A microfluidic stiffness gradient gel platform for probing cancer cell invasion into the extracellular matrix

Introduction:: Tumors are stiffer than healthy tissues due to alterations in the extracellular matrix (ECM), solid stress, and interstitial pressure, which can promote cancer progression, metastasis, and resistance to therapy [1]. Most studies on tumor stiffness and cancer progression use culture systems of defined stiffness; however, the stiffness of the tumor microenvironment is heterogeneous [2]. Thus, an urgent need exists to develop in vitro models capable of mimicking this ECM complexity. Several methods for making stiffness gradient hydrogels have been developed, including using heat gradients, reinforcement additions to polymers, microfluidic mixing devices, photolithography, and two-step polymerization [3]. Yet, some of these methods can be complex and/or do not allow for real-time monitoring and manipulation of tumor cell invasion. Here, we developed a versatile, non-toxic, and highly reproducible stiffness gradient hydrogel system in a controllable microfluidic device that allows for 3D investigation of breast cancer cell invasion and behavior. To do this, we fabricated Gelatin Methacrylate (GelMA) stiffness gradient hydrogels spanning ~0.5 – 18 kPa, mimicking healthy to tumor tissue stiffness, into microchips, which were crosslinked using a photomask and visible light sensitive initiator Ruthenium with sodium persulfate (SPS). The microchip used in this study (Dax-1, Aim Biotech Pte. Ltd.) is readily available in the market, and thus is widely applicable to other biological systems. The fabricated devices were applied to understand the role of ECM stiffness in breast cancer cell invasion, chemoresistance, and immune cell infiltration. The insights gained will aid in the development of novel therapeutic strategies to combat cancer.

Materials and Methods:: High degree of methacrylation ( > ~80%) GelMA was synthesized as previously described [4]. To prepare the stiffness gradients, GelMA was solubilized in PBS and then mixed with SPS and Ruthenium. The resulting solution was pipetted into the central channel of the Dax-1 microchip (Aim Biotech Pte. Ltd.) and polymerized under a visible light source (400-450 nm) with a transparency gradient photomask that creates a stiffness gradient in the resulting hydrogel. Stiffness was measured with a Nanoindenter and critical point drying (CPD) of the gels followed by scanning electron microscopy (SEM) was used to examine gel porosity along the stiffness gradient. MDA-MB-231 metastatic breast cancer cells and MCF-7 non-metastatic breast cancer cells were separately seeded into one of the side channels of the device. Media with or without epidermal growth factor (EGF) was added into the opposite channel of the device, and the cells were cultured for five days at 37^oC with 5% CO₂ in a humidified incubator. Real-time monitoring was used to understand the dynamics of invasion. At day 5, the devices were fixed and immunostained for cell and nuclear morphology (F-actin and DAPI), adhesion formation (Paxillin), mechanotransduction (yes-associated protein 1, YAP1), and proliferation (EdU). Live and fixed imaging was carried out using a Zeiss Celldiscoverer 7 and both 2D and 3D image analysis was done in Imaris.

Results, Conclusions, and Discussions:: Results and discussion

We produced GelMA linear stiffness gradient hydrogels in a controlled microfluidic device using Ruthenium/SPS visible light photo-initiator with a gradient photomask. Nanoindentation showed that the stiffness gradient ranged from ~0.5 to 18 kPa over 10.5 mm. The device was used to study the invasion of breast cancer cells into hydrogels as a function of stiffness. While non-metastatic cells (MCF-7) do not invade the hydrogel, we found that as stiffness increased, invasion of metastatic MDA-MB-231s increased (Fig. 1). The addition of EGF - which activates the EGF receptor, initiates intracellular signaling, and in turn regulates cancer invasion and migration - into the opposite chamber of the device enhanced cell invasion up through day 5 of culture. Moreover, the study revealed the time-dependent influence of stiffness on cell area, with larger cells found in softer regions on day 3, but larger cells found in stiffer regions on day 5. These results coincide with morphological changes at day 3, including in cellular ellipticity. Our findings align with previous studies that reported cancer cell migration is enhanced with material stiffness (3.7 vs. 1.6 GPa) [5], although our system is able to recapitulate a continuous regime of physiologically relevant stiffness. The differences we observed in cancer invasion and morphology vs. stiffness at day 5 could be due to the pore size of the hydrogels, where pores are larger in softer regions than in the stiffer regions, rates of proliferation, degradability of the gels, as well as specific modes of cell migration. Further research is required to understand the mechanisms driving the invasion of MDA-MB-231s and other metastatic cancer cell types that have been reported to have altered mechanosensitive machinery [6].

Conclusion

In this study, we developed a microfluidic device capable of mimicking the heterogeneity of the tumor ECM stiffness using a GelMA-based stiffness gradient hydrogel. The system was utilized to examine the invasion and behavior of non-metastatic (MCF-7) and metastatic (MDA-MB-231) breast cancer cells as a function of stiffness. Future work aims to explore matrix stiffness impact on immune cell tumor infiltration, immune cell-cancer cell interactions, cancer metastasis, and chemoresistance.


Acknowledgements (Optional): : This work was supported by the Ministry of Education under the Research Centres of Excellence programme through the Mechanobiology Institute at the National University of Singapore and the Biomedical Engineering Department at the National University of Singapore.

References (Optional): :

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