Tumor protrusion fluctuations as a signature of 3D cancer invasiveness

INTRODUCTION: Tumor progression follows a complex cascade of events, including changes in the morphodynamics of invading protrusions. Recent evidences have shown that tumor cells modulate protrusion type to optimize their invasion in 3D [1]. However, the role of protru-sion fluctuations in this mechanism is unclear [2]. Herein, we analyze their role and define novel parameters (signature) of tumor invasiveness.
METHODS: Human A549 lung adenocarcinoma and MCF7 breast cancer cells (ATCC) were encapsulated for 3 days into a collagen I (Gibco) 3D matrix at low density (105 cells·ml−1) to form u-spheroids (Fig.1A). For drug experiments, cells were incubated with 0.1, 1.0 and 10 ug·ml-1 of doxorubicin (DOX; Sigma) and 1.0 ug·ml-1 C3 Rho inhibitor (Cytoskeleton, Inc). Tumor protrusion fluctuations were monitored by time-lapse. Biophysical parameters: frequency of probing (nu_p) – # of protrusions that elongate and retract over time; stabilization lifetime (tau_s) – dwelling time of an extended tumor protrusion.
RESULTS: Protrusion fluctuations displayed a distinct morphodynamics, which depended on tumor metastatic capability. Non-metastatic A549 u-spheroids showed a lower nu_p and larger tau_s compared to more-metastatic MCF7. The addition of DOX perturbed protrusion fluctuations, increasing (decreasing) nu_p (tau_s) in both cell types (Fig.1B-C), resulting in an enhanced tumor invasiveness. Interestingly, we found a linear correlation between both parameters, which correlated with the invasive potential of cells (Fig.1D). We also found that Rho inhibition modulated nu_p and tau_s, which abolished the invasive capacity of tumors. Next, we used a tumor-on-chip model integrating our u-spheroids and endothelial cells to mimic the native scenario. The endothelial cells affected protrusion dynamics and orientation, suggesting a crosstalk between both cell types. Finally, all these observations were encoded into a phase diagram, which provided a novel landscape capable to ´predict´ the invasiveness of tumors based on their fluctuations.

DISCUSSION & CONCLUSIONS: These data illustrate that protrusion fluctuations are key players in the physicochemical mechanism of tumor invasion, and are governed by nu_p and tau_s.

ACKNOWLEDGEMENTS: H2020 (FoReCaST – no 668983); Portuguese FCT (PTDC/BTM-ORG /28070/2017 and PTDC/BTM-ORG/28168/2017) funded by Norte2020 supported by FEDER.

REFERENCES
[1] Friedl P et al. J Cell Biol, 2010. 188(1):11-19
[2] Caballero D et al. Biophys J. 2014; 107:34-42