Esophageal Cancer Organoids Associate Chemotherapy Resistance with Antioxidant Programs and Identify a Drug Candidate
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.
A Tumor Library with Matched Normal References
Researchers at the Institute of Science Tokyo and collaborating hospitals developed organoids from resected esophageal squamous cell carcinomas. Tumor-culture attempts from 40 patients produced 24 cancer organoid lines, a 60% establishment rate. Normal epithelium from tumor-free margins yielded 40 normal organoid lines under a different, more extensively supplemented medium.
Most cancer cultures could be maintained for more than three months, whereas normal cultures lasted around 30 days. Exome sequencing of 11 matched sets supported retention of many parental tumor mutations, and selected lines formed tumors after transplantation into immunodeficient mice. Some cultures nevertheless carried additional or enriched variants relative to the sampled tissue, leaving culture selection or acquired changes as possibilities.
The resource separates a model-establishment result from subsequent functional testing. The 24 successful tumor lines are not the denominator for every molecular comparison: transcript profiling, chromatin analysis and single-cell studies used their own subsets.
Drug Exposure and Recovery Define the Resistant Phenotype
The main chemotherapy assay added 11 μM cisplatin and 2.46 μM 5-fluorouracil from culture days 5 to 10, followed by five days without drug. ATP-based viability was measured on day 15. Across the 24 lines, seven were classified as resistant and 17 as sensitive, with considerable variation in the response.
Resistance did not significantly change between early passages or after the tested exposure-and-regrowth comparison. Five of 14 lines from previously treated patients were resistant, compared with two of ten from patients without prior chemotherapy. The small, nonrandomized groups did not establish that clinical treatment caused resistance.
Bulk and single-cell profiles associated resistance with increased expression of antioxidant genes regulated by the transcription factor NRF2. In six single-cell-profiled cultures, these genes were distributed across cell clusters rather than confined to one obvious resistant subclone. Resistant cultures also showed greater accessibility at selected antioxidant-associated loci, consistent with a pre-existing transcriptional state.
Antioxidant Association Does Not Establish Uniform Dependence
Only three of six resistant lines with exome data carried an NFE2L2 mutation, so mutation status alone did not separate resistant from sensitive cultures. The study also found no simple KEAP1 or CUL3 alteration explaining the remaining cases. Selected NRF2 target loci were more accessible, but this pattern was not identical for every antioxidant gene.
The accessibility pattern also had gene-specific limits: SPP1, UCHL1 and ABCC2 loci showed constitutively open states in resistant cultures, whereas ALDH3A1 and TXNRD1 did not show the same pattern. Elevated antioxidant expression therefore did not correspond to one identical chromatin change across all candidate markers.
Functional knockdown provided a stricter test. Lowering NFE2L2 reduced NRF2-related expression in three resistant lines, yet enhanced chemotherapy sensitivity only in ESCCO15. ESCCO17 and ESCCO34 did not show the same sensitization. The shared expression signature therefore does not demonstrate the same pathway dependence in every resistant tumor model.
A separate five-day cisplatin dose-response analysis linked higher ALDH3A1 expression to a larger response-curve area, with a reported Spearman correlation of 0.84. RNAscope then detected higher ALDH3A1, SPP1 and TXNRD1 signals in tissue from two patients whose organoids were resistant than in two whose organoids were sensitive. This is a focused tissue comparison supporting biomarker candidates, rather than an independent clinical diagnostic-accuracy study.
Fedratinib Activity Points Beyond the Initial Signature
The researchers screened 27 kinase inhibitors at source-defined maximum plasma concentrations in four resistant organoid lines. Fedratinib reduced survival more strongly than the cisplatin–5-fluorouracil comparison in all four. However, it did not reduce the measured NRF2 target-gene expression, so its activity was not attributed to direct suppression of that program.
The authors discuss other activities, including BRD4 inhibition, as possible explanations. Those possibilities remain mechanistic hypotheses in this study. The library supports drug testing across distinct resistant tumors and helps distinguish a useful molecular association from a uniformly actionable target. Fedratinib’s activity remains an organoid finding, without demonstrated treatment benefit or prospective patient-response prediction in esophageal cancer.