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1

Organoid Chemotherapy and Immunotherapy Evaluation

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Organoids were subsequently treated after 7 days of culture. Chemotherapies included cisplatin (1, 10, 100 µM) (232120, Sigma Aldrich), doxorubicin (0.1, 1, 10 µM) (S1208, Selleckchem), vincristine (0.1, 1, 10 µM) (V8879 Sigma Aldrich), doxorubicin with cisplatin (0.1/1, 1/10, 10/100 µM), cisplatin with etoposide (S1125, Selleckchem) (1/0.1, 10/1, 100/10 µM) (Selleckchem), and doxorubicin with vincristine (0.1/0.1, 1/1, 10/10 µM). For immunotherapy treatment, 100 nM of pembrolizumab (A2002, Selleckchem), ipilimumab (A2001, Selleckchem) or nivolumab (A2005, Selleckchem) was used on both iPTOs and PTOs in parallel treatments. Media was removed from the wells and drug solutions mixed in culture media were added. Organoids remained in treated media solution for 72 h prior to endpoint viability assessment.
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2

Sarcoma Organoid Drug Screening

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Therapy screening was initiated on day 7 following organoid biofabrication. Treatments were tailored for each sarcoma subtype based on preoperative biopsies or final surgical pathology. The number of therapies and doses involved varied based on available cells. The therapies used for sarcoma organoids were doxorubicin (0.1, 1, 10 μM) [S1208, Selleckchem, Houston, TX, USA], ifosfamide (2, 20, 200 μM) [I4909, Sigma-Aldrich], temozolomide (10, 100, 1000 μM) [T2577, Sigma-Aldrich], imatinib mesylate (1, 10, 100 μM) [STI571, Selleckchem], regorafenib (0.1, 1, 10 μM) [S1178, Selleckchem], gemcitabine (1, 10, 100 μM) [G6423, Sigma-Aldrich], olaparib (0.1, 1, 10 μM) [S1060, Selleckchem], 100 nM pembrolizumab (A2005, Selleckchem,), 100 nM nivolumab (A2002, Selleckchem), and 100 nM ipilimumab (A2001, Selleckchem). Drug containing-media was added to organoid wells for 72 h, followed by viability assays.
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