Periodic Reporting for period 1 - ORION (HydrOdynamics & biomechanics of canceR cell mIgration in heterOgeNeous media)
Okres sprawozdawczy: 2023-04-01 do 2025-03-31
Metastasis—the spread of cancer from a primary tumor to distant organs—remains one of the most complex and least understood aspects of cancer progression, responsible for over 90% of cancer-related deaths. A key step in this process is the migration of circulating tumor cells (CTCs) through the heterogeneous and mechanically complex environments of the extracellular matrix (ECM) and microvasculature. Despite its clinical significance, the physical and mechanical factors governing CTC transport, deformation, and retention in these environments remain largely unexplored. Traditional cancer research has focused predominantly on genetic and biochemical signaling pathways, leaving a gap in our understanding of the physical forces and fluid dynamic conditions that influence metastasis.
The ORION project was designed to fill this critical knowledge gap by investigating how hydrodynamic forces, microstructural heterogeneity, and mechanobiological properties of tumor cells interact to govern their migration behavior. The overarching objective was to develop a fundamental understanding of CTC transport through confined porous environments, using a combination of microfluidic experimentation, computational fluid dynamics (CFD), rheological modeling, and mechanobiology. By integrating these approaches, ORION aimed to reveal the physical principles underlying key metastatic behaviors such as cell trapping, deformation, collective motility, and the response to chemotactic gradients like TGF-β.
The expected impact of ORION extends across multiple domains. Scientifically, the project contributes a novel and quantifiable framework for studying cancer cell transport in complex microenvironments. Biomedically, the insights gained can help building new strategies for early detection of metastatic potential, improve therapeutic targeting, and inspire bioengineered platforms for drug testing or cell sorting. Industrially, the findings offer pathways toward the development of microfluidic systems for diagnostic or therapeutic use. Societally, understanding and ultimately disrupting the physical mechanisms of metastasis could lead to measurable improvements in patient survival and reduce the burden on healthcare systems.
Situated within the broader strategic landscape of Horizon Europe and the EU’s Mission on Cancer, ORION supports the goal of reducing the societal impact of cancer by contributing to upstream prevention and more effective treatment strategies. Its interdisciplinary and translational nature aligns with Europe's commitment to cross-sectoral, high-impact biomedical innovation. While social sciences and humanities were not explicitly integrated into this project, ORION’s focus on public health relevance and its potential to influence clinical practice make it highly aligned with societal priorities in healthcare.
The ORION project was designed to fill this critical knowledge gap by investigating how hydrodynamic forces, microstructural heterogeneity, and mechanobiological properties of tumor cells interact to govern their migration behavior. The overarching objective was to develop a fundamental understanding of CTC transport through confined porous environments, using a combination of microfluidic experimentation, computational fluid dynamics (CFD), rheological modeling, and mechanobiology. By integrating these approaches, ORION aimed to reveal the physical principles underlying key metastatic behaviors such as cell trapping, deformation, collective motility, and the response to chemotactic gradients like TGF-β.
The expected impact of ORION extends across multiple domains. Scientifically, the project contributes a novel and quantifiable framework for studying cancer cell transport in complex microenvironments. Biomedically, the insights gained can help building new strategies for early detection of metastatic potential, improve therapeutic targeting, and inspire bioengineered platforms for drug testing or cell sorting. Industrially, the findings offer pathways toward the development of microfluidic systems for diagnostic or therapeutic use. Societally, understanding and ultimately disrupting the physical mechanisms of metastasis could lead to measurable improvements in patient survival and reduce the burden on healthcare systems.
Situated within the broader strategic landscape of Horizon Europe and the EU’s Mission on Cancer, ORION supports the goal of reducing the societal impact of cancer by contributing to upstream prevention and more effective treatment strategies. Its interdisciplinary and translational nature aligns with Europe's commitment to cross-sectoral, high-impact biomedical innovation. While social sciences and humanities were not explicitly integrated into this project, ORION’s focus on public health relevance and its potential to influence clinical practice make it highly aligned with societal priorities in healthcare.
