Characterization of a TP53 Somatic Variant of Unknown Function From an Ovarian Cancer Patient Using Organoid Culture and Computational Modeling

Abstract

In our proof-of-concept study of 1 patient with stage IIIC carcinosarcoma of the ovary, we discovered a rare mutation in the tumor suppressor, TP53, that results in the deletion of N131. Immunofluorescence imaging of the organoid culture revealed hyperstaining of p53 protein. Computational modeling suggests this residue is important for maintaining protein conformation. Drug screening identified the combination of a proteasome inhibitor with a histone deacetylase inhibitor as the most effective treatment. These data provide evidence for the successful culture of a patient tumor and analysis of drug response ex vivo.

Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib over Bevacizumab: Cell Cycle Abrogation and Synergy with Chemotherapy

“…Patient tumor specimens were obtained within 30 min of surgical resection. Organoids were created per our protocol for ascites fluid [ 19 ], with modifications to digest tissue and isolate single cells. Specifically, freshly resected tissue was washed with 10 mL PBS and cut into small pieces.…”

“…The following antibodies were used at the indicated dilutions; all antibodies were purchased from Cell Signaling (Danvers, MA, USA): anti-p-AKT-S473 (1:1000, #4060), anti-AKT (1:1000, #4685), anti-p-p44/42 MAPK (Erk1/2) -Thr202/Tyr204 (1:1000, #4370), anti-p44/42 MAPK (Erk1/2) (1:1000, #4695), anti-p-p38 MAPK-Thr180/Tyr182 (1:1000, #9211), anti-p38 MAPK (1:1000, #8690), anti-p-cdc2-Tyr15 (1:1000, #4539), anti-cdc2 (1:1000, #9116), antip-CDC25C-Ser216 (1:1000, #4901), anti-CDC25C(1:1000, #4688), anti-p-Histone H3-ser10 (1:1000, #53348). Viability of the tumor organoids following drug treatment was performed as previously described [19]. Briefly, patient-derived organoids were collected with organoid harvesting solution (Cultrex, Trevigen, Gaithersburg, USA), and then digested to single cells with TrypLE Express supplemented with 4 µL 10 mg/mL DNAse I stock and 4 µL 10 mM Y-27632 stock.…”

“…These cell lines are models of more aggressive endometrial cancer and harbor distinct mutations in TP53 that are frequently detected in patient tumors [ 18 ]. This study also includes patient-derived organoid models of endometrial cancer whereby freshly resected tumor specimens are cultured in a three-dimensional model system [ 19 , 20 ]. As compared to patient-derived xenograft models, which also use fresh patient tumor tissue, patient-derived organoids are much more cost-effective with a higher rate of successful culture [ 17 ].…”

Advantages of Tyrosine Kinase Anti-Angiogenic Cediranib Over Bevacizumab: Cell Cycle Abrogation and Synergy With Chemotherapy

“…Patient tumor specimens were obtained within 30 min of surgical resection. Organoids were created per our protocol for ascites uid [16], with modi cations to digest tissue and isolate single cells. Speci cally, freshly resected tissue was washed with 10 ml PBS and cut into small pieces.…”

“…Organoids: Viability of the tumor organoids following drug treatment was performed as previously described [16]. Brie y, patient-derived organoids were collected with organoid harvesting solution (Cultrex, USA), and then digested to single cells with TrypLE Express supplemented with 4 µl 10 mg/ml DNAse I stock and 4 µl 10 mM Y-27632 stock.…”