Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies.
Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies.
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DOI：
10.3791/52801
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发表时间：
2015-06-20
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影响因子：
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通讯作者：
Heeschen C
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文献类型：
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作者：
Lonardo E;Cioffi M;Sancho P;Crusz S;Heeschen C
胰腺导管腺癌 (PDAC) 含有专门致瘤性癌症干细胞 (CSC) 的一个子集，这些细胞已被证明可驱动肿瘤的发生、转移以及对放疗和化疗的抵抗。在这里，我们描述了一种在不依赖锚定的条件下将原代人胰腺 CSC 培养为肿瘤球的具体方法。细胞在无血清、非贴壁条件下生长，以富集 CSC，而其分化程度更高的后代在单细胞接种后的初始阶段无法存活和增殖。该测定可用于估计肿瘤细胞群中存在的 CSC 百分比。形成的肿瘤球的大小（范围为 35 至 250 微米）和数量都代表了大量培养的癌细胞或新鲜收获和消化的肿瘤中所蕴藏的 CSC 活性 1,2。通过这种测定，我们最近发现二甲双胍选择性地消融胰腺 CSC；随后通过证明多能性相关基因/表面标记物的表达减少以及二甲双胍处理的细胞的体内致瘤性降低进一步证实了这一发现。作为临床前开发的最后一步，我们用二甲双胍治疗携带已形成肿瘤的小鼠，发现存活时间显着延长。目前正在进行测试二甲双胍在 PDAC 患者中的使用的临床研究（例如 NCT01210911、NCT01167738 和 NCT01488552）。从机制上讲，我们发现二甲双胍通过增强活性氧（ROS）的产生和降低线粒体跨膜电位，在 CSC 中引发致命的能量危机。相比之下，二甲双胍治疗并没有消除非CSC，而是经历了可逆的细胞周期停滞。因此，我们的研究是体外球体形成作为筛选工具的潜力的成功范例，以识别潜在靶向CSC的化合物，但该技术需要进一步的体外和体内验证，以消除错误的发现。
Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.