A10 OVEREXPRESSION OF ASCL2 ALTERS DIFFERENTIATION, CELL CYCLE AND RESISTANCE TO ANTI-CANCER TREATMENT IN ESOPHAGEAL ORGANOIDS
Bibliographic record
Abstract
Abstract Background The esophagus is in constant contact with the austere environment caused by food and gastric reflux. It is protected by a squamous epithelium which maintenance is provided by a rare subpopulation of basal cells: Keratin 15+ (Krt15+) stem cells. However, little is known about the mechanisms underlying the expansion and the function of these stem cells. It was shown that the transcription factor ASCL2 is strongly upregulated in Krt15+ cells compared to Krt15- cells. Interestingly, ASCL2 is a gene target of the Wnt/β-catenin pathway, which acts as a regulator of proliferation and maintenance of the stemness state. Purpose The ultimate goal of my research project is to determine the role of ASCL2 in the maintenance of esophageal stem cells. To do so, I will investigate the role of ASCL2 in esophageal epithelial biology. Method Lentiviral infection approach was used to obtain mouse esophageal organoids overexpressing ASCL2 (ASCL2OE). Organoid culture, immunostaining (such as IF and H&E), qPCR, WB, mass spectrometry and proliferation assay were used to characterize the effect of ASCL2OE on morphology, differentiation, proliferation, self-renewal, and gene expression. Result(s) ASCL2OE severely altered the morphology of organoids, which were smaller and less differentiated. Defects in differentiation was investigated by IF which showed that some cells expressed both p63 and K13, respectively basal and suprabasal markers. Thus, cells seem to be blocked in an intermediate state of differentiation suggesting a default in cell fate decision. Mass spectrometry analysis confirmed a change in biological processes related to differentiation of keratinocytes and of epithelial cells. We also investigated the role of ASCL2 in self-renewal and observed that organoid formation rate (OFR) was reduced in ASCL2OE organoids. Furthermore, proliferation was also reduced in WST-1 and EdU assays. We then observed significant changes in the cell cycle by flow cytometry: there is an increased in the number of cells in G0/G1 and a major decrease in G2/M cells, suggesting a blockade in G1. Interestingly, CDNK2a (p16INK4a), an inhibitor of cell cycle progression, was increased in our mass spectrometry results. Finally, ASCL2 could also play a role in radio and chemoresistance of Krt15+ stem cells, as ASCL2OE organoids are less sensitive to radiation and chemotherapy agents than control. Conclusion(s) ASCL2 could play a role in orchestrating cell fate decision in the esophageal epithelium as ASCL2OE organoids showed alteration in differentiation, proliferation, and cell cycle. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; NSERC, Canada Research Chairs and CRCHUS scolarship Disclosure of Interest None Declared
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".