Vetspon absorbable hemostatic gelatin 1 cm3 sponges

Human metastatic UM tissue was collected following patient consent at Thomas Jefferson University Hospital under an IRB-approved protocol (#02.9014R). Less than 16 hours post-surgery, excess adipose and stromal tissue was removed and the tumor (Explant of Patient No.4/Ex-Pt#4) was cut into 1 mm³ pieces. Vetspon absorbable hemostatic gelatin 1 cm³ sponges (Novartis, Basel, Switzerland) were pre-soaked in 12-well plates for 15 minutes at 37°C in 500 µL of DMEM medium containing 10% FBS, penicillin-streptomycin and drugs. DMSO was used as a vehicle control. To avoid concerns of intra-tumoral heterogeneity, up to four ~1 mm³ pieces from different locations of the original tumor were placed per sponge per treatment condition. Samples were treated for 48 hours with medium being replaced after 24 hours. Tumor pieces for western blotting were homogenized in lysis buffer with phosphatase and protease inhibitors (PhosSTOP and cOmplete tablets, Roche, Basel, Switzerland). Laemmli sample buffer was added and samples were heated at 99°C for 5 minutes.

Tumors were collected following informed patient consent at Thomas Jefferson University Hospital under an IRB-approved protocol (#10D.341). Less than 16 hours post-surgery, excess adipose and stromal tissue was removed and tumors were cut into 1 mm³ pieces. Vetspon absorbable hemostatic gelatin 1 cm³ sponges (Novartis; Basel, Switzerland) were pre-soaked in 12-welled plates for 15 minutes at 37°C in 500 μL of DMEM/10% FBS containing drugs or DMSO as a vehicle control. To avoid concerns of intratumoral heterogeneity, up to three 1 mm³ pieces from different locations of the original tumor were placed per sponge per treatment condition. Similarly, xenograft tumors were dissected into 1 mm³ pieces and placed on medium/drug-soaked sponges. Medium was replaced every 24 hours. Tumor pieces for western blotting were homogenized in modified RPPA lysis buffer (29 (link)) with phosphatase and inhibitors (PhosSTOP and cOmplete tablets Roche, Basel, Switzerland). Laemmli sample buffer was added and samples were heated at ≥95°C for 5 minutes. For IHC analysis, tumor pieces were fixed in formalin for 24 hours. Two of the samples (TJU-MEL-27A and TJU-MEL-27B) were different lesions from the same patient and combo treatment was not assayed for TJU-MEL-30.