Monocytes Use Protrusive Forces to Generate Migration Paths in Viscoelastic Collagen-Based Extracellular Matrices

Immunology Bioengineering Biophysics Cell Biology Oncology Life Sciences Medicine
monocyte cell migration extracellular matrix viscoelasticity tumor microenvironment collagen protrusive force

New Mechanism of Immune Cell Migration Revealed: How Monocytes “Pioneer Their Path” in the Tumor Microenvironment

I. Academic Research Background and the Central Question

Cell migration is a crucial biological process in life, encompassing embryonic development, tissue repair, immune responses, and the progression of various diseases. In the context of the tumor microenvironment, immune cells—particularly monocytes—enter tumor tissue from the bloodstream and can further differentiate into macrophages, playing a key regulatory role in tumor progression. However, the extracellular matrix (ECM) of tumor tissues not only features significant differences in mechanical properties (such as stiffness and viscoelasticity) but also becomes increasingly dense and complex as tumors develop.

In recent years, many studies have shown that elevated matrix stiffness and increased viscoelasticity in the tumor microenvironment are closely related to tumor progression. These changes in the mechanical properties of the matrix not only affect the migratory and proliferative capacity of tumor cells themselves, but also greatly affect the infiltration and function of immune cells. Yet, the specific migration mechanisms of monocytes within highly viscoelastic, densely packed three-dimensional matrices, as well as the effects of changes in matrix mechanics on their migration process, have remained largely unclear.

Previous research on cell migration has mostly focused on two-dimensional or three-dimensional systems with preset migration channels, making it difficult to simulate the highly compact spaces of the real tumor microenvironment. Much of the current understanding of immune cell migration, especially for monocytes, also derives from simple in vitro models. Consequently, this has led to an inadequate understanding of the actual process of immune response in tumors, and limits a theoretical basis for designing immunotherapy strategies based on changes in matrix mechanics. Therefore, revealing the authentic migration patterns of monocytes in dense three-dimensional matrices and clarifying the specific regulatory effects and mechanisms of matrix stiffness and viscoelasticity changes has both fundamental research and clinical application value.

II. Source and Institutional Affiliations of the Paper

This is an original research article published in the Proceedings of the National Academy of Sciences (PNAS) on June 16, 2025, Volume 122, Issue 25, DOI: 10.1073/pnas.2309772122. The research team is primarily from Stanford University, involving departments such as Chemical Engineering, Mechanical Engineering, Bioengineering, Biology, and Genetics, plus collaborators at the University of California San Diego, University of Texas at Austin, and University of Wisconsin-Madison. The corresponding author is Ovijit Chaudhuri (chaudhuri@stanford.edu).

The main authors include Kolade Adebowale, Cole Allan, Byunghang Ha, among others, who contributed significantly to research design, experimental operation, data analysis, and tool development. The study was handled by editor Peter Friedl (Radboudumc, Nijmegen/University of Texas MD Anderson Cancer Center) and accepted by editorial board member Herbert Levine on May 7, 2025.

III. Experimental Workflow and Technical Approach

1. Overall Research Workflow

This study aims to simulate the type I collagen–rich matrix structure of the tumor microenvironment, using collagen–alginate interpenetrating network (IPN) hydrogels with tunable mechanical properties to systematically investigate how matrix stiffness and viscoelasticity regulate the three-dimensional migratory behavior of monocytes. The research primarily includes:

  • Development and characterization of IPN matrix materials: achieving independently tunable stiffness and viscoelasticity, closely simulating mechanical changes in the tumor matrix.
  • Three-dimensional migration experiments of monocytes (U937 cell line and human primary monocytes), including quantitative and qualitative observations of cellular morphology and migratory capacity.
  • Dissection of molecular mechanisms related to migration, covering both adhesion molecules and intracellular cytoskeletal components (such as actin, myosin), through functional intervention experiments, analysis of cell polarity, and mechanical deformation.
  • Use of gene editing (CRISPR KO) and specific pharmacological interventions to reveal key signaling pathways.
  • Matrix deformation tracking and observation of channel formation to explore the “pioneering” mechanism of migration.
  • Data analysis and modeling.

