Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue
Abstract
:1. Introduction
2. Results
2.1. Establishment and Morphology of 3D Organoids From Endoscopic Biopsies from Rectal Cancer Patients
2.2. Gene Profiling of Normal Colon and Normal Rectum Organoids
2.3. Gene Profiling of Normal Rectum and Rectal Tumor Organoids
2.4. Transcriptomic Profiles of Rectal Tumor and Colon Tumor Organoids
2.5. Effect of Calcitriol on Organoids from Normal and Tumor Tissues
3. Discussion
4. Materials and Methods
4.1. Human Samples and Ethical Guidelines
4.2. Establishment of 3D Organoid Cultures from Endoscopy Biopsies
4.3. Growth and Expansion of Organoid Cultures
4.4. Histology and Imaging
4.5. Mutational Status
4.6. RNA-Sequencing
4.7. Coefficient of Determination and Correlation Zone
4.8. Gene Set Enrichment Analysis (GSEA)
4.9. Data Availability
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Patient | Gender | Age (y) | Sample | T a | N b | M c |
---|---|---|---|---|---|---|
#40 | Female | 65 | Normal: Colon + Rectum | pTis | pN0 | cM0 |
#41 | Male | 88 | Normal: Colon + Rectum | cT3, ypT0 | cN2a, ypN0 | cM1a |
#42 | Male | 70 | Normal: Colon + Rectum | cT2-3, ypTis | cN1a, ypN0 | cM0 |
#43 | Male | 64 | Normal: Colon + Rectum | pT4b | pN0 | cM0 |
#44 | Male | 65 | Normal: Colon + Rectum Tumor: Lower rectum | cT3, ypT2 | cN2, ypN0 | cM0 |
#45 | Male | 72 | Normal: Colon + Rectum | cT3, ypT3 | cN2, ypN0 | cM0 |
#46 | Male | 69 | Tumor: Lower rectum | cT3, ypT2 | cN1-2, ypN0 | cM0 |
#47 | Male | 88 | Tumor: Lower rectum | cT3-4, ypT3 | cN1, ypN0 | cM0 |
#48 | Female | 69 | Tumor: Medial rectum | cT4 | cN2b | cM1 |
#49 | Female | 51 | Tumor: Upper rectum * | NA | cN+ | cM1 |
#50 | Female | 88 | Tumor: Upper rectum | pT3 | pN1b | cM0 |
Buffer | Reagent | Concentration | Supplier |
---|---|---|---|
Washing buffer | Advanced DMEM/F12 | 100% | Thermo Fisher Scientific |
Hepes | 10 mM | Thermo Fisher Scientific | |
Glutamax | 10 mM | Thermo Fisher Scientific | |
Normal culture medium | Advanced DMEM/F12 | 50% | Thermo Fisher Scientific |
Wnt3a-Conditioned medium | 50% | Previously described [33] | |
Hepes | 10 mM | Thermo Fisher Scientific | |
Glutamax | 10 mM | Thermo Fisher Scientific | |
Nicotinamide | 10 mM | Sigma-Aldrich, MD, USA | |
N2 | 1× | Thermo Fisher Scientific | |
B27 | 1× | Thermo Fisher Scientific | |
N-acetyl-L-cysteine | 1 mM | Sigma-Aldrich | |
Primocin | 1:500 | Invivogen | |
Noggin | 0.1 μg/mL | Peprotech, NJ, USA | |
Gastrin | 1 μg/mL | Tocris, Bristol, UK | |
Y-27632 | 10 μM | Tocris | |
RSPO1 | 1 μg/mL | Sinobiological, Beijin, China | |
EGF | 50 ng/mL | Peprotech | |
PGE2 | 0.02 μM | Sigma-Aldrich | |
LY-2157299 | 1 μM | Axon-Medchem, Groningen, The Netherlands | |
SB-202190 | 10 μM | Sigma-Aldrich | |
Lysis buffer | TrisHCL pH 8.0 | 50 mM | Sigma-Aldrich |
EDTA pH 8.0 | 100 mM | Sigma-Aldrich | |
NaCl | 100 mM | Sigma-Aldrich | |
SDS | 1% | Sigma-Aldrich | |
Proteinase K | 20 mg/mL | Merck-Millipore, MA, USA |
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Costales-Carrera, A.; Fernández-Barral, A.; Bustamante-Madrid, P.; Domínguez, O.; Guerra-Pastrián, L.; Cantero, R.; del Peso, L.; Burgos, A.; Barbáchano, A.; Muñoz, A. Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue. Cancers 2020, 12, 2302. https://doi.org/10.3390/cancers12082302
Costales-Carrera A, Fernández-Barral A, Bustamante-Madrid P, Domínguez O, Guerra-Pastrián L, Cantero R, del Peso L, Burgos A, Barbáchano A, Muñoz A. Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue. Cancers. 2020; 12(8):2302. https://doi.org/10.3390/cancers12082302
Chicago/Turabian StyleCostales-Carrera, Alba, Asunción Fernández-Barral, Pilar Bustamante-Madrid, Orlando Domínguez, Laura Guerra-Pastrián, Ramón Cantero, Luis del Peso, Aurora Burgos, Antonio Barbáchano, and Alberto Muñoz. 2020. "Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue" Cancers 12, no. 8: 2302. https://doi.org/10.3390/cancers12082302
APA StyleCostales-Carrera, A., Fernández-Barral, A., Bustamante-Madrid, P., Domínguez, O., Guerra-Pastrián, L., Cantero, R., del Peso, L., Burgos, A., Barbáchano, A., & Muñoz, A. (2020). Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue. Cancers, 12(8), 2302. https://doi.org/10.3390/cancers12082302