Organotypic Murine Tumor Slice Assay

Cores 4 mm in diameter were punched from sacrificed murine tumors using a biopsy punch (#7424; RoyalTek, TWN) and cut into 1 mm organotypic tumor slices. Tumor slices were cultured individually on 12 mm Transwell with 0.4 μm pore polycarbonate membrane inserts (#3401; Corning) using 12-well plates. Slices were cultured in 1× Advanced Dulbecco’s Modified Eagle Medium (#12491023; Thermo Fisher) supplemented with 5% FBS, 1× GlutaMAX (#35050061; Thermo Fisher), 0.5× Penicillin-Streptomycin, 1× Insulin-Transferrin-Selenium supplement (#41400045; Thermo Fisher), and 15 mM HEPES (#15630130; Thermo Fisher) as previously described [38 (link)] and maintained at 37° in 10% CO₂. After 24 h in culture, slices were exposed to vehicle or 40 μM HMA for 24 h. Tumor slice viability was measured using RealTime Glo (#G9711; Promega) according to the manufacturer’s instructions. Images were taken before (day 0) drug treatment and 24 h after drug treatment with a ChemiDoc MP Imaging System (BioRad) and analyzed with Image Lab software version 1.2 to quantify the RealTime Glo signal.

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Berg A.L., Rowson-Hodel A., Hu M., Keeling M., Wu H., VanderVorst K., Chen J.J., Hatakeyama J., Jilek J., Dreyer C.A., Wheeler M.R., Yu A.M., Li Y, & Carraway KL I.I.I. (2022). The Cationic Amphiphilic Drug Hexamethylene Amiloride Eradicates Bulk Breast Cancer Cells and Therapy-Resistant Subpopulations with Similar Efficiencies. Cancers, 14(4), 949.

Publication 2022

pore polycarbonate membrane inserts Advanced Dulbecco’s Modified Eagle Medium GlutaMAX Insulin-Transferrin-Selenium supplement HEPES RealTime Glo ChemiDoc MP Imaging System

Biopsy Drug Eagle Hepes Insulin Membrane Murine Penicillin Polycarbonate Promega Selenium Streptomycin Supplement Transferrin Tumor

Corresponding Organization : University of California, Davis

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Variable analysis

independent variables

Drug treatment (vehicle or 40 μM HMA)

dependent variables

Tumor slice viability

control variables

Murine tumor cores (4 mm in diameter)
Tumor slice thickness (1 mm)
Culture medium (1× Advanced Dulbecco's Modified Eagle Medium supplemented with 5% FBS, 1× GlutaMAX, 0.5× Penicillin-Streptomycin, 1× Insulin-Transferrin-Selenium supplement, and 15 mM HEPES)
Culture conditions (37°C, 10% CO2)
Culture duration (24 h after drug treatment)

controls

Positive control: Not explicitly mentioned.
Negative control: Vehicle treatment

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