2. Specific Experimental Procedures and Innovative Techniques

a) Matrix Material System Development and Tuning

  • The team developed double-network IPN hydrogels wherein type I collagen simulates the matrix structure of solid tumors, and unmodified alginate provides non-adhesive, tunable mechanical scaffolding. By varying the amount of crosslinker (calcium ion), stiffness was modulated from 1 kPa to 2.5 kPa; by adjusting alginate molecular weight, stress relaxation time was tuned from ~100 s (fast-relaxing) to ~1000 s (slow-relaxing).
  • All material parameters (storage modulus, loss modulus, relaxation time, etc.) were quantitatively measured using a rheometer (TA Instruments AR2000ex), matrix collagen fiber architecture was analyzed in detail using confocal reflectance microscopy and the CT-FIRE software, ensuring highly consistent collagen fiber structure across different material parameter sets.
  • A key innovation is that this system allows independent adjustment of stiffness and viscoelasticity with the collagen structure essentially unchanged, providing an excellent platform for realistically simulating the mechanical ecology of the tumor.

b) Three-Dimensional Monocyte Migration Model and Experimental Design

  • Migration experiments used the human U937 monocytic cell line and human peripheral blood–derived primary monocytes, embedded in various IPN hydrogels. Real-time, long-term (over 20 hours) dynamic confocal microscopy (20x or 40x/1.15 oil objective) was used to monitor migratory behavior and morphological changes.
  • Cell automatic tracking and analysis were conducted using self-developed analysis scripts based on the Imaris system; a migratory cell was defined as one with net displacement of >20μm in 3 hours.
  • Key parameters such as migration speed, migration probability, and mean squared displacement (MSD) were quantitatively analyzed using large sample sizes (n > 1000), ensuring reliability of the data.
  • For various matrix conditions, migration tracks and cellular morphological parameters (aspect ratio, circularity, etc.) were systematically evaluated across multiple replicates.

c) Molecular Mechanisms and Signaling Pathways Analysis

  • Targeted CRISPR/Cas9 knockouts or pharmacological inhibition were performed for cytoskeletal and adhesion molecules (e.g., DDR1, β1/β2 integrin, talin-1) and their effector proteins to clarify their roles during migration. For example, knockout of β2-integrin even enhanced migratory ability, whereas knockout of talin-1 impaired migration speed, indicating complex regulation and uncoupling of adhesion.
  • Pharmacological interventions included:
    • Latrunculin A (Lat. A) to inhibit actin polymerization;
    • CK-666 to inhibit ARP2/3 complex;
    • Y-27632 and Fasudil to inhibit ROCK signaling;
    • MLCK to inhibit myosin activation.
  • Real-time cytoskeletal remodeling and protein localization were observed using Spy650-FastAct (cytoskeletal dye); in addition, WASP-GFP fusion protein expression allowed dynamic tracking of WASP spatial distribution, revealing the molecular basis for cell polarity.
  • All functional interventions were accompanied by cell viability assays to ensure scientific validity.

d) Tracking of Cell–Matrix Mechanical Interaction and Component Distribution

  • Fluorescent microbeads were mixed in the collagen–alginate gels; combined with three-dimensional digital volume correlation (DVC) and digital image correlation (DIC), this enabled real-time 2D/3D visualization of the matrix deformation fields induced by single-cell migration.
  • To verify whether cells mechanically “push open” the front matrix to form a path during migration, mean deformation field overlays were used to locate the main force application site and form.
  • Continuous confocal imaging tracked the formation of cell migration channels, confirming the physical creation of micron-sized channels left after migration.

e) Cell Volume Change Experiments During Migration

  • Cells were long-term labeled with SPY650-FastAct dye and 3D reconstructed to accurately measure single-cell volume before and during migration.
  • Inhibitors for the TRPV4 channel (GSK205) and Na+/H+ pump (EIPA) were used to intervene in cell volume changes, clarifying their distinct impacts on migratory ability.

3. Data Analysis and Statistical Methods

  • All major experiments were performed with 2–3 biological replicates. Statistical tests included Kolmogorov–Smirnov, Kruskal–Wallis, and Fisher’s exact test, with p-values corrected for multiple comparisons to ensure reliability.
  • Analysis items such as migration speed, morphological distributions, and matrix force fields were visualized via large-sample statistics, heatmaps, and population trajectory analysis.

IV. Main Research Findings

1. Regulation by Matrix Stiffness and Stress Relaxation Significantly Enhances 3D Migration of Monocytes

Experiments clearly showed that both increased stiffness and accelerated stress relaxation of the matrix can independently and significantly enhance the migration speed and probability of monocytes. Under different parameter combinations, average migration rate and overall “diffusive” capacity (MSD curve slope) were elevated. Faster matrix relaxation also shifted cells towards a more stretched (ellipsoidal) morphology, with more “ellipsoid-head and tail” shapes observed, indicating structural adaption during migration.

2. Mode of Migration: Amoeboid-Like, Cytoskeleton-Dependent but Adhesion-Independent

In both three-dimensional IPN and pure alginate (with no ECM adhesive motifs) matrices, monocytes displayed round/ellipsoid morphologies, with no obvious pseudopodia, invadopodia, or filopodia. Functional experiments showed that most classic adhesion receptors (such as DDR1, β2-integrin) had no significant effect on migration when inhibited, while only combined targeting of talin-1 and β1-integrin reduced migratory ability—demonstrating that monocytes can employ a migration mode that is almost entirely adhesion-independent in highly dense matrices. This finding overturns the canonical perception of cell–ECM adhesion-dependence.

