Spatial Cues from Tumor ECM Patterns Regulate CD8⁺ T Cell Motility and Deformation

Introduction: : The extracellular matrix (ECM) in the tumor microenvironment (TME) not only provides structural support but also actively regulates cell behavior. Compared to healthy tissues, tumor-associated ECM is characterized by increased collagen fiber density and directional alignment, giving rise to distinct architectures known as tumor-associated collagen signatures (TACS). Immune cells—particularly cytotoxic CD8+ T cells—play a critical role in antitumor immunity. However, their infiltration and activity can be significantly influenced by ECM topography. Prior studies have demonstrated that cell behavior, including migration and function, can be regulated by micropatterns and topographical features. Despite the progress of current immunotherapies, immune evasion remains a major hurdle. The physical barrier posed by altered ECM topology may impair immune cell infiltration and reduce their effectiveness. Most current in vitro cancer models fall short in replicating the anatomical, immunological, and mechanical features of the tumor ECM. To bridge this gap, we designed a three-dimensional, ring-shaped ECM model to simulate the spatial confinement observed in tumors. Our goal was to investigate how CD8+ T cells respond to this pattern in terms of migration and morphology.

Materials and

Methods: : A concentric circle-shaped micropattern was designed and fabricated using 3D printing, then transferred onto a PDMS (polydimethylsiloxane) substrate. CD8⁺ T cells were isolated from human peripheral blood mononuclear cells (PBMCs), activated using anti-CD3 and anti-CD28 antibodies, and seeded onto surface-treated PDMS micropatterns to enhance cell adhesion. Real-time migration and morphological changes of the T cells interacting with the patterned substrate were monitored. Live-cell imaging was performed one hour after seeding, and immunofluorescence staining was conducted after one day of culture to observe T cell migration and behavior. Nuclei were labeled using DAPI or Hoechst, and actin was visualized using phalloidin.

Results, Conclusions, and Discussions::

Results: On the ring-shaped PDMS pattern with curved microgrooves and sidewalls (A–B), activated CD8⁺ T cells exhibited two distinct modes of migration: localized rotational movement and faster, more randomized migration (C–D). The presence of concentric curvature appeared to guide T cell trajectories, compelling them to follow the groove pathways. Live-cell imaging revealed that a subset of T cells successfully crossed the sidewalls of the grooves—or attempted to, but failed and returned (E). These cells elongated and significantly deformed their bodies, suggesting active efforts to overcome the physical barrier presented by the topography. The ability to deform and migrate across these structures appeared to correlate with cellular activity levels, indicating that more motile cells may be better equipped to infiltrate topographically complex environments.

Conclusion:
This study demonstrates that ECM topography—particularly ring-like confinement structures that mimic tumors surrounded by collagen fibers—significantly influences the migration behavior and morphology of CD8⁺ T cells. The physical architecture of the ECM affects immune cell infiltration by modulating how cells adapt to topographic constraints. By integrating human ECM cues, topographical design, and immune components, our model provides a robust in vitro platform for investigating immune–ECM interactions in TME.

Discussion:
To investigate the distinct migration modes of T cells and their ability to cross ECM sidewalls, we hypothesize that the ring-shaped ECM patterns commonly observed in tumors may act as physical barriers that impede or delay immune cell infiltration. This structural confinement may also increase the energy expenditure required for T cells to navigate the tumor microenvironment, potentially diminishing their functional capacity over time. In future studies, we aim to refine our model by incorporating biomimetic ECM through decellularized matrices to enhance physiological relevance. Additionally, we will investigate the metabolic costs associated with T cell migration in topographically confined environments. Understanding how ECM-induced mechanical constraints affect immune cell energetics and contribute to immune exhaustion could provide valuable insights for the development of more effective immunotherapies.

Acknowledgements and/or References (Optional)::