News and Reviews (April 2016)

3D Microcages to Analyze the Mechanical Properties of Cancer Cells

(Barbara Spagnolo)  Tumor cells that disseminate from a tumor can migrate into the vascular system and build metastasis. This process involves cell migration across micrometer-sized pores along the vessel walls [1, 2].

In order to build a model to analyze tumor cell migration across pores significantly smaller than the cell itself, we have conceived and engineered three-dimensional cage-like structures (Figure 1) realized by means of two-photon polymerization (2PP) [3]. They consist of four cylindrical pillars and five suspended porous walls with tunable pore areas (3, 18 or 85µm²). Structures were made in IP-L photoresist which was demonstrated to be fully biocompatible [3].

SEM images of cage-like structures obtained by means of 2PPFig. 1:  SEM images of cage-like structures obtained by means of 2PP with small, medium and large pore area: 3 µm2, 18 µm2 and 85 µm2 pores. Ridges in-plane thickness is ~ 500 nm and out-of-plane thickness is ~ 1 µm.

We analyzed three different cells lines derived from the human breast (non tumorigenic, tumorigenic and metastatic). These lines are characterized by increasing invasive potential and decreasing stiffness values. We found that the cages with the smallest pore area allow metastatic cells to enter into the cage while keeping outside non tumorigenic cells. These data suggest a selection based on the cells mechanical properties (Figure 2, panels A-A' and C-C'). Immunostaining analysis also revealed that the cytoskeletal protein myosin accumulates at the points were cells are applying force during the invasion process, showing that cage invasion is an active mechanism (Figure 2, panels B and D). The work, published on Scientific Reports (DOI: 10.1038/srep10531) [3] show how 2PP microfabricated cage-like scaffolds with tunable pores can be exploited to investigate tumor cell transmigration in three-dimensional systems and, more in general, how they can be a useful tool to study cell mechanics.

Confocal reconstruction of cage-like structures and DAPI staining of cell nucleiFig. 2: Confocal reconstruction of cage-like structures and DAPI staining of cell nuclei. Panel A (bottom view) and A' show non tumorigenic cells accumulating around a cage with small pores. Tumorigenic (not shown) and metastatic cells (C and C') enter into the cage exploiting 3 µm2 pores. In both cases myosin forms aggregates at the cell-cage contact points were cells are applying force (panels B and D, blue box)

This article and images were provided by our customer IIT Arnesano, Barbara Spagnolo

[1] Pellegrino, T., Parak, W.J., Boudreau, R., Gerion, D., Alivisatos, A.P., and Larabell, C.A.: ‘Quantum dot-based cell motility assay’, Differentiation, 2003, 71, (9), pp. 542-548
[2] Chen, M.B., Whisler, J.A., Jeon, J.S., and Kamm, R.D.: ‘Mechanisms of tumor cell extravasation in an in vitro microvascular network platform’, Integrative Biology, 2013, 5, (10), pp. 1262-1271
[3] Spagnolo, B., Brunetti, V., Leménager, G., De Luca, E., Sileo, L., Pellegrino, T., Pompa, P.P., De Vittorio, M., and Pisanello, F.: ‘Three-dimensional cage-like microscaffolds for cell invasion studies’, Scientific reports, 2015, 5