Jan 22, 2026
Version 2
Directed Developmental Reprogramming of Prostate Cancer Cells to Stem-Like Cells V.2
- Mannan Nouri1,
- Ralph Buttyan1,
- Josselin Caradec1,
- Amy Anne Lubik1,
- Na Li1,
- Sarah Truong1
- 1Vancouver Prostate Centre, Vancouver, Canada & Department of Urologic Sciences, University of British Columbia, Vancouver, Canada
Protocol Citation: Mannan Nouri, Ralph Buttyan, Josselin Caradec, Amy Anne Lubik, Na Li, Sarah Truong 2026. Directed Developmental Reprogramming of Prostate Cancer Cells to Stem-Like Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldrxj7g5b/v2Version created by Mannan Nouri
Manuscript citation:
Nouri, M., Caradec, J., Lubik, A.A., Li, N., Hollier, B.G., Takhar, M., Altimirano-Dimas, M., Chen, M., Roshan-Moniri, M., Butler, M., et al. (2017). Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance. Oncotarget. DOI: 10.18632/oncotarget.14850.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: January 22, 2026
Last Modified: January 22, 2026
Protocol Integer ID: 239193
Keywords: Biological techniques, Prostate cancer cells, reprogramming, stem cells, pextocheck, expressed neural crest stem cell gene, neural crest stem cell gene, prostate cancer cell, neural crest stem cell, netd of prostate adenocarcinoma, neural crest lineage, prostate cancer, prostate adenocarcinoma, neuroendocrine transdifferentiation, stem cell, like pca cell, neuroendocrine, developmental reprogramming of prostate cancer cell
Disclaimer
Please note this protocol was originally published on the Protocol Exchange repository; a PDF of the protocol as it appeared there is attached. An automated process was used to import the protocol onto protocols.io. If there are internal references to other steps in the protocol they may not be accurately reflected, please refer to the attached PDF of the protocol to check. Please email us at [email protected] with the DOI of the protocol and any errors that you identify as we can fix these if necessary.
Abstract
A significant hindrance to pre-clinical prostate cancer (PCa) research is the lack of models necessary to study the disease and it’s many presentations. Prostate cancer is a highly heterogeneous disease. That heterogeneity is only magnified under treatment in patient tumours. Upon detailed microarray examination of a classical model of Neuroendocrine Transdifferentiation (NEtD), a notable amplification of genes associated with neural/neural crest lineage, development and function were observed. Previously, NEtD of prostate adenocarcinoma to neuroendocrine-like cells was thought to occur without a stem cell intermediate. In the below publication we demonstrated that NEtD may occur through a transient neural/neural crest stem cell-like intermediate, and developed a method to capture and study that intermediate in a number of PCa cell models. Four different AR+/PSA+ PCa cell lines cultured in Stem Transition Media (STM) underwent a similar morphological transition, readily formed spheroids, over-expressed neural crest stem cell genes, became highly invasive, metastatic, and tumour initiating. Through the culture-mediated protocol, described below, we were able to capture these “Developmental Reprogrammed” stem-like PCa cells, and add four unique PCa models to the arsenal of PCa researchers.
This protocol is associated with the following reference:
Nouri, M., Caradec, J., Lubik, A.A., Li, N., Hollier, B.G., Takhar, M., Altimirano-Dimas, M., Chen, M., Roshan-Moniri, M., Butler, M., et al. (2017). Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance. Oncotarget. DOI: 10.18632/oncotarget.14850.
