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