3. WASP-Mediated Cytoskeletal Polymerization at the Leading Edge is Responsible for “Pushing Open” the Matrix, Myosin is Distributed Throughout and Aids Rear Retraction

  • During migration, actin mainly forms dense puncta at the cell rear, while WASP (Wiskott–Aldrich syndrome protein) congregates at the leading edge; real-time live imaging revealed a “polymerization–retrograde flow” mechanism, establishing classic polarity.
  • WASP knockout (CRISPR KO) significantly reduced migratory ability, while knockout of another cytoskeletal promoter, WAVE, had little effect. Inhibition of cdc42 GTPase also greatly impeded migration—indicating that the cdc42–WASP–ARP2/3 axis is the key driver of leading edge cytoskeletal polymerization and motility.
  • Matrix mechanical deformation mapping revealed that cells “push” the matrix at the front to generate leading forces, with the rear and sides serving as reaction points. Cells leave sustained micron-scale migration channels in the matrix.

4. Changes in Cell Volume Are Not Essential for Migration

Although there was a trend for cell volume to increase during migration, blocking the volume increase with EIPA (Na+/H+ pump inhibitor) did not significantly affect migration, suggesting that change in cell volume is not the inherent mechanism for “path creation.” While TRPV4 inhibition slowed migration, this might reflect broad downstream effects of calcium signaling and requires a more complex explanation.

V. Research Conclusions and Significance

This study systematically reveals a new mode of monocyte migration in dense, high-viscoelasticity tumor matrices. The central conclusion is that monocytes can mechanically open up the matrix in front of them via “pushing” forces created by leading edge WASP-dependent actin polymerization, forming their own migration channels. This mode is also effective in three-dimensional, virtually adhesion-free environments, breaking the paradigm of adhesion-dependent migration. Myosin acts throughout the cell to promote rear cytoskeletal contraction, further driving forward movement.

Scientifically, this study enriches the fundamental theory of mechanical regulation and immune cell migration behavior within the tumor immuno-microenvironment, providing exceptionally valuable physical and biological insights for understanding tumor progression and immune evasion. Furthermore, it provides a robust theoretical basis for designing immunotherapeutic and tumor microenvironment–modifying strategies that target matrix properties.

In terms of application, the findings indicate that changes in matrix stiffness and viscoelasticity may influence the recruitment and functional competence of monocytes and macrophages, suggesting that differentiated macrophages and monocytes in immunotherapy can be subjected to distinct regulatory strategies. This supports the data and rationale for optimizing combined approaches of matrix remodeling and immunotherapy in solid tumors.

VI. Research Highlights and Innovations

  1. First high-fidelity 3D matrix model with independent tuning of stiffness and stress relaxation: Lays a new material foundation for advanced tumor microenvironment simulation.
  2. Discovery of a novel “adhesion-free, protrusive force-driven” mechanism for monocyte migration: Overturns the traditional adhesion-dependent paradigm and deepens our understanding of immune cell locomotion in extremely dense environments.
  3. Identification of the dominant role of the cdc42–WASP–ARP2/3 axis in 3D migration: Offers new molecular targets for understanding migration mechanisms of primary immune cells versus differentiated macrophages.
  4. Integration of digital volume correlation and real-time 3D imaging for quantitative tracking of matrix mechanical deformation and dynamic observation of migration channels: Provides a methodological paradigm for “in-situ reconstruction” of cellular mechanical behaviors.
  5. Validation of monocyte migration in non-degradable, non-adhesive nanoporous environments, proposing that migration paths in the tumor microenvironment are not pre-existing but are actively “pioneered” by the cells.

VII. Other Noteworthy Content

  • The Materials and Methods section details the preparation of CRISPR–KO stable cell lines and various fluorescence imaging and data analysis strategies, greatly informing subsequent related research.
  • This study also offers new ideas for mechanical intervention in immune cell motility disorders associated with hereditary adhesion deficiencies (e.g., leukocyte adhesion deficiency-1).
  • The open-source analysis algorithms and experimental platforms used in the paper are highly reproducible, and are expected to promote systematic research in the biomechanics of cell migration.

VIII. Conclusion

This study is a comprehensive, cross-disciplinary, and original work that deeply reveals a brand-new mechanism for immune cell migration under highly dense, viscoelastic matrix conditions, holding significant scientific implications for tumor immuno-microenvironment research and clinical interventions. It not only enriches the theoretical foundations of cellular biomechanics but also provides a solid basis for the development of new immunotherapeutic strategies and tumor microenvironment modulation.