Solid evidences have shown that during tumor dissemination cancer cells modulate the morphodynamics of invasive protrusions to optimize their migration efficiency. However, it is yet unclear how the stochastic fluctuations of tumor-associated protrusions regulate the early invasion events in more complex multi-cellular tumors, and how they correlate with their metastatic potential. In this work, we used a reductionist model based on tumor micro-spheroids with increasing invasion capability to investigate the role of fluctuating protrusions in breast cancer progression . To quantitate fluctuations, we defined a new set of key biophysical parameters that precisely correlated with the invasive potential of tumors. We showed that by perturbing protrusion activity using chemotherapeutics (Doxorubicin) and pharmacological inhibitors (C3 transferase) of key signaling pathways (Rho pathway), tumor invasiveness was significantly altered. Next, we defined a novel quantitative index encoding a minimal set of biophysical parameters and the relative levels of cell-cell/ECM interactions, which was capable of assessing tumor invasion capability. Finally, to better investigate how tumor-associated protrusion fluctuations invade the tumor microenvironment and initiate metastasis, we have created a new organ-on-a-chip model of the human microcirculation embedding blood and lymphatic vasculature . Overall, this work provides a new biophysical framework showing how protrusion fluctuations regulate tumor cell invasion, suggesting that they may be employed as an early indicator – signature – of the metastatic potential of tumors.
Acknowledgements: D.C. acknowledges the financial support from the Portuguese Foundation for Science and Technology (FCT) under the program CEEC Individual 2017 (CEECIND/00352/2017). D.C., A.C.L., C.M.A., and S.C.K. also acknowledge the support from FCT under the 2MATCH project (02/ SAICT/2017—028070) funded by the Programa Operacional Regional do Norte supported by FEDER. Finally, all the authors acknowledge the financial support from the EU Framework Programme for Research and Innovation Horizon 2020 on Forefront Research in 3D Disease Cancer Models as in vitro Screening Technologies (FoReCaST—no. 668983).
 Caballero, D., Brancato, V., Lima, A. C., Abreu, C. M., Neves, N. M., Correlo, V. M., Oliveira, J. M., Reis, R. L., Kundu 2101019, S. C., Tumor-Associated Protrusion Fluctuations as a Signature of Cancer Invasiveness. Adv. Biology 2021, 5, 2101019.
 Luque-González MA, Reis RL, Kundu SC, Caballero D, Adv. Biosys. 2000045, 2020