Q5 - Substrate Stiffness Modulates Doxorubicin-Induced Apoptosis and Fractional Killing in Cancer Cells

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Natalie Calahan

Undergraduate Researcher
Colorado State University
Fort Collins, Colorado, United States

Co-Author(s)

SB

Scott Burlingham

Graduate Student
Colorado State University, United States
AP

Ashok Prasad

Professor
Colorado State University, United States

Primary Investigator(s)

Soham Ghosh

Associate Professor
Colorado State University
Timnath, Colorado, United States

Introduction: : In cancer treatment, one of the major clinical challenges lies in the phenomenon of fractional killing, where a fraction of cancer cells survives therapeutic interventions. These surviving cells frequently display an increase in drug resistance and lead to tumor relapse. Fractional killing is often linked to heterogeneous cellular mechanisms that enhance cancer cell resilience and enable evasion of apoptosis [1]. However, there has been minimal exploration into how targeting these mechanisms could lead to increased therapeutic outcomes. One such area of interest lies within the tumor microenvironment, particularly the extracellular matrix (ECM) stiffness, and its distribution within a tumor. Tumors often exhibit stiffness gradients, with softer cores and increased stiffness in the peripheral regions. This micro-architecture creates treatment challenges as cell behavior and responses to therapy can be attributed to microenvironment mechanics [2]. Substrate stiffness has been shown to alter cell morphology, with stiffer substrates promoting more spread-out morphologies and softer substrates resulting in rounder cell shapes [3]. These morphological differences may influence intracellular processes such as drug uptake and sensitivity to therapy, which could potentially contribute to fractional killing. The objective of this study is to find (1) if substrate stiffness defines the doxorubicin-induced cell death, and (2) if the stiffness-driven cell cycle status affects the cell death.

Materials and

Methods: : HeLa cells were cultured in DMEM with 10% FBS and 1X anti-anti and seeded on mechanically soft (sylgard 527 PDMS), stiff (8:1, sylgard 527: sylgard 184 PDMS) or plastic substrate. Doxorubicin (DOX) (5 µg/mL) was to understand how cancer cell apoptosis dynamics changes based on substrate stiffness. Cytochalasin-D (Cyto-D) (0.4 µM) and Y27632 (Y27) (10 µM) were used to inhibit the actin structure and the actomyosin contraction to determine actomyosin’s role in drug uptake into the nucleus. Cells were cultured on 8-well ibidi plates with/without PDMS for imaging.
Live cell imaging was performed using NucBlue Live ReadyProbes Reagent (1 drop/mL) and NucView caspase-3 substrate (2 µM). Cells were imaged every hour using a 10× objective for 5 hours starting at DOX application. A live/dead assay was completed using ethidium homodimer-1 (4 µM) and calcein, AM (2 µM) to distinguish live and dead cells after 3 hours of DOX exposure. To determine DOX uptake into the nucleus, cells were fixed before imaging with a confocal microscope using a 40× oil objective. The autofluorescence of cells and DOX was used for this.
Flow cytometry of DNA content was obtained using NucRed (1 drop/4 mL), with calcein AM used to identify live cells. A double thymidine block (2mM) was used for cell cycle synchronization. Thymidine was applied for 16 hrs, released for 8 hrs, followed by a second 16 hr application, which was released simultaneously to DOX application.

Results, Conclusions, and Discussions:: We investigated how the activation of the apoptosis indicator Caspase-3 changes based on substrate stiffness. When performing live-cell imaging, we found that there was a greater and more consistent activation of Caspase-3 when cells were seeded on a stiff substrate and tracked on a cell-by-cell basis (Fig. 1A, B). This reveals that Caspase-3 activation is not identical, regardless of substrate stiffness, indicating that fractional killing is still occurring irrespective of stiffness (Fig.1B). Translation to cell death was confirmed using a live/dead assay (Fig. 1C). This data shows that substrate stiffness plays a key role in mediating apoptosis and there is a mechanical sensing component to programmed cell death.

There are distinct morphological differences between cells cultured on a soft vs a stiff substrate. We investigated how this morphology could lead to changes in F-actin structure. Through measuring fluorescent intensity, we found that cells cultured on a stiff substrate displayed a more robust F-actin structure (Fig. 2A). Nuclear localization of DOX suggests that there was more accumulation of DOX in cells on a stiff substrate compared to soft, prompting questions about how actomyosin structure may contribute to the ability of DOX to enter the nucleus by altering nuclear pore size. The treatment of cells on a stiff substrate with Cyto-D and Y27 did not result in any decrease of DOX uptake (Fig. 2B). Overall, this data indicates that while less DOX accumulated in the nucleus of cells on a soft substrate, this cannot be attributed to actomyosin contraction or cytoskeletal F-actin structure.

Flow cytometry of DNA content reveals that after 12 hrs of DOX application, there is a significant decrease in the S-phase cell population (Fig. 3A). This confirms that DOX is targeting cells as they are replicating their DNA. Synchronization to the boundary between G1 and S-phase before DOX application reveals more consistent activation of Caspase-3 across substrates than cells in their natural states (Fig. 3B). This is evidence that proliferation rate due to substrate stiffness is a major determinant of apoptosis dynamics.

Acknowledgements and/or References (Optional): : [1] Roux, J et al. Molecular Systems Biology, 11:803, 2015.
[2] Liu, C et al. Acta Biomaterialia, 131:326-340, 2021.
[3] Bell, S et al. Biophysical Journal, 3:551-559, 2019.