Cancer invasion involves the growth of the tumor and the directional migration of cancer cells. During growth, the tumor typically elongates and retracts highly dynamic protrusion-like structures prior the spreading and invasion. Recent evidences using individual cells have shown the importance of protrusion fluctuations in the physicochemical mechanism of directed cell motion, a key event in cancer metastasis. However, the role of protrusion fluctuations during tumor progression and cancer cell invasion is still not yet well understood. In this work, we investigate the role of protrusion fluctuations in the invasiveness capability of – non-metastatic – lung and – more-metastatic – breast tumor μ-spheroids embedded into a 3D collagen matrix as a model. By using this reductionist model, we identified different types of fluctuating protrusions, whose number, frequency, and lifetime was depended on the type of cells and tumor origins. Next, we found that the perturbation of protrusion dynamics using low doses of a conventional anti-cancer drug (doxorubicin) and an inhibitor of the Rho-pathway affected the morphodynamics of protrusion fluctuations, and importantly, the invasion capability of cells. Finally, we used a vessel-on-a-chip approach integrating our tumor model and human endothelial cells to mimic the native tumor microenvironment. We found that protrusions extended mainly towards the endothelial cells, whereas on isotropic environments, protrusions were distributed stochastically, suggesting a crosstalk between endothelial and tumor cells. Overall, these results demonstrate that fluctuations of protrusions are key players in the physicochemical mechanism of tumor invasion. This may have important implications for the development of therapeutic approaches targeting protrusions activity.