Q17 - Investigating the Effects of Heat on Chromosome Loss in Ovarian Cancer Cells

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

SH

Shiori W. Harima

Undergraduate Student
Johns Hopkins University
Baltimore, Maryland, United States

Co-Author(s)

William Nebeker

Undergraduate Student
Vanderbilt University
Austin, Texas, United States

Co-Author(s)

Markus T. Sprenger, MS

Graduate Student
University of Pennsylvania, United States
JK

Jude Khatib

Postdoctoral researcher
University of Pennsylvania, United States

Primary Investigator(s)

Dennis Discher

Robert D Bent Professor
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Introduction: : Chromosomal losses and gains are central to cancer evolution, particularly in malignant and recurring tumors, and are largely driven by chromosome instability (CIN).1 CIN, characterized by the ongoing gain and loss of chromosomes, contributes to tumor heterogeneity and promotes more aggressive, therapy-resistant cancer cells.2 However, too much instability leads to cell death, while too little limits adaptability, both of which can be effectively targeted with cytotoxic or precision therapies.3
Heat shock has been shown to disrupt mitotic progression, resulting in chromosome segregation errors that closely align with increased CIN.4 Because cancer cells rely on a delicate balance of genomic instability, this vulnerability can be exploited through heat stress. Thermotherapy, a developing cancer treatment, uses elevated temperatures to push tumors beyond their viable threshold, enhancing the efficacy of chemotherapy and radiation.5
In this study, we examined the effects of heat stress on chromosomal stability and tumor growth in M2 mouse ovarian cancer cells using both 2D cultures and 3D spheroid models. We developed GFP Chromosome Reporter (ChReporter) that allows us to detect chromosome loss with high sensitivity.6 By quantifying chromosome loss after heat stress, we aim to uncover how common physical stressors, such as heat and confinement, drive chromosomal instability and, in turn, promote tumor heterogeneity.

Materials and

Methods: : Cell Line and Reporter System
GFP chromosome reporter (ChReporter) were knocked into M2 mouse ovarian carcinoma cells at the Lamin-B1 locus on chromosome 18. This system enables detection of chromosome loss through GFP signal quantification via imaging and flow cytometry.
2D Heat Shock Experiments
GFP-positive M2 cells were sorted by flow cytometry and seeded into six-well plates. After a 24-hour adhesion period at 37°C, cells were exposed to heat shock at 42°C for 3 and 6 hours. DMSO was used as a negative control and Reversine as a positive control (6 hours). Following heat treatment, cells were returned to 37°C in fresh media for a 41-45-hour recovery. Cells were then stained for DNA content and analyzed by flow cytometry or fixed for fluorescence microscopy. Micronuclei were quantified using ImageJ.
3D Spheroid Heat Shock Experiments
GFP-sorted M2 cells were cultured in anti-adhesive U-bottom 96-well plates at 37°C. After 24 hours, 1% methylcellulose was added to promote spheroid formation and simulate mechanical pressure akin to extracellular matrix stiffness. Spheroids were allowed an additional 24 hours to fully form before treatment. They were then exposed to heat shock (42°C for 3 hours), Reversine (24 hours), or DMSO (24 hours), followed by a 3-day recovery at 37°C. Spheroids were then disassembled and prepared for flow cytometry. Images were captured at spheroid formation (Day 1), Day 3, and Day 5. ImageJ and FlowJo were used to assess spheroid growth and GFP ChReporter loss.

Results, Conclusions, and Discussions:: In 2D experiments, flow cytometry revealed that cells exposed to 3 hours of heat exhibited significantly greater GFP loss compared to both negative (p ≤ 0.0001) and positive controls (p ≤ 0.01) (Figure 1A). GFP indicates chromosome loss when absent; thus, increased GFP loss is a direct marker of chromosomal instability. Analysis of micronuclei formation, a hallmark of chromosome missegregation, reveals an increase with heat exposure (Figure 1B), and longer durations of heat stress correlated with higher micronuclei counts. The 6-hour heat condition also showed significantly elevated GFP loss, but microscopy revealed apoptotic cell death, suggesting that prolonged exposure exceeds the stress threshold compatible with viability.
In contrast, 3D experiments showed no difference in GFP loss between heat-treated spheroids and the negative control. This suggests that heat-induced instability is parallel to instability induced by matrix stiffness.7 GFP loss may not be as readily detected in 3D conditions (potentially due to altered cell cycling or technical constraints), chromosomal instability is still occurring.
One 3D condition involving reversine treatment was inconclusive. Additionally, differences in proliferation rates may account for the sensitivity between 2D and 3D conditions. While M2 typically have a doubling time of 13-15 hours in 2D, spheroids showed slower growth, with doubling times of two days. Interestingly, heat exposure in 2D reduced doubling time to one day, reflecting stress-induced proliferation in response to damage.
The combined increase in micronuclei formation and GFP loss strongly supports the conclusion that heat induces chromosomal instability. These markers demonstrate that thermal stress compromises genome integrity by promoting chromosome loss. Moreover, increased GFP loss in 3D compared to 2D suggests that confinement may amplify effects of heat stress, although further investigation is needed to confirm this.

These findings demonstrate that heat acts as a potent inducer of chromosomal instability in M2, particularly in 2D environments where proliferation is rapid. Increased GFP loss, elevated micronuclei formation, and altered doubling times collectively reveal the destabilizing effect of thermal stress on the genome. This study contributes to a broader understanding of how common physical stressors, such as heat and confinement, drive chromosomal instability and, ultimately, promote tumor heterogeneity.

Acknowledgements and/or References (Optional): : This research was primarily/partially supported by NSF through the University of Pennsylvania Materials Research Science and Engineering Center (MRSEC) (DMR-2309043)
1. Bakhoum, S. F., & Cantley, L. C. (2018). The multifaceted role of chromosomal instability in cancer and its microenvironment. Cell, 174(6), 1347–1360. https://doi.org/10.1016/j.cell.2018.08.027
2. Dhital, B., & Rodriguez-Bravo, V. (2023). Mechanisms of chromosomal instability (CIN) tolerance in aggressive tumors: Surviving the genomic chaos. Chromosome Research, 31(2), 15. https://doi.org/10.1007/s10577-023-09724-w
3. Bakhoum, S. F., & Landau, D. A. (2017). Chromosomal Instability as a Driver of Tumor Heterogeneity and Evolution. Cold Spring Harbor Perspectives in Medicine, 7(6), a029611. https://doi.org/10.1101/cshperspect.a02961
4. Giordano, M., Infantino, L., Biggiogera, M., Montecucco, A., & Biamonti, G. (2020). Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs. International Journal of Molecular Sciences, 21(8), 2812. https://doi.org/10.3390/ijms21082812
5. Hyperthermia to Treat Cancer—NCI (nciglobal,ncienterprise). (2011, September 9). [cgvArticle]. https://www.cancer.gov/about-cancer/treatment/types/hyperthermia
6. Hayes, B. H., Zhu, P. K., Wang, M., Pfeifer, C. R., Xia, Y., Phan, S., Andrechak, J. C., Du, J., Tobin, M. P., Anlas, A., Dooling, L. J., Vashisth, M., Irianto, J., Lampson, M. A., & Discher, D. E. (2023). Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters. Journal of cell science, 136(11), jcs260753. https://doi.org/10.1242/jcs.260753
7. Anlaş, A. A., Sprenger, M. T., Wang, M., Ontko, N., Phan, S., & Discher, D. E. (2025). Matrix stiffness induces heritable changes in chromosome numbers, consistent with solid tumor heterogeneity. bioRxiv: the preprint server for biology, 2025.01.28.635370. https://doi.org/10.1101/2025.01.28.635370