Abstract 5309: Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells
Bibliographic record
Abstract
Abstract Extracellular vesicles (EVs) are nano-sized (30-1000 nm) membranous particles released by nearly all cell types. EV biology is a rapidly growing field, however its relative novelty and the intrinsic heterogeneity of EVs result in a lack of standardized and reproducible protocols. We sought to develop and validate an EV isolation method using rhabdomyosarcoma cell lines, with multiple characterization/validation methods, to improve the reproducibility and reliability of EV research in pediatric cancer research. EVs were isolated and analyzed in triplicate from 4 rhabdomyosarcoma cell lines (RH4, RH18, RH30, RD), of varying clinical characteristics including subtype, TP53 status and FOXO1 fusion status. Cells were grown to 70% confluency in six 175cm2 flasks, to ensure sufficient EV content for all downstream analyses, then incubated for 48 hours in media containing 10% EV-depleted FBS. The resultant 120mL of conditioned media (CM) per cell line was collected and subject to differential ultracentrifugation to isolate EVs. CM was centrifuged twice, then concentrated to less than 10mL using 3kDa MWCO filter tubes. Concentrated CM was centrifuged twice before passing through a 0.22μm pore-sized syringe filter prior to the final ultracentrifugations. The sample was ultracentrifuged and the EV pellet was carefully washed, re-centrifuged and resuspended in 100μL of PBS. Protein analysis by Western blot confirmed the presence of EV markers (syntenin-1, TSG101) and absence of non-EV markers (calnexin, histone H2Ax). EV quantification and size distributions were evaluated by NanoSight nanoparticle tracking analysis and visualized by transmission electron microscopy. All isolations were positive for EV markers and negative for non-EV markers by Western Blotting. All isolations showed similar size distributions by nanoparticle tracking analysis, within the range of 30-250nm. EVs in each sample with the typical cup-like shape were observed in electron microscope images of EVs. Overall, our method demonstrates good reproducibility of EV isolations from multiple rhabdomyosarcoma cell lines. This methodology can be used as a baseline for other cell lines to provide greater consensus within the field, allowing further discoveries analyzing the secretome of patient-derived cell lines with the potential to translate into more specific diagnostic tools. Citation Format: Paula R. Quaglietta, Ashby Kissoondoyal, Ethan Malkin, Salvador Mejia, Ann Gong, David Malkin, Reto M. Baertschiger. Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5309.
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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 | 0.002 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.002 |
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".