The elongation of invasive protrusions in tumors mediates the initial steps of metastasis. These tumor-associated protrusions play a key role on the invasion of individual cancer cells . However, less is known about how they regulate cancer progression on more complex multi-cellular systems, such as tumor spheroids. In addition, it is not understood how their fluctuations correlate with their metastatic potential . In this work, we analyzed the role of tumor-associated protrusion fluctuations in cancer invasion using as a model non-metastatic and more metastatic tumor microspheroids encapsulated into a collagen matrix. We defined novel biophysical indicators characteristic of tumor invasiveness: probing frequency (np) and stabilization lifetime (ts) of protrusions. We found that these morphodynamic indicators depended on the metastatic potential of tumors. In particular, non-metastatic A549 m-spheroids showed a lower np and a larger ts compared to more-metastatic MCF7 cells. The addition of doxorubicin, an anti-cancerous drug, perturbed protrusion fluctuations, increasing (decreasing) np (ts) in both tumor types. Interestingly, we identified a linear correlation between both parameters, which was in agreement with the invasive potential of the tumors. We also observed that the inhibition of the Rho pathway modulated np and ts, and abolished the invasive capacity of tumors. All these observations were used to define an invasion index integrated into a phase diagram, which provided a novel landscape to assess the invasiveness potential of tumors based solely on their protrusion activity. Overall, the obtained results illustrate that protrusion fluctuations are key players in the mechanism of tumor invasion, which may be employed as early indicators of the metastatic potential of tumors.
Keywords: cancer, invasiveness, protrusions.
Acknowledgements: The financial support from the Portuguese Foundation for Science and Technology (FCT) under the program CEEC Individual 2017 (CEECIND/00352/2017 to D.C.); FCT under the scope of the project 2MATCH (PTDC/BTM-ORG/28070/2017 to D.C. and S.C.K.) funded by the Programa Operacional Regional do Norte supported by FEDER; and the EU Framework Programme for Research and Innovation H2020 under grant agreement nº 668983 – FoReCaST.
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