Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics
Abstract
:1. Introduction
2. Results
2.1. Organoid Raft Cultures Retain the Morphological Features of Colorectal Cancer
2.2. Colorectal Primary Tissues Express Varying Levels of BCL-2 Family Proteins
2.3. Effect of ABT-263 Treatment on BCL-2 Family Proteins in Organoid Raft Cultures
2.4. ABT-263 Treatment of Organoid Raft Cultures Decreases Cell Proliferation and Increases Apoptosis
3. Discussion
4. Materials and Methods
4.1. Materials Required for Organoid Raft Cultures (ORCs)
- Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and 1% Penicillin Streptomycin Antibiotic (100×, Corning, Cat # 30-002-CI, Fisher Scientific, Hampton, NH) was used for culturing feeder cells.
- We used Ham’s F12:F12 mix (1X) (1 L, package, GIBCO, Cat # 21700-0075, Thermo Fisher Scientific, Waltham, MA, USA). For reconstitution, directions on the package were followed, and 1.176 g of sodium bicarbonate was added. The pH was adjusted to 7.0 with 1 N HCl before filtration. F12, not more than a month old, should be used, as the pH fluctuates swiftly.
- Insulin stock (1000X) (Sigma–Aldrich, Cat # I-1882, Millipore Sigma, Burlington, MA, USA) was made by dissolving 100 mg in 20 mL sterile ultrapure water inside a cell culture hood. Aliquots were stored at −20 °C.
- Apo-transferrin stock (1000X) (Sigma–Aldrich, Cat # T1147, Millipore Sigma, Burlington, MA, USA) was made by dissolving 100 mg in 20 mL of sterile ultrapure water inside a cell culture hood. Aliquots were stored at −20 °C.
- Hydrocortisone-21-hemisuccinate stock (10,000X) (Sigma–Aldrich, Cat # H-488, Millipore Sigma, Burlington, MA, USA) was made by dissolving 100 mg in 25 mL of 100% ethanol inside a cell culture hood. Aliquots were stored at −20 °C.
- Human epidermal growth factor stock (10,000X) MilliporeSigma Calbiochem, Cat # 32-483-1200UG, Fisher Scientific, Hampton, NH, USA) was made by diluting 100 µL in 20 mL of sterile ultrapure water inside a cell culture hood. Aliquots were stored at −20 °C.
- Cholera toxin stock (10,000X, 1 mM) (Cayman Chemical, Cat # NC1425699, Fisher Scientific, Hampton, NH) was made by dissolving 1 mg in 2 mL of sterile ultrapure water inside a cell culture hood. Of this solution, 174 µL was added to 826 µL of sterile ultrapure water. Aliquots were stored at 4 °C. This solution should not be frozen.
- We used Type I Rat Tail Collagen (Corning, Cat # CB-40236, Fisher Scientific, Hampton, NH, USA).
- F12 stock (10X) (GIBCO, Cat # 21700-0075, Thermo Fisher Scientific, Waltham, MA, USA) was made by dissolving one 1-L package of Ham’s F12 medium in 100 mL of sterile ultrapure water. In this stock, pH was not adjusted, and sodium bicarbonate was not added. Filter-sterilized aliquots were then stored at −20 °C.
- We used phosphate-buffered saline (PBS) (10X) (Fisher BioReagents, Cat # BP39920, Fisher Scientific, Hampton, NH, USA).
- We used Antibiotic and Antimycotic mixture (Anti–Anti) (100X) (GIBCO, Cat # 15240062 Thermo Fisher Scientific, Waltham, MA, USA.
- We used 3T3-J2 mouse fibroblasts cells (Cat#EF303, Kerafast, Boston, MA, USA).
- Stainless steel grids (Edward J. Darby Inc, Philadelphia, PA, USA) measuring 1”× 1” (L × B) consisted of squares of wire mesh with square pores (size, 0.25 mm). The corners of the wired mesh were bent to make rafts/boats (approximate height 2 mm).
4.2. Generation of Patient-Derived Xenografts (PDXs)
4.3. Collection of the Patient-Derived Xenograft Tissue
4.4. Preparing Collagen Dermal Equivalent
- 400 µL of 10× Reconstitution Buffer (RB Buffer: 2.2 gm sodium bicarbonate, 0.2 gm sodium hydroxide (Sigma, Cat # 1310-73-2, Millipore Sigma, Burlington, MA, USA), and 4.76 gm HEPES (free acid; Sigma, Cat# H 3375) per 100 mL. Aliquots of 10 mL were stored at −20 °C);
- 400 µL 10× F12;
- 4 mL Type-1 Rat tail collagen.
4.5. Seeding on Collagen Bed/Boat
4.6. Generation of Organoid Raft Cultures (ORCs)
4.7. Treatment with Navitoclax (ABT-263)
4.8. Harvesting the Organoid Raft Cultures (ORCs)
4.9. Immunohistochemical (IHC) Analysis
4.10. RNA Extraction and Quantitative-PCR
4.11. TUNEL Staining
4.12. Immunofluorescence Microscopy
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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CRC Patient # | Sex | Age | Body Mass Index | Sample Origin | Tumor (T), Nodes (N), and Metastases (M) | Degree of Differentiation | |
---|---|---|---|---|---|---|---|
1 | CRC-18 | Female | 73 | 21.92 | Colon | T3N0Mx | Moderate |
2 | CRC-59 | Male | 40 | 26.42 | Sigmoid Colon | T3N0 | Moderate |
3 | CRC-82 | Male | 65 | 34.94 | Ascending Colon | T3N1 | Moderate |
Genes | Primer Sequence (5′→ 3′) | |
---|---|---|
1 | BCL2-F | GGA TTG TGG CCT TCT TTG AG |
2 | BCL2-R | GCC GGT TCA GGT ACT CAG TC |
3 | BCL-XL-F | AGT TTG AAC TGC GGT ACC GG |
4 | BCL-XL-R | GCA TTG TTC CCA TAG AGT TC |
5 | BCL W-F | GCG GAG TTC ACAGCT CTA TAC |
6 | BCL W-R | AAA AGG CCC CTA CAG TTA CCA |
7 | β-Actin-F | CTG CTT GCT GAT CCA CAT CTG |
8 | β-Actin-R | ATC AAG ATC ATT GCT CCT CCT GAG |
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Bajpai, P.; Banerjee, N.S.; Moore, D.W.; Kim, H.-G.; Afaq, F.; Contreras, C.M.; Heslin, M.J.; Reddy, V.B.; Peter, S.; Varambally, S.; et al. Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics. Int. J. Mol. Sci. 2022, 23, 14392. https://doi.org/10.3390/ijms232214392
Bajpai P, Banerjee NS, Moore DW, Kim H-G, Afaq F, Contreras CM, Heslin MJ, Reddy VB, Peter S, Varambally S, et al. Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics. International Journal of Molecular Sciences. 2022; 23(22):14392. https://doi.org/10.3390/ijms232214392
Chicago/Turabian StyleBajpai, Prachi, Nilam Sanjib Banerjee, Dianne W. Moore, Hyung-Gyoon Kim, Farrukh Afaq, Carlo M. Contreras, Martin J. Heslin, Vishnu B. Reddy, Shajan Peter, Sooryanarayana Varambally, and et al. 2022. "Developing 3D Organoid Raft Cultures from Patient-Derived Xenografts as Rapid Models to Screen Efficacy of Experimental Therapeutics" International Journal of Molecular Sciences 23, no. 22: 14392. https://doi.org/10.3390/ijms232214392