Guidelines
See attached Article file: "Nouri et al. Prot. Exchange 2017":http://www.nature.com/protocolexchange/system/uploads/5363/original/Articlefile-NourietalProtocolExchange2017.docx?1490972676
Materials
Reagents
- Trypsin (Hyclone, SH3004201)
- Defined Trypsin Inhibitor (Gibco, R-007-100)
- DMEM/F12, Phenol-Red Free (Gibco, 21041-025)
- GlutaMAX‱-I Supplement (Gibco, 35050061)
- bFGF (Life Technlogies, PHG0024)
- EGF (Life Technologies, PHG0314)
- StemPro‱ Neural Supplement (Gibco, A10508-01)
- Poly-ethylenimine (Sigma, 408727-100ML)
Equipment
- CO2 incubator (humidified, CO2 at 5%, 37°C)
- Tissue culture hood
- Tissue culture centrifuge
Troubleshooting
Problem
Inconsistent Results from Gene Expression Assays
Solution
Developmentally reprogrammed cells form spheroids quickly, and tend to form clusters if not properly maintained. Cells should be monitored diligently to ensure large spheroids do not develop. Large spheroids tend to differentiate, and may not be held in the metastable stem-like state in 1X STM. Therefore, only single cells and small rosettes should be used in experimental protocols.
Problem
Slow/No Grwoth of VCaP Cells
Solution
VCaP cells require DMEM modified to contain 4mM L-glutamine, 4500mg/L glucose, 1mM sodium pyruvate, and 1500mg/L sodium bicarbonate to remain viable in a 5% CO2 incubator.
Developmentally reprogrammed VCaP cells should be maintained at high density as they require the paracrine signalling of their sister cells for viability.
Problem
Poor Attachment/Attachment Needed for Functional Analysis
Solution
If necessary developmentally reprogrammed cells may be seeded onto Poly-ethylenimine coated dishes/flasks, and will attach. No differences in gene expression or drug response have been noted between attached and unattached stem-like developmentally reprogrammed cells.
Procedure
LNCaP, VCaP, 22Rv1 (ATCC, Manassas, VA) and LAPC4 cell line (obtained from Dr. Charles Sawyer), should be maintained in phenol red-free ATCC-recommended media prior to experimental protocols.
Begin by producing 1X Stem Transition media (STM). STM is formulated from phenol red-free DMEM/F12 supplemented with 2mM GlutaMAX, 20ng/mL rhbFGF, 20ng/mL rhEGF and 2% StemPro Neural Supplement.
For reprogramming, seed AR+ LNCaP, LAPC4, VCaP or 22Rv1 prostate cancer cell lines at 30%, 50%, 50%, or 20% respectively in Poly-ethylenimine coated culture tissue culture dishes/flasks.
After attachment (24-48 hours), Suction culture media, and wash cells gently in 1X PBS, then suction PBS. Add 1X STM to culture and return to incubator.
Refresh media every two-three days during transition.
Monitor cells and passage when they reach 80-90% confluence OR after 7-days in culture.
- Passage cells using Trypsin and Defined Trypsin Inhibitor. DO NOT use serum containing media to dilute the trypsin as this may cause cells to re-differentiate before the reprogramming process is complete.
- For LNCaP, LAPC4 or 22Rv1, seed cells in large tissue culture flasks to ensure rosettes and spheroids do not clump/attach to each other.
- For VCaP cells, a high seeding density is necessary for cell viability, this will require consistent monitoring of cultures to ensure large spheroids do not form from cell clustering.
Monitor cells and passage if needed between days 7-14. At Day 14 cells are ready for analysis or experimental manipulation.
- If necessary developmentally reprogrammed cells may be seeded onto Poly-ethylenimine coated dishes/flasks, and will attach. No differences in gene expression or drug response have been noted between attached and unattached stem-like developmentally reprogrammed cells.
Timing
- Day -2: Seed prostate cancer cells.
- Day 0: Replace medium with 1X STM.
- Days 0-7: Refresh media every two-three days. Monitor cells and passage when they reach 80-90% confluence or after 7-days in culture.
- Day 7: Passage cells using Trypsin and Defined Trypsin Inhibitor.
- Day 7-14: Monitor cells and passage if needed.
- Day 14: Cells are ready for analysis or experimental manipulation.
Anticipated Results
Unlike parental PCa cells that tend to separate until confluent, cells placed in STