A 2025 bioRxiv preprint (Ha et al., Stanford; DOI 10.1101/2025.10.03.680352) reported that when T cells migrate through confining, collagen-rich extracellular matrices (ECMs), the matrix property that best predicts their motility is not stiffness, viscoelasticity, or pore size but shear strength — the stress at which the matrix material fails. To move, T cells extend thin actin-rich protrusions that undergo a divergent, breaststroke-like motion that physically tears the matrix open, generating a migration path. The work was performed entirely in in vitro hydrogels (collagen, alginate, and their interpenetrating networks). The preprint's discussion explicitly leaves open whether matrix shear strength actually regulates T cell infiltration in vivo — in fibrotic organs and the dense stroma around tumors, where T cell exclusion predicts poor immunotherapy outcomes — and the molecular machinery that coordinates the tearing motion (formin-mediated actin polymerization, Rho/myosin-driven contractility, integrin adhesions) remains unresolved. This problem asks for that in vivo test and the coupled mechanistic dissection: establish whether shear strength causally governs T cell infiltration in living fibrotic or tumor tissue, determine the molecular coordination between formin-mediated actin polymerization and myosin-driven contractility that produces the breaststroke motion, and establish whether the tearing mechanism is conserved across T cell subsets and other immune cells.

Background

T cells constantly patrol soft tissues to find infected, stressed, or cancerous cells, so their ability to enter tissue — T cell infiltration — is a prerequisite for immune surveillance and for the success of cancer immunotherapies such as checkpoint blockade and CAR-T cells. In many tumors and fibrotic organs, however, T cells are physically excluded from the tissue interior by a dense extracellular matrix (ECM), the network of collagen and other structural proteins surrounding cells.

Most of what is known about T cell motility comes from settings where a migration path already exists: microfluidic channels, collagen gels with micron-scale pores, or pre-existing tissue tracks. There, T cells use a fast 'amoeboid' migration mode — blob-like, bleb-and-squeeze locomotion that requires little adhesion and no enzymatic matrix digestion. How T cells (or any immune cell) create a path where none exists — in matrices whose mesh pores are smaller than the cell — was far less understood.

Ha et al. (2025) addressed this by encapsulating Jurkat T cells (a standard human T-cell line) and primary human T cells in hydrogels whose mechanical properties were independently tuned: collagen gels, alginate gels (a polysaccharide matrix), and collagen–alginate interpenetrating networks (IPNs, where a collagen network is interwoven with an alginate network). Across gels spanning a range of stiffness (elastic modulus), viscoelasticity (time-dependent, fluid-like stress relaxation), mechanical plasticity (irreversible deformation), and shear strength (the shear stress at which the material fails — here, the stress needed to physically rupture the matrix), only shear strength strongly correlated with T cell migration. Imaging showed T cells extending thin, actin-rich, finger-like protrusions into the matrix that then moved apart in a divergent 'breaststroke-like' motion, applying outward stresses that rupture the matrix and open a path. Pharmacology pointed to the molecular machinery: migration was strongly impaired by inhibitors of myosin contractility (ML7, blebbistatin) and of the Rho/ROCK pathway (the small GTPase Rho and its effector kinase ROCK, which drive actomyosin contraction; inhibited by Y27632), and partially by the formin inhibitor SMIFH2 (formins are actin nucleators that build unbranched actin filaments, as in these protrusions), but not by the Arp2/3 inhibitor CK666 (Arp2/3 builds branched actin networks); β1-integrin-mediated adhesions to collagen helped cells exert force in strong matrices. The authors further measured human pancreatic ductal adenocarcinoma (PDAC) tissue and found its shear strength to be more than 20 times that of normal pancreas, suggesting that elevated matrix shear strength could physically explain the notorious failure of T cells to infiltrate pancreatic tumors.

Adjacent work makes the question pointed rather than settled. Monocytes generate migration paths in viscoelastic collagen matrices using protrusive — not divergent, tearing — forces (Adebowale et al., PNAS 2025). Migrating immune cells in vivo globally coordinate protrusive forces (Reis-Rodrigues et al., Nature Immunology 2025). T cells in microfluidic confinement use a central actin pool dependent on DOCK8 (dedicator of cytokinesis 8, an actin-cytoskeleton regulator whose loss causes human immunodeficiency) to widen channels and shield the nucleus (Science Immunology 2025), and tissue-resident memory CD8+ T cells use bleb-led mechanosensing migration in salivary glands (Science Immunology 2023). None of these address matrix tearing by shear-strength-limited failure, and none test shear strength as a regulator of infiltration in vivo. The preprint's discussion leaves open the in vivo relevance of shear strength (fibrosis, tumor microenvironment) and whether the breaststroke motion is conserved across T cell subsets and other immune cells. The study did not resolve the molecular coordination between formin-mediated actin polymerization and myosin-driven contractility beyond the coarse pharmacology, and it examined only collagen and alginate matrices.

Problem Statement

Does the shear strength of the extracellular matrix — the stress at which the matrix physically fails — causally regulate T cell infiltration in vivo, and what molecular machinery produces the matrix-tearing breaststroke motion? Concretely, building on Ha et al. (bioRxiv 2025, DOI 10.1101/2025.10.03.680352), where only in vitro hydrogels were examined: (1) test in a living fibrotic or tumor context (or ex vivo human tissue) whether manipulating or stratifying matrix shear strength changes T cell infiltration, establishing (or refuting) shear strength as an in vivo regulator of infiltration and thereby a mechanistic explanation for T cell exclusion in diseases such as pancreatic cancer; (2) dissect the molecular coordination between formin-mediated actin polymerization and Rho/myosin-driven contractility that produces the divergent breaststroke-like motion of actin-rich protrusions and the resulting matrix rupture, going beyond the coarse pharmacology (SMIFH2, blebbistatin/ML7, Y27632) and the verbal model of myosin-driven retrograde actin flow (rearward movement of actin filaments from the leading edge) diverging at the leading edge; and (3) establish whether this tearing mechanism is conserved across T cell subsets (e.g., CD4+ versus CD8+ versus regulatory T cells) and other immune cells, or whether it is unique to T cells, and whether ECM components beyond collagen and alginate modulate shear strength and the tearing mechanism. Answering the in vivo question requires the mechanistic and cell-type results to interpret what is being tested, so these parts constitute one coherent experimental program and are jointly required rather than independently resolving.

References

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