Figure 3.
FNA-Based Patient-Derived Organoids are Readily Adapted to High-throughput Screening
(A) FNAs directly plated in semisolid organoid culture can be tested for drug sensitivity. Using 24-well plates, BRAFV600E-mutant (left) and BRAF-wild-type (right) thyroid cancer organoids were treated with dabrafenib, a BRAF inhibitor (30 nM), for 20 days. BRAFV600E-mutant organoids show decreased organoid size and number following treatment. As expected, BRAF-wild-type organoids show no observable response. Images representative of three replicates. ∗∗p < 0.05, Mann Whitney test.
(B) FNA-based patient-derived gastric signet ring organoids were assayed for drug sensitivity using disc-organoid MTT assay in 96-well plates. Images representative of four replicates; error bars represent standard deviation. ∗ = p < 0.05 (Student’s t test). The x axis indicates the concentrations of each drug (alone or in combination); the y axes are MTT signals normalized to the highest absorbance value within each experiment.
(C) FNA-PDOs from aggressive anaplastic thyroid cancer were assessed for viability following doxorubicin treatment using an automated high-throughput 384-well assay. The thyroid cancer FNA-PDOs showed response to doxorubicin. Automated high content imaging also confirms organoid growth in the 384-well format, as seen in these untreated melanoma FNA-PDOs. Images representative of three replicates; error bars represent standard error of the mean (SEM).
(D) Using a high-throughput fluorescent assay, patient-derived melanoma organoids were labeled with Calcein AM (green fluorescence, labels live cells) and propidium iodide (red fluorescence, labels dead cells). To visualize both live and dead cells, a cell-permeable DNA stain Hoechst 33342 was used. Although the overall organoid structure remained intact, the organoid decreased in size and cells in the organoids were killed following 3 days of treatment with MEK inhibitor, trametinib (0.1 μM), and BRAF inhibitor, dabrafenib (10 μm). This is in agreement with clinical data showing that dabrafenib and trametinib combination is effective for treatment of BRAF V600E melanoma.31 Images representative of three replicates; scale bar, 100 μm.
(E) Organoids from indicated human melanoma tumors were generated using FNA of corresponding PDX tumors grown in BALB/c nu/nu mice. Organoids were plated in semisolid media into 96-well low-attachment plates and treated with 5 μm AMG-232, 1 μm debrafenib, 0.1 μm trametinib, or combination of three drugs for 3 days. Calcein M/Propidium Iodide viability assay was performed as shown in (C). The ratio of the intensity of red (dead cells) and green (live cells) fluorescent signal in individual organoids was plotted. Each dot represents an individual organoid. We analyzed 9–23 organoids per group for PDO2552, 11–24 for PDO1668, 17–43 for PDO 9164, and 8–14 for PDO 2132. Unpaired t test was used to determine the significance of differences between indicated treatment groups. Error bars represent standard deviation.