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

项目摘要 僵硬与柔软环境中基因变化的活细胞报告者——原因和后果 与“液体”造血肿瘤相比，实体瘤通常明显僵硬，且 3D 生长受到更多限制。 对数十种癌症类型的广泛测序进一步表明，僵硬组织内的实体瘤表现出 比液体和软组织肿瘤更多的遗传变化 [Pfeifer 2017]。我们的第一个假设是 机械遗传学假说，即遗传变化部分是由肿瘤或肿瘤的力学引起的 组织微环境。当前测序方法的一个关键限制是它们需要杀死细胞 分离 DNA，防止在基因改变之前、期间和之后追踪细胞。一种新方法是 需要跟踪不同生物物理压力下活细胞的遗传变化。我们的第二个假设是 基因编辑可用于实时跟踪单细胞遗传学的一些变化。 新方法的初步结果已经支持这两个假设。 RFP（红色荧光 蛋白质）与癌细胞或正常细胞中丰富的组成基因的单个等位基因融合。对于 适当的基因，我们发现 RFP-neg 细胞已经丢失了全部或部分编辑的染色体，使用方法 范围从单细胞 DNA 测序到等位基因特异性 PCR。对于已编辑的一条染色体 研究最深入的（三者中），RFP-neg 细胞分裂并传递基因变化，并且它们还表现出 “生长”表型与关键肿瘤抑制因子的部分丧失一致。在实体瘤异种移植物中 从新分选的 RFP-pos 细胞开始，RFP-neg 细胞的比例随细胞数量的增加而强烈变化 与 2D 培养物不同，3D 成像进一步表明 (i) 分裂细胞在体内变平，并且 (ii) 具有高曲率的间期核容易破裂并表现出高 DNA 损伤。在还原论 3D 文化中 研究表明，限制和收缩同样会增加 GFP-neg 细胞的数量。直接得出初步结果 支持我们的机械遗传学假设。 我们将在体外和体内复制和扩展我们的初步结果，最终目标是 识别染色体丢失的机械调节途径及其对表型的影响。对于 与患者相关，体内研究将包括肝癌患者来源的异种移植物（PDX） 基因编辑并在肝脏以及较软和较硬的部位生长。

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.