The ORION project was structured around three core work packages (WP1–WP3), each addressing a critical question related to circulating tumor cell (CTC) behavior in complex microenvironments. The activities integrated advanced microfluidic experimentation, computational modeling, and cellular rheology to uncover the physical and biomechanical drivers of metastasis.
Work Package 1 focused on quantifying CTC transport and retention in heterogeneous porous networks. A custom-designed microfluidic device mimicking capillary-scale heterogeneity was fabricated, incorporating a wide range of pore throat sizes (10–120 μm). Fluorescence microscopy and high-speed imaging were used to track the migration of breast cancer cell lines (MCF-7 and MDA-MB-231) under different flow rates. A key achievement was the discovery of dynamic two-way coupling between cell trapping and fluid flow: less invasive MCF-7 cells became trapped more frequently, significantly altering local flow paths and leading to downstream acceleration of other cells. Only ~20% of MCF-7 cells exited the network, compared to ~60% of MDA-MB-231, establishing a mechanical marker of metastatic potential. COMSOL simulations supported these findings by mapping evolving flow heterogeneity caused by local clogging.
Work Package 2 examined how scalar gradients—specifically TGF-β—modulate cell motility and EMT activation within ECM-like 3D matrices. A 3D matrix-integrated microfluidic platform was developed to impose both interstitial flow and TGF-β gradients on A549 cancer spheroids. Fluorescent reporter assays demonstrated that flow enhanced TGF-β–induced Smad signaling and increased motility, confirming the synergy between biochemical cues and fluid forces in promoting EMT. Further 3D invasion assays with A549 and MV3 spheroids revealed that matrix porosity influenced the transition from non-invasive to gas-like invasion states. These results link matrix structure and cytokine signaling to metastatic behaviors in a physiologically relevant environment.
Work Package 3 focused on the viscoelastic properties of tumor spheroids as mechanical indicators of metastatic potential. Using a dynamic compression setup with a pressure transducer and microfluidic constriction, we measured strain evolution and recovery times for MCF-10A, MCF-7, and MDA-MB-231 spheroids. A modified Maxwell model was used to extract elasticity and viscosity parameters. Benign MCF-10A spheroids were found to be stiffer and more elastic, recovering quickly after compression, while malignant spheroids exhibited slower, incomplete recovery and greater residual deformation. Confocal imaging of F-actin confirmed that cytoskeletal disorganization and weaker intercellular adhesion underpinned these mechanical differences, providing direct evidence for RQ5. This approach revealed measurable biomechanical signatures linked to malignancy.
To support experimental data, a phase-field simulation was developed to model single-cell motion through constrictions, allowing the prediction of breakthrough curves based on statistical distributions of transit time. These simulations validated experimental observations and offered a theoretical framework for CTC transport in disordered geometries.
Overall, the ORION project produced three peer-reviewed publications, two publications under peer-review at the closure of this project, advanced new microfluidic and modeling methodologies, and established multiple quantitative markers of metastatic behavior based on physical, chemical, and mechanical cues. The outcomes offer translational potential in diagnostics and therapeutic targeting and lay the foundation for follow-up research under new academic leadership.
Work Package 1 focused on quantifying CTC transport and retention in heterogeneous porous networks. A custom-designed microfluidic device mimicking capillary-scale heterogeneity was fabricated, incorporating a wide range of pore throat sizes (10–120 μm). Fluorescence microscopy and high-speed imaging were used to track the migration of breast cancer cell lines (MCF-7 and MDA-MB-231) under different flow rates. A key achievement was the discovery of dynamic two-way coupling between cell trapping and fluid flow: less invasive MCF-7 cells became trapped more frequently, significantly altering local flow paths and leading to downstream acceleration of other cells. Only ~20% of MCF-7 cells exited the network, compared to ~60% of MDA-MB-231, establishing a mechanical marker of metastatic potential. COMSOL simulations supported these findings by mapping evolving flow heterogeneity caused by local clogging.
Work Package 2 examined how scalar gradients—specifically TGF-β—modulate cell motility and EMT activation within ECM-like 3D matrices. A 3D matrix-integrated microfluidic platform was developed to impose both interstitial flow and TGF-β gradients on A549 cancer spheroids. Fluorescent reporter assays demonstrated that flow enhanced TGF-β–induced Smad signaling and increased motility, confirming the synergy between biochemical cues and fluid forces in promoting EMT. Further 3D invasion assays with A549 and MV3 spheroids revealed that matrix porosity influenced the transition from non-invasive to gas-like invasion states. These results link matrix structure and cytokine signaling to metastatic behaviors in a physiologically relevant environment.
Work Package 3 focused on the viscoelastic properties of tumor spheroids as mechanical indicators of metastatic potential. Using a dynamic compression setup with a pressure transducer and microfluidic constriction, we measured strain evolution and recovery times for MCF-10A, MCF-7, and MDA-MB-231 spheroids. A modified Maxwell model was used to extract elasticity and viscosity parameters. Benign MCF-10A spheroids were found to be stiffer and more elastic, recovering quickly after compression, while malignant spheroids exhibited slower, incomplete recovery and greater residual deformation. Confocal imaging of F-actin confirmed that cytoskeletal disorganization and weaker intercellular adhesion underpinned these mechanical differences, providing direct evidence for RQ5. This approach revealed measurable biomechanical signatures linked to malignancy.
To support experimental data, a phase-field simulation was developed to model single-cell motion through constrictions, allowing the prediction of breakthrough curves based on statistical distributions of transit time. These simulations validated experimental observations and offered a theoretical framework for CTC transport in disordered geometries.
Overall, the ORION project produced three peer-reviewed publications, two publications under peer-review at the closure of this project, advanced new microfluidic and modeling methodologies, and established multiple quantitative markers of metastatic behavior based on physical, chemical, and mechanical cues. The outcomes offer translational potential in diagnostics and therapeutic targeting and lay the foundation for follow-up research under new academic leadership.
The ORION project delivered a set of scientifically robust and technically innovative results that significantly advance the understanding of circulating tumor cell (CTC) migration in complex microenvironments. The core outcomes include:
- Quantitative demonstration of two-way coupling between CTC migration and evolving fluid flow in microfluidic constriction networks, establishing a new framework for understanding dynamic clogging and preferential flow paths in metastatic transport.
- Experimental evidence of the synergistic effect of interstitial flow and cytokine gradients (e.g. TGF-β) on EMT activation and enhanced cell motility, supported by real-time fluorescent reporter assays in 3D matrix-embedded spheroids.
- Mechanical profiling of tumor spheroids under dynamic compression, revealing measurable viscoelastic signatures that correlate with metastatic potential, supported by cytoskeletal imaging and biophysical modeling.
- Development of a phase-field simulation framework to predict breakthrough dynamics based on transit-time distributions, providing a computational tool that complements experimental results.
These findings have important implications for scientific advancement, biomedical innovation, and potential translational applications. On the scientific side, ORION introduces a multi-scale, mechanistic understanding of cell migration that bridges fluid dynamics, mechanobiology, and cancer cell behavior—opening up new lines of inquiry for researchers in biophysics and oncology.
- Quantitative demonstration of two-way coupling between CTC migration and evolving fluid flow in microfluidic constriction networks, establishing a new framework for understanding dynamic clogging and preferential flow paths in metastatic transport.
- Experimental evidence of the synergistic effect of interstitial flow and cytokine gradients (e.g. TGF-β) on EMT activation and enhanced cell motility, supported by real-time fluorescent reporter assays in 3D matrix-embedded spheroids.
- Mechanical profiling of tumor spheroids under dynamic compression, revealing measurable viscoelastic signatures that correlate with metastatic potential, supported by cytoskeletal imaging and biophysical modeling.
- Development of a phase-field simulation framework to predict breakthrough dynamics based on transit-time distributions, providing a computational tool that complements experimental results.
These findings have important implications for scientific advancement, biomedical innovation, and potential translational applications. On the scientific side, ORION introduces a multi-scale, mechanistic understanding of cell migration that bridges fluid dynamics, mechanobiology, and cancer cell behavior—opening up new lines of inquiry for researchers in biophysics and oncology.