## 4 Discussion  
Many different organoid systems enable researchers to study cells in a tumor environment in vitro [4; 5], but the development of methods for understanding organoid formation is needed to study tumor initiation. Cell motility is an important feature as it is suggestive of invasiveness and, when monitored along organoid formation, arguably informs about the metastatic capacity of tumor cells [17]. Nonetheless, quantitative methods for studying these early events are scarce, as conventional migration assays such as wound-healing and transwell assays do not evaluate growth in 3D [18]. In turn, in vitro 3D cultures are commonly imaged using bright-field, fluorescence, or phase-contrast microscopy [19], where organoid formation is not continuously monitored. Bright-field and phase-contrast images show qualitative data rather than quantitative, although in precisely focused, high-quality images, some structures can be measured [20]. Live fluorescence-microscopy monitoring is useful since it enables the visualization of cell subsets in a complex mixture, but it causes phototoxicity [20; 21] that could compromise obtained results. In this study, we presented a method for generating organoids from cells in suspension and monitoring their formation by measuring optical properties. Collected data using quantitative phase imaging were based on the phase shift of the light, making the images fully quantifiable [20]; since light intensity in HoloMonitor M4 is 0.2 mW/cm\({}^{2}\), it did not cause any measurable phototoxicity [22]. We showed that organoids could be obtained from cell concentrations of 100 to 1000 cells/\(\upmu\)L in hydrogel within 15 h. Optical thickness and volume increased with organoid size, highlighting the homogeneity of 3D structures. However, inaccurate measurements of optical parameters were observed in structures with an optical volume above 10\({}^{5}\)\(\upmu\)m\({}^{3}\), which highlighted the limitations of the method. By tracking multiple single cells and establishing a threshold on the basis of size, we observed that aggregation events took place up to 8 h after seeding, acquiring fully defined sphere shapes from that point onwards. To show an application of the method, we evaluated the association between cell motility and organoid-formation capacity on the basis of the expression of CSC markers CD44 and NGFR. Here, we showed that CD44-enriched CSCs had greater ability to move and form organoids, something that may be correlated with the higher tumorigenic potential of this subset [17; 23]. CD44\({}^{+}\) tumor cells were linked to a stem-like phenotype because of their expression of stemness-related genes Sox2 and Nanog, as well as epithelial-mesenchymal-transition markers, such as vimentin, which are governed by increased signaling through the Wnt/\(\beta\)-catenin pathway [24]. In summary, the present method could be a useful tool for evaluating organoid initiation, and thus be implemented in drug screenings attempting to target early stages of tumor initiation in a label-free manner. To this end, cell density should be carefully examined for each cell type, and the tested substances should be able to diffuse into the hydrogel without causing structural changes in the extracellular matrix. Conceptualization, D.G.J., K.A., L.G., and M.L. ; methodology, D.G.J., S.C.S., K.A., and M.L. ; formal analysis, D.G.J., S.C.S., K.A., and M.L. ; resources, K.A., L.G., and M.L. ; writing--original-draft preparation, D.G.J., S.C.S., and K.A. ; writing--review and editing, D.G.J., S.C.S., L.G., K.A., and M.L. ; project administration, K.A. and M.L. ; funding acquisition, M.L. All authors have read and agreed to the published version of the manuscript. This research was funded by EU-H2020-MSCA-COFUND-2016-754299 and the Cancer Foundation. The authors would like to acknowledge [PERSON] and [PERSON] for their contribution to the present work by supplying primary OPSCC cell lines. [PERSON], [PERSON], [PERSON], and [PERSON] declare no conflict of interest.