Method Profile
Classify patient-derived esophageal squamous-cancer organoids by chemotherapy response and compare molecular signatures with target knockdown and candidate-drug activity.
Investigate stable resistance-associated states and screen drug candidates across resistant patient models.
Twenty-four tumor organoid lines from 40 patient sampling attempts; 40 normal epithelial cultures provide a separate reference with different medium requirements.
Cisplatin 11 μM and 5-fluorouracil 2.46 μM from day 5 to 10, then drug-free culture until day-15 ATP measurement. The separate cisplatin dose-response uses day 10. For the 27-kinase-inhibitor screen, single cells are seeded on day 0, inhibitors at source-defined Cmax concentrations are added from day 1 to 4, and CellTiter-Glo viability is measured on day 4. Individual Cmax values are specified in source Table S3.
Cultures and response
Matrigel cultures with Noggin, EGF, R-spondin-1 and Y-27632; tumor/normal references and CellTiter-Glo at assay-specific time points.
Mechanistic comparisons
Bulk/single-cell RNA sequencing, ATAC sequencing, matched-tissue mutations, RNAscope, NFE2L2 knockdown and fedratinib screening.
Use the actual denominator for each experiment. Separate resistance-associated expression, direct target dependence and a compound response through potentially different mechanisms.
24 tumor lines: seven resistant and 17 sensitive. NFE2L2 knockdown uses three resistant lines; the kinase screen four. RNAscope compares four patient tissues, two from resistant-source and two from sensitive-source organoids, with five technical measurements per sample. The candidate-inhibitor screen was performed in two independent experiments.
Tumor/normal cultures; resistant/sensitive organoids; early passages and prior chemotherapy exposure; NFE2L2 knockdown versus control; kinase inhibitors versus cisplatin–5-fluorouracil.
Resistance was associated with antioxidant/NRF2 target programs and selected open chromatin regions. NFE2L2 knockdown enhanced chemotherapy sensitivity in only one of three tested lines. Fedratinib reduced viability more than the chemotherapy comparison in four resistant models without reducing the tested NRF2 target expression.
Primary study comparisons of the named models, exposures and measured outcomes.
A 24-line esophageal squamous-cancer organoid library contained seven chemotherapy-resistant lines with elevated antioxidant-associated programs. Fedratinib reduced viability in four resistant models, but NRF2 knockdown sensitized only one of three tested lines, limiting a single-pathway explanation.
