱导缺氧的治疗不总是增加上皮可塑性和肿瘤侵袭性（Ostapoff等人，2013; Cenik等人，2013）。我们已经使用哌莫硝唑来评价肿瘤中的缺氧，并且在本文中为这种有用的工具提供详细的方案来询问体内缺氧水平。 Hypoxyprobe TM （哌莫硝唑盐酸盐）免疫组织化学分析方法的实用性允许评估不同组织以及细胞类型中的缺氧。哌莫硝唑是2-硝基咪唑，其在缺氧细胞中特异性地被还原活化，并与蛋白质，肽和氨基酸中的巯基形成稳定的加合物（Cenik等人，2013; Arnold等人。，2010; Raleigh和Koch，1990; Raleigh等人，1998）。此外，检测到的吡莫硝唑的量与肿瘤内的缺氧水平成正比