Noninvasive evaluation of tumor spheroids with different sizes based on FLIM

Recently, three-dimensional (3D) cultured tumor spheroid models have been widely used in biological research fields to address the limitations of 2D cultured cells. Tumor spheroids can exhibit remarkable variability in multiple parameters including morphology, size, growth rate, metabolism, and cytotoxicity, which may caused by the different culture conditions. Therefore, novel methods to characterize tumor spheroids are in high demand to overcome the limitations of conventional methods, such as time-consuming procedures and unsatisfactory reproducibility in living cells. Fluorescence lifetime imaging microscopy (FLIM) is an effective method to quantitatively analyze cellular metabolism and molecular environment. In this study, we applied FLIM method to classify tumor spheroids into large spheroids and small spheroids. Results showed that both types of tumor spheroids had inner and outer layers. The fluorescence lifetime values of the outer layer and the inner layer of the small tumor spheroids were in the range of 2500-2800 ps and 2300- 2500 ps, respectively. The fluorescence lifetime values of the outer layer of the large tumor spheroids were in the range of 2500-2800 ps, close to those of the small tumor spheroids. By contrast, the lifetime values of the inner layer of the large tumor spheroids were around 2500-2800 ps, which were significantly higher than those of the inner layer from the small ones. The difference of the fluorescence lifetime values between the inner layers of the small and the large spheroids may indicate the different metabolic states of these two types of tumor spheroids, suggesting FLIM method can provide quantitative data to characterize tumor spheroids with different sizes, leading to a non-invasive real-time evaluation method in further drug development of anti-tumor therapy.