Fig 1.
Scheme of the research strategy.
Experimental outline of the research work, from spheroid culture and treatment with CZB to setting up the analyses on fixed and live samples. The LoVo cells were cultured in Ultra-Low Attachment Microcavity Plate in presence of 250 and 500 nM of CZB. At 10 days fully mature and organized spheroids were harvested, for the analysis procedures. Live and fixed samples were evaluated with a new fluidic device (W8) for the accurate, simultaneous, and rapid measurement of mass density, weight, and size. These parameters were correlated with protein quantification (NanoOrange Assay) and deep imaging analysis performed on clarified samples.
Fig 2.
Measurement of mass density, weight, and diameter of LoVo spheroids treated with CZB.
Measurements of mass density (A and D), weight (B and E), and diameter (C and F) of live (top panels) and fixed (bottom panels) LoVo spheroids after 10 days of treatment with CZB 250 nM (shown in red) and 500 nM (shown in blue) and relative controls (shown in green). Data are graphically depicted in box-and-whisker plots and the lines, extending from the boxes, indicate variability outside the upper and lower quartiles. **p< 0.01 and ***p< 0.001. The "r" Pearson correlation coefficients, able to measure the degree of relationship between the value of mass density, weight and diameter and the different drug concentrations, are indicated for each graph. The F-ratio value obtained from one-way ANOVA test estimating the total effect of fixation on W8 output data, is 0.00027, the result is not significant at p < 0.01.
Fig 3.
Protein quantification and imaging analysis of CZB treated LoVo spheroids.
A) Protein quantification performed by NanoOrange assay of CZB 250 nM and 500 nM, and relative control. Data are presented as mean± SD. Statistical analysis was performed using two-tailed unpaired Student’s t-test. *p< 0.05, **p< 0.01, and ***p< 0.001. B) Representative images of LoVo spheroids exposure to different doses of CZB after clearing procedures. The upper panel shows bright field imaging from control and treated spheroids at low magnification. The middle panel exhibits confocal imaging of DAPI (in blue) and phalloidin (in red) staining for nuclei and actin respectively. The lower panel provides volume view of DAPI signal in 3D z-depth confocal rendering reported as a scale of colors (palette on the right). Scale bars 50 μm. C) Optical density of fixed LoVo spheroids, calculated as ratio between the mode of absorbance (subtracted to the background) and the spheroid area after 10 days of treatment with CZB 250 nM (histogram in black), 500 nM (histogram in gray) and relative control (histogram in white). Statistical analysis was performed using two-tailed unpaired Student’s t-test. *p< 0.05, **p< 0.01, and ***p< 0.001. D) Total cell nuclei counting of fixed LoVo spheroids after 10 days of treatment with CZB 250 nM (histogram in black), 500 nM (histogram in gray) and relative control (histogram in white). Statistical analysis was performed using two-tailed unpaired Student’s t-test. *p< 0.05, **p< 0.01, and ***p< 0.001. E) Volume of fixed LoVo spheroids after 10 days of treatment with CZB 250 nM (histogram in black), 500 nM (histogram in gray) and relative control (histogram in white). Statistical analysis was performed using two-tailed unpaired Student’s t-test. *p< 0.05, **p< 0.01, and ***p< 0.001. F) Volumetric cell nuclei density of fixed LoVo spheroids, calculated as ratio between the total number of nuclei and the spheroid volume after 10 days of treatment with CZB 250 nM (histogram in black), 500 nM (histogram in gray) and relative control (histogram in white). Statistical analysis was performed using two-tailed unpaired Student’s t-test. *p< 0.05, **p< 0.01, and ***p< 0.001.