Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures
Abstract
:1. Introduction
2. Material and Methods
2.1. Preparation of Human Pancreas Organoid Feeding Medium
2.2. Cultivation and Passaging of Human Pancreas Organoid Cultures
2.3. Isolation of Total RNA from Organoid Cultures
2.4. Synthesis of cDNA from RNA Templates
2.5. Design of Primer Sequences for qPCR
2.6. Quantitative PCR (qPCR) Analysis
2.7. Isolation of DNA from Organoid Cultures
2.8. Droplet Digital PCR (ddPCR)
2.9. Embedding Organoids for Immunohistochemistry (IHC) Analysis
2.10. Immunohistochemistry Staining
2.11. Organoid Harvest and Metabolite Extraction
2.12. Metabolite Extraction from PDO Conditioned Medium
2.13. GC/MS Based Metabolic Profiling
2.14. Statistical Analysis
2.15. Data Avialbility
2.16. Compliance with Ethical Standards
3. Results
3.1. Patient and Clinical Data of Organoid Lines Used in This Study
3.2. Analysis of Pancreas and Fibroblast Lineage Marker Expression
3.3. Metabolic Analysis of PDOs Derived from Early and Late Recurrent PDAC Patients
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Organoid Line | Sex | Age/Years | TNM | CRM | Tumor Size/cm | Tumor Location | PDAC Grading | KRAS Mutation | Time to, Location of Recurrence/days | Survival (Last Follow Up) after Resection/days |
---|---|---|---|---|---|---|---|---|---|---|
ER-1 | Male | 32 | pT3, pN1 (3/14). L0. V0. Pn1 | R1 | 6.5 | Pancreas head | G2 | G12R | 101, Liver | (1172) |
ER-2 | Male | 67 | pT3, pN1 (7/27). L1. V0. Pn1 | R1 | 4.5 | Pancreas head | G2 | G12D | 69, Liver | (1106) |
ER-3 | Female | 66 | pT3, pN2 (5/37). L1. V1. Pn1 | Narrow | 5.0 | Pancreas body | G2 | G12D | 42, Liver | 94 |
LR-1 | Male | 77 | pT3, pN2 (6/16). L1. V0. Pn1 | Narrow | 5.0 | Pancreas head | G3 | G12R | 538, Lung | 886 |
LR-2 | Male | 72 | pT2, pN2 (4/22). L1. V0. Pn1 | Narrow | 3.8 | Pancreas head | G3 | G12D | 431 | (431) |
LR-3 | Male | 58 | pT2, pN1 (1/19). L1. V0. Pn0 | R1 | 3.2 | Pancreas head | G2 | G12D | 492, Liver local | (543) |
Pathway Abbreviation | Pathway | Pathway Impact | −log(p) |
---|---|---|---|
(a) | Alanine, aspartate and glutamate metabolism | 0.6234 | 9.4709 |
(b) | beta-Alanine metabolism | 0.39925 | 7.5625 |
(c) | Pyruvate metabolism | 0.32192 | 7.0445 |
(d) | Histidine metabolism | 0.22131 | 5.7457 |
(e) | TCA cycle | 0.24338 | 4.7773 |
(f) | Glutamine and glutamate metabolism | 0.5 | 4.8607 |
(g) | Starch and sucrose metabolism | 0.55921 | 4.4369 |
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Braun, L.M.; Lagies, S.; Klar, R.F.U.; Hussung, S.; Fritsch, R.M.; Kammerer, B.; Wittel, U.A. Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures. Cancers 2020, 12, 1440. https://doi.org/10.3390/cancers12061440
Braun LM, Lagies S, Klar RFU, Hussung S, Fritsch RM, Kammerer B, Wittel UA. Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures. Cancers. 2020; 12(6):1440. https://doi.org/10.3390/cancers12061440
Chicago/Turabian StyleBraun, Lukas M., Simon Lagies, Rhena F. U. Klar, Saskia Hussung, Ralph M. Fritsch, Bernd Kammerer, and Uwe A. Wittel. 2020. "Metabolic Profiling of Early and Late Recurrent Pancreatic Ductal Adenocarcinoma Using Patient-Derived Organoid Cultures" Cancers 12, no. 6: 1440. https://doi.org/10.3390/cancers12061440