(31d) Matrix Shear Strength Regulates T Cell Migration through Confining Microenvironments

T cells adeptly infiltrate various soft tissues to combat pathogens, despite the challenging microenvironments they encounter, characterized by pore sizes much smaller than their own diameter. Extracellular matrices (ECMs) in tissues are often nanoporous (Fig. 1a) [1,2]. For cells to migrate through such confining microenvironments, they must create micron-sized openings to translocate their bodies and stiff nuclei. Additionally, ECMs exhibit matrix mechanical plasticity, undergoing irrecoverable deformation when subjected to cellular forces [3,4], and can fail locally at sufficiently high cellular forces. Both in vitro studies and in vivo observations indicate that T cells do not rely on enzymatic degradation to migrate, suggesting the use of physical forces to mold migration pathways [5]. However, the biophysical mechanisms that drive T cell migration through confining microenvironments remain unknown [6]. Here, we sought to understand how ECM mechanics regulates T cell migration, and the biophysical mechanisms driving T cell migration through confining ECMs.

Materials and Methods

Interpenetrating networks (IPNs) of alginate and collagen with as a tunable model of collagen-rich matrices. We developed interpenetrating networks of type-1 collagen and alginate (IPNs) to emulate type-1 collagen-rich tissues, such as stromal matrices, which T cells traffic through. The collagen not only offers structural support but also presents cell-adhesion ligands to T cells to facilitate integrin-mediated bindings. Meanwhile, the alginate network fills in the micropores of collagen, making the IPNs nanoporous. A pivotal feature of alginate is that it facilitates the tuning of the mechanical properties of the overall IPN. Importantly, alginate lacks any cell adhesion motifs and is resistant to degradation by mammalian proteases. This forces T cells to physically open a path for migration. IPNs are formed by blending type-1 collagen (1.5 mg/ml) with alginate (6.0 mg/ml), followed by crosslinking the alginate using calcium ions (Fig. 1b).

Migration assay. Live-cell confocal microscopy and image analysis software were used to track the migration of fluorescently labeled Jurkat cells, used here as a model of T cells, encapsulated in the IPNs.

Results, Discussion, and Conclusion

We first developed a set of IPNs with a range of stiffness, viscoelasticity, plasticity and shear strength. The stiffness or Young’s modulus of the IPNs was modulated from 500 Pa to 4 kPa by varying the degree of calcium crosslinking (Fig. 1c). By adjusting the molecular weight (MW) of the alginate and calibrating the calcium crosslinking density to keep the initial Young’s modulus or storage modulus consistent and pore sizes, the viscoelasticity, plasticity, and shear strength were modulated independent of the stiffness. For instance, the loss tangent, plasticity, and shear strength can range from respectively 0.06, 0.1, 600 Pa in low MW alginate IPNs to 0.08, 0.17, 100 Pa in high MW IPNs, all while preserving an initial Young’s modulus of approximately 1.4 kPa (Fig. 1d-f). Overall, we formed a set of gels with varying stiffness, viscoelasticity (loss tangent or stress relaxation), plasticity, and shear strength. Importantly, no two parameters were perfectly coupled, allowing us to explore the independent contribution of each to regulation of migration.

Using the tunable IPNs, we examined the impact of various matrix mechanical properties on T cell migration. These properties represent material behaviors spanning from linear viscoelastic (i.e., Young’s modulus, loss tangent, stress relaxation) to plastic (i.e., yield stress, plasticity) and failing (i.e., shear strength). Jurkat human T lymphocytes were encapsulated within seven distinct 3D matrices. Their movements were observed for a duration of 6 hours through time-lapse microscopy experiments (Fig. 1g). To quantitatively describe motility, several metrics were considered: the mean and maximum speeds of cell migration, track length (the cumulative distance of all trajectory segments), track displacement length (the straight-line distance from the starting to the ending point of the entire trajectory), and the fraction of motile cells. Subsequent correlation analysis aimed to discern the influence of hydrogel mechanical properties on these motility descriptors (Fig. 1h). Notably, among the various metrics representing matrix mechanical properties, the shear strength emerged as the strongest predictor of robust cell migration (Fig. 1h,i). Considering that linear viscoelasticity corresponds to 100% strain recovery, plasticity to 75-90%, and shear strength to 0%, the results indicate that cell-induced IPN remodeling was associated with a fully irrecoverable material failure, rather than a partially recoverable deformation. Additionally, in IPNs exhibiting shear strength above a certain threshold, T cells were immobilized. This suggests that when cells are not able to apply stresses that exceed the shear strength of the gel, they are unable to migrate.

We next examined the shear strength in soft tissues during cancer to explore the significance of these findings. Analysis of human pancreas tumors revealed significantly higher shear strengths compared to normal bowel tissues (Fig. 1j). This suggests that changes in shear strength during cancer progression could play a key role in mediating immune cell infiltration and activity. Our study implies that T cells can migrate through a nanoporous matrix that is sufficiently weak such that cells can destructively rearrange the polymer networks and enlarge the pores for migration.

Further, we measured the motility of T cells encapsulated in pure alginate gels and compared against their IPN counterparts. Two types of alginate hydrogels were prepared: one weak and one strong, with shear strengths of about 100 and 200 Pa, respectively (Fig. 1k). Corresponding IPNs contained identical molecular weight and concentration of alginate and demonstrated equivalent levels of shear strength. Notably, T cells exhibited no migratory activity within the strong alginate gels, while demonstrating moderate migration within the strong IPN counterparts (Fig. 1l). Conversely, T cells displayed robust migration within the weak alginate gel, with even faster migration compared to cells within the weak IPN (Fig. 1m). This suggests that T cells may exert stronger forces to establish migration pathways within the IPNs, possibly by interacting with the collagen-1 network, albeit at a slower pace due to increased adhesion formation. Ongoing studies are aimed at elucidating the molecular mechanism of T cell migration.

In conclusion, these findings establish shear strength as the critical feature of the matrix governing Jurkat cell migration through nanoporous meshes and implicate changes in shear strength during pancreatic cancer progression. Moreover, the work highlights the potential involvement of collagen in providing adhesion motifs for Jurkat T cells, enabling the cells to exert greater forces when navigating through soft tissues. Consequently, these results have significant implications for understanding immune cell infiltration into tumors or fibrotic regions, and for informing the design of biomaterials aimed at enhancing therapeutic T cell delivery.

Acknowledgments: This work was supported by the National Science Foundation under Postdoctoral Fellowship in Biology grant 2209411 and National Science Foundation CAREER award (CMMI 1846367).

References

[1] Piérard, G. E. & Lapière, C. M. Microanatomy of the dermis in relation to relaxed skin tension lines and Langer's lines. The American journal of dermatopathology 9, 219-224 (1987).

[2] Wolf, K. et al. in Semin. Cell Dev. Biol. 931-941 (Elsevier).

[3] Wisdom, K. M. et al. Matrix mechanical plasticity regulates cancer cell migration through confining microenvironments. Nature communications 9, 4144 (2018).

[4] Chaudhuri, O., Cooper-White, J., Janmey, P. A., Mooney, D. J. & Shenoy, V. B. Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature 584, 535-546 (2020).

[5] Wolf, K., Müller, R., Borgmann, S., Brocker, E.-B. & Friedl, P. Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases. Blood 102, 3262-3269 (2003).

[6] Lee, J. Y. & Chaudhuri, O. Modeling the tumor immune microenvironment for drug discovery using 3D culture. APL bioengineering 5, 010903 (2021).

[7] Wolf, K. et al. Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J. Cell Biol. 201, 1069-1084 (2013).