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

僵硬与柔软环境中遗传变化的活细胞报告 - 原因

• 批准号: 10092733
• 负责人: Dennis E. Discher
• 金额: $ 91.66万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10092733
• 关键词: 3-Dimensional; Alleles; Biophysics; Biopsy; Brain; Cell Count; Cell Fraction; Cell Nucleus; Cell division; Cells; Chromosomes; DNA; DNA Damage; DNA Repair; DNA sequencing; Defect; Engineering; Engraftment; Exhibits; Flow Cytometry; Genes; Genetic; Genetic Processes; Goals; Growth; Hematopoietic Neoplasms; Heritability; Human; Image; Immune checkpoint inhibitor; Immunodeficient Mouse; In Vitro; Incidence; Injections; Interphase; Karyotype determination procedure; Link; Liquid substance; Liver; Liver neoplasms; Malignant Neoplasms; Malignant neoplasm of liver; Measures; Mechanics; Methods; Mitosis; Mitotic spindle; Modernization; Mutation; Normal Cell; Nuclear Envelope; Palpable; Pathway interactions; Patients; Pediatric Neoplasm; Pharmaceutical Preparations; Phenotype; Physics; Point Mutation; Process; Public Health; Reporter; Risk Factors; Rupture; Shapes; Site; Soft Tissue Neoplasms; Solid Neoplasm; Squash; Stress; Suspensions; Testing; Therapeutic; Three-Dimensional Imaging; Time; Tissues; Tumor Suppressor Proteins; Tumor Tissue; cancer cell; cancer imaging; cancer type; cell killing; cell motility; chromosome loss; constriction; experimental study; in vivo; induced pluripotent stem cell; intraperitoneal; knock-down; malignant breast neoplasm; novel strategies; patient derived xenograft model; pressure; prevent; red fluorescent protein; soft tissue; subcutaneous; three dimensional cell culture; trend; tumor; tumor xenograft

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.

项目摘要 硬环境与软环境中遗传变化的活细胞报告者-原因和后果 实体肿瘤通常明显僵硬，并且比“液体”造血肿瘤在3D生长中受到更多限制。 对数十种癌症类型的广泛测序进一步表明，坚硬组织中的实体瘤表现出 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 机械遗传学假说，即遗传变化部分是由肿瘤的力学引起的， 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， 分离DNA，这可以防止在基因变化之前，期间和之后跟踪细胞。了一种新的方法 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组成型基因的单个等位基因融合。为 适当的基因，我们发现RFP阴性细胞已经失去了全部或部分编辑的染色体，使用方法， 从单细胞DNA测序到等位基因特异性PCR。对于一个编辑过的染色体， 研究最深入的（三个），RFP阴性细胞分裂并传递遗传变化，它们也表现出 一种与关键肿瘤抑制基因部分缺失一致的“继续生长”表型。在实体瘤异种移植物中， 从新鲜分选的RFP-pos细胞开始，RFP-neg细胞的分数与细胞的数量密切相关， 与2D培养物不同，3D成像进一步显示（i）分裂细胞在体内是扁平的，以及（ii） 具有高曲率间期核倾向于破裂并表现出高DNA损伤。在简化的3D文化中 研究表明，限制和收缩同样增加了GFP阴性细胞的数量。初步结果直接 支持我们的机械遗传假说 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 鉴定染色体丢失的机械调节途径和表型的后果。为 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。