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. 2020 Jul 13;23(8):101365. doi: 10.1016/j.isci.2020.101365

Figure 7.

Figure 7

Detecting Antitumor Activities of Targeted Therapy and On-Target Activity in Live GBM-PDOs

(A) Left, model depicting detection of activated caspase-3 (Casp-3) activity by DEVD-NucView488 and live GBM-PDO cell population-averaged apoptosis, migration, and invasion assays. Right, the diagram displays the PDO-activated caspase-3 assay. GFP-positive cells within the organoids indicate apoptotic cells. GBM cells that invade the semisolid extracellular Matrigel represent invading cells, whereas cells that migrate outside of the organoid represent migrated cells.

(B) Comparison of cell viability and activated caspase-3 between untreated, DMSO, TMZ, and targeted therapy groups was determined by two-way ANOVA with Bonferroni post-hoc test. Graph indicates significance versus TMZ with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Violin plot on the top right represents real-time measurement of apoptotic cells in GBM-PDOs from GBM#46 and GBM#50 as untreated, DMSO-treated, or in response to treatment with TMZ, BEZ (targeting mTOR/PI3K), and combination therapy.

(C) GBM PDO cell migration captured by phase contrast using the IncuCyte HD system. Frames were captured at 4-h intervals from time 0 to 72 h from two separate regions/well using a 10× objective. Image processing involved fluorescent color channel separation, data resizing, illumination adjustment, threshold setting, and data extraction for either GBM#46 PDOs or GBM50 PDOs. Representative results of three separate experiments are shown for GBM#46 and GBM#50 as percentages of cell migration relative to 72-h values of untreated PDO cells.

(D) GBM-PDO cell invasion images were captured similarly with the IncuCyte HD system and shown as percentage of migrated cells relative to 72-h values of untreated GBM-PDO cells. Values determined by two-way ANOVA with Bonferroni post-hoc test (∗∗∗p < 0.001).

(E and F) (E) Three key on-target activity markers were detected in PDOs in response to drug treatments: GFAP (hallmark of GBM), BMI1 (GSCs), and phospho-S6 (PI3K/mTOR activity). Representative confocal images were taken after the indicated treatments in fixed PDOs and represent overlay of green (GFAP on the right, BMI1 in center, and pS6 on the left), red (phalloidin), and blue (DAPI for nuclei). (F) Data represent quantitation of marker expression upon treatment from six independent experiments utilizing GBM-PDOs, GBM#46, and GBM#50. Results are mean ± SD of eight fields of at least 1,000 cells. Comparison of marker expression between combined versus single therapy groups was determined by two-way ANOVA with Bonferroni post-hoc test (∗∗∗p < 0.001). Scale bars, 100 μm in (A and C).