Live cell reporters of genetic changes in stiff vs soft surroundings - Causes & Consequences

项目摘要 硬环境与软环境中基因变化的活细胞记录器--因果 实体肿瘤通常明显僵硬，与“液体”血液肿瘤相比，3D生长更受限制。 对数十种癌症类型的广泛测序进一步表明，僵硬组织中的实体瘤表现为 比液体和软组织肿瘤更多的基因变化[Pfeifer 2017]。我们的第一个假设是 机制遗传学假说，即遗传变化部分是由肿瘤的机制或 组织微环境。目前测序方法的一个关键限制是它们需要杀死细胞来 分离DNA，防止在基因改变之前、期间和之后追踪细胞。一种新的方法是 需要跟踪活细胞在不同生物物理压力下的遗传变化。我们的第二个假设是 基因编辑可以用来实时跟踪单细胞遗传学的一些变化。 一种新方法的初步结果已经支持了这两种假设。RFP(红色荧光 蛋白质)与癌细胞或正常细胞中丰富的构成基因的单个等位基因融合。为 适当的基因，我们发现RFP-neg细胞已经丢失了编辑的全部或部分染色体，使用方法 范围从单细胞DNA序列到等位基因特异性聚合酶链式反应。一条编辑过的染色体 研究最深入的(三个中)，RFP-neg细胞分裂并传递遗传变化，它们还表现出 一种与部分失去关键抑癌基因一致的“继续生长”的表型。在实体瘤异种移植中 从新分离的RFP-pos细胞开始，RFP-neg细胞的比例随着细胞数量的增加而显著增加 与2D培养不同，分裂和3D成像进一步表明(I)分裂细胞在体内是扁平的，以及(Ii) 高曲率的间期核容易破裂，表现出较高的DNA损伤。在简约主义3D文化中 研究、限制和限制同样增加了GFP-neg细胞的数量。初步结果直接 支持我们的机械遗传学假说。 我们将在体外和体内复制和推广我们的初步结果，最终目标是 识别染色体丢失的机械调制途径和对表型的影响。为 与患者相关的体内研究将包括肝癌患者来源的异种移植(PDX) 基因在肝脏中编辑和生长，以及更软和更硬的部位。

Project Abstract Live cell reporters of genetic changes in stiff vs soft surroundings – causes & consequences Solid tumors are often palpably stiff and more constrained in 3D growth than ‘liquid’ hematopoietic tumors. Extensive sequencing of dozens of cancer types further indicates that solid tumors within stiff tissues exhibit many more genetic changes than liquid and soft-tissue tumors [Pfeifer 2017]. Our first hypothesis is a mechano-genetics hypothesis, namely genetic changes are caused in part by the mechanics of the tumor or tissue micro-environment. A key limitation of current sequencing methods is that they require killing cells to isolate the DNA, which prevents tracking a cell before, during, and after a genetic change. A new method is needed to track genetic changes in living cells under diverse biophysical stresses. Our second hypothesis is that gene editing can be used to enable tracking some changes in the genetics of single cells in real-time. Preliminary results from a new approach already support both hypotheses. RFP (red fluorescent protein) is fused to a single allele of an abundant constitutive gene in cancer cells or normal cells. For appropriate genes, we find that RFP-neg cells have lost all or part of the edited chromosome, using methods that range from single cell DNA-seq to allele-specific PCR. For the one edited chromosome that has been studied most deeply (of three), the RFP-neg cells divide and pass on the genetic change, and they also exhibit a ‘go-and-grow’ phenotype consistent with partial loss of a key tumor suppressor. In solid tumor xenografts that start with freshly sorted RFP-pos cells, the fraction of RFP-neg cells scales strongly with the number of cell divisions, unlike 2D cultures, and 3D imaging further shows that (i) dividing cells are flattened in vivo, and (ii) interphase nuclei with high curvature tend to rupture and exhibit high DNA damage. In reductionist 3D culture studies, confinement and constriction likewise increase GFP-neg cell numbers. The preliminary results directly support our mechano-genetics hypothesis. We will replicate and extend our preliminary results both in vitro and in vivo with the ultimate goals of identifying mechanically modulated pathways of chromosome loss and consequences for phenotype. For relevance to patients, the in vivo studies will include liver cancer patient derived xenografts (PDX) that are gene edited and grown in liver as well as softer and stiffer sites.