Establishment and analysis of zebrafish xenografts from LGSOC cells. (a) Schematic workflow for the generation of zebrafish xenografts from LGSOC cells. Early passage cell line was labeled with Vybrant CM-DiI dye (red) and microinjected in the PVS of 2 dpf larvae. One dpi larvae were screened and randomly distributed into treatment groups. After 96 h of treatment, xenografts were euthanized and divided for postmortem analysis based on immune staining. (b,b’) Assessment of tumor progression through live-cell imaging in zebrafish xenograft at 1 dpi and 5 dpi. Top row shows brightfield picture; bottom row shows RFP filter with tumor cells expressing red fluorescence. Anatomic structures are indicated in pictures; dashed box indicated tumor area. (c,c’) Hematoxylin eosin staining and (d) immunohistochemistry for Ki67 were performed in histological sections to observe morphology and proliferative capacity of the cancer cells in the zebrafish larvae. Dashed box indicates area of zoom. (e) Representative whole mount immunofluorescence staining for Ki67, all images are at 25× magnification (scale bar, 50 µm). Nuclei stained with DAPI in blue, anti-Ki67 in green and fluorescently labeled cancer cells in red. Tumor area is indicated with a white line. All images are anterior to the left, dorsal up.

Whole mount immune fluorescence staining for Ki67. Vybrant CM-DiI labeled LGSOC cells (red), nuclei stained with DAPI (blue) and anti Ki-67 (green). (a–d) The number of xenografts analyzed for Ki67 is indicated in the figures; all images are at 25X magnification (scale bar 50 µM). Tumor area is indicated with a white line. (e–h) Quantification of Ki67 expression in zebrafish xenografts using ImageJ. Generation of Maximum Intensity Projections (MIPs) from relevant Z-stacks (e), transformation of 8-bit files to RGB file (f), binary masking of Ki67 staining using fixed threshold (g), select ROI of tumor (h), percentage of stained area in ROI represents Ki67 expression. (i) Percentages of different treatment conditions are presented as AVG ± SEM and each dot represents a xenograft. Multiple comparisons between treatment groups were determined by One-way ANOVA; Tukey procedure was used to adjust p-values. p-values of 0.05 were considered as statistically significantly different and output was represented as: not-significant (ns) p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001.

Whole mount immune fluorescence staining cleaved caspase-3. Vybrant CM-DiI labeled LGSOC cells (red), nuclei stained with DAPI (blue) and anti-cleaved caspase-3 (violet). (a–d) The number of xenografts analyzed for cleaved caspase-3 is indicated in the figures; all images are at 25× magnification (scale bar 50 µM). Tumor area is indicated with a white line. (e,f) Quantification of cleaved caspase-3 expression in zebrafish xenografts using Cell Counter plugin in ImageJ. Cells positive for cleaved caspase-3 are indicated by yellow arrows. (g) Ratios of different treatment conditions are presented as AVG ± SEM and each dot represents one xenograft. Multiple comparisons between treatment groups were determined by One-way ANOVA; Tukey procedure was used to adjust p-values. p-values of 0.05 were considered as statistically significantly different and output was represented as: * p ≤ 0.05, *** p ≤ 0.001.