Non-destructive in situ monitoring of structural changes of 3D tumor spheroids during the formation, migration, and fusion process

Reviewer #1 (Public review):

Summary:

The ingenious design in this study achieved the observation of 3D cell spheroids from an additional lateral view and gained more comprehensive information than the traditional one angle of imaging, which extensively extended the methods to investigate cell behaviors in the growth or migration of tumor organoids in the present study. I believe that this study opens an avenue and provides an opportunity to characterize the spheroid formation dynamics from different angles, in particular side-view with high resolution, in other organoids study in the future.

Reviewer #2 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Strengths:

The system allows the in situ observation of the 3D structures along the 3 axes (x,y and z) without disrupting the integrity of the spheroids; in a time-lapse manner it is possible to follow the formation of the 3D structure and the spheroids fusion from multiple angles, allowing a better understanding of the cell aggregation/growth and kinetic of the cells.

Interestingly the system allows the analysis of cell migration/ escape from the 3D structure analysing not only the morphological changes in the periphery of the spheroids but also from the inner region demonstrating that the proliferating cells in the periphery of the structure are more involved in the migration and dissemination process. The application of the system in the study of the effects of doxorubicin and NK cells would give new insights in the description of the response of tumor 3D structure to killing agents.

Author response:

Reviewer #1:

We sincerely thank you for your thoughtful review and constructive comments on our work and we appreciate your positive assessment of our study’s innovative design, which allows for improved observation of 3D cell spheroids from an additional lateral view. Your comments underscore the importance of our approach in advancing methods for investigating cell behaviors in tumor organoid studies.

In response to your suggestions, we will first add a detailed image of the ‘First surface mirror’ in Fig. 1 to provide a reference for readers and other researchers, thereby facilitating broader use of this method in similar observations. Regarding the suitable sample sizes for this device, as the spheroid sizes are relatively small compared to the mirror and culture dish, we have been able to image samples up to 5 mm in height, which provides ample capacity for most spheroids under 1 mm. We will include additional experiments and explanations in the manuscript to clarify this further.

Concerning the ring-shaped seeding pattern of spheroids, we have conducted extensive culture experiments to optimize this method. The agarose microwells-based method has proven to be highly tolerant of variations. Within these microwells, cells have a propensity to self-aggregate, leading to the formation of spheroid structures. We will add a discussion in the revised manuscript to address this issue.

Lastly, this device can accommodate the fluorescence imaging of 3D spheroid samples. We will supplement the discussion with a schematic illustrating the principles of fluorescence imaging using this device, providing a foundation for future work in this area. We will also regarding language improvements to enhance the overall quality of the manuscript.

Thank you once again for your valuable insights, which have greatly contributed to the strengthening of our manuscript.

Reviewer #2:

We sincerely thank you for your detailed and supportive review of our manuscript. Your recognition of our system’s capabilities for in situ observation of 3D structures along multiple axes, as well as its potential applications in studying therapeutic effects, is highly encouraging. Your comments on the advantages of this system for analyzing cell migration, morphological changes, and responses to therapeutic agents are especially appreciated.

Thank you again for your thoughtful feedback and for highlighting the contributions of our work. Your insights have been invaluable in refining the focus and clarity of our study, and we hope that our revisions meet your expectations.