2nd Edition of Cell & Gene Therapy World Conference 2026

Abstract

Background: Intratumoral heterogeneity is a major determinant of chemotherapy efficacy and patient prognosis. Within a single tumor, genetically and morphologically distinct cell subpopulations can respond very differently to the same drug. Recent work, including our own, points to substrate stiffness — the mechanical elasticity of the tissue on which cells grow — as an underappreciated modifier of this heterogeneity. Adherent cells sense substrate elasticity through integrin receptors and relay this mechanical information via the cytoskeleton to the nucleus, altering their biochemical response. Compliant polyacrylamide hydrogels reproduce this mechanical microenvironment in vitro across a physiologically relevant stiffness range (~2 kPa, brain tissue, to ~40 kPa, muscle tissue). Preliminary observations show that some cytostatic agents active on rigid plastic dishes lose efficacy on compliant substrates, and vice versa.

Aim: We are investigating how substrate elasticity shapes the response of the heterogeneous rat mammary carcinoma cell line WC256 — chosen for its several distinct, bone-metastasis-competent subpopulations — to chemotherapeutics with different mechanisms of action: taxol (microtubule disruption) and a DNA-complexing agent (doxorubicin; cyclophosphamide in the ongoing continuation study).

Methods: WC256 cells are cultured on polyacrylamide hydrogels of tunable stiffness (2–40 kPa) coated with extracellular-matrix proteins (fibronectin, collagen I, collagen IV, laminin, and combinations thereof), selected to optimize viability and morphology. Live-cell brightfield imaging documents morphology; immunofluorescence staining of the actin/microtubule cytoskeleton, focal adhesions, and nuclei allows high-throughput quantification via tile-scan fluorescence microscopy and detailed confocal analysis. Drug dose–response is bounded between vehicle control and the minimal dose producing 75% cell death, cross-validated using three complementary assays: MTT, LDH release, and FDA/PI staining.

Results to date: Substrate-stiffness parameters and extracellular-matrix coating composition have been optimized for WC256. IC50 values for both chemotherapeutics were established on rigid substrates; comparing MTT, LDH, and FDA/PI assays revealed assay-dependent differences in the estimated IC50, underlining the importance of assay choice in cytotoxicity screening.

Conclusion: This methodological groundwork now enables the direct, ongoing comparison of drug response across WC256 subpopulations as a function of substrate elasticity. Characterizing this mechanobiological dimension of chemotherapy response may help explain clinically observed variability in treatment outcomes and support the design of more physiologically relevant in vitro drug-screening platforms.

Keywords: tumor heterogeneity; mechanobiology; substrate stiffness; polyacrylamide hydrogel; chemotherapy; WC256