Fluorescent Image Analysis–Cell Division in Spheroids

Cell division inside spheroids can be visualized by staining cell nuclei and microtubules. The number of cells in mitosis in spheroids can be quantitatively evaluated using images captured by a laser confocal microscope and NoviSight™ software's counting module.

Objectives

The degree of malignancy of cancer tissue is evaluated by measuring the number of cells in mitosis or cells in atypical mitosis. The number of cancer cells in mitosis is usually determined by flow cytometry. In that technique, cancer tissue is dissociated and the nuclei stained to count the number of the cells in mitosis, regardless of whether it is a 2D or 3D cell culture. However, since flow cytometry cannot visualize the original 3D structures of tissues, it is impossible to identify the localization of cells in mitosis within the cancer tissue. It is also difficult to analyze the relationship between morphological information such as tumor size and cell division. In this study, we successfully visualized the mitotic cells inside an intact tumor spheroid by staining the nucleus and microtubule followed by tissue clearing. This technique, coupled with NoviSight™ software, enabled us to count the number of cells in mitosis. Quantitative  analysis of the images made it possible to identify the location of the cells in mitosis and those in atypical mitosis.

Preparation of samples

A cell suspension of HT-29 was seeded into a PrimeSurface® 96U plate (SUMITOMO BAKELITE CO., LTD) at 500 cells/well. After 5 days culture, cells formed a spheroid structure, and we treated the spheroids with various concentrations of Paclitaxel. After the addition of Paclitaxel, spheroids were incubated for 48 hours and fixed with 4% paraformaldehyde. Cell nuclei and microtubules were stained with Hoechst33342 (DOJINDO) and Alexa Fluor 488-conjugated anti-Tubulin antibody (eBioscience), respectively. Stained spheroids were treated with a tissue clearing reagent, ScaleS, overnight at 37 °C.

Conclusion
Acquisition and analysis of fluorescent images

Fluorescent images of spheroids were obtained using the FV3000 confocal laser scanning microscope. In the untreated control group, spindle formation by microtubules, a specific feature of cells in mitosis, were observed in three cell layers from the spheroid surface (A). NoviSight™ software was used to count the number of cells in mitosis in the control and Paclitaxel treated groups (B,C). The results show that Paclitaxel increased the number of cells in mitosis in a concentrationdependent manner (D). The results clearly show the effect of Paclitaxel as a microtubule depolymerization inhibitor.

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