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

> **NIH NIH U01** · UNIVERSITY OF PENNSYLVANIA · 2024 · $756,522

## Abstract

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.

## Key facts

- **NIH application ID:** 10818403
- **Project number:** 5U01CA254886-04
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Dennis E. Discher
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $756,522
- **Award type:** 5
- **Project period:** 2021-04-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10818403

## Citation

> US National Institutes of Health, RePORTER application 10818403, Live cell reporters of genetic changes in stiff vs soft surroundings - Causes & Consequences (5U01CA254886-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10818403. Licensed CC0.

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