Utilization of 3D micropatterned collagen models to study lung adenocarcinoma invasion

摘要

Introduction: Cancer cells modulate their migration and metastatic activity in response to the tumor stroma microarchitecture. Although lung cancer accounts for the majority of cancer deaths worldwide, little focus has been placed on the effects of stroma structure on lung cancer migration. Reconstituted collagen hydrogels are commonly used as 3D models to study cancer cell migration in vitro. Despite collagen's biomimetic properties, there is a need for a model that allows for better control of the matrix's microstructure. We have developed a 3D in vitro model of lung cancer cell migration using laser micropattemed collagen hydrogels, which allows for controlled matrix pore size encountered by tumor cells in their tumor microenvironment. This model will identify the physical determinants of the matrix space that can enhance or impede lung tumor cell migration, and define how matrix structure and mechanical properties affect invasive behaviors of lung cancer cells. Methods: Metastatic mouse lung adenocarcinoma tumor spheroids are embedded in a non-pepsinized collagen I hydrogel of a various desnties. Confocal 2-photon laser scanning microscopy (2P-LSM) is used to laser-pattern microtunnel voids using 800 nm excitation wavelength at a maximum focal power of 340 mW with a 20X/1.0 NA objective. The laser ablates collagen fibers as it scans over regions of interest defined by a user-created photomask (Fig. 1a). The laser repeats the photomask pattern in each focal plane in an optical stack to create a 3D void within the gel (Fig. 1b). Microtunnels 5 and 15 μm in diameter are laser patterned adjacent to the spheroids. 2P-LSM is used to image the collagen's second harmonic signal and tumor cell migration into the 3D microtunnels. Results and Discussion: We are able to generate feature sizes as small as 0.83 μm in collagen hydrogels of various densities using the 2P-LSM patterning method (Fig. 1). When microtunnels are ablated adjacent to tumor spheroids in 6 mg/mL gels, metastatic lung tumor cells are able to migrate into and expand tunnels as small as 5 μm in diameter within 24 hours (Fig 2a,b). Tumor cells migrate collectively into the tunnels, maintaining cell-cell contact as they push against the matrix in order to invade their local environment (Fig 2c). These preliminary results suggest that lung adenocarcinoma cells primarily mechanically deform, rather than proteolytically remodel, the surrounding stroma as they invade. This also suggests that the architecture of the tumor stroma space can influence the direction and robustness of lung cancer cell migration. Conclusion: These initial results demonstrate how 2P-LSM laser ablation can generate microtunnels of defined geometries in a collagen hydrogel, allowing the user to precisely manipulate the microarchitecture the cells experience. Using this micropattemed gel in vitro, we can determine how the physical constraints of the microenvironment affect lung cancer cell migration and invasion, leading to a better understanding of lung cancer metastasis in different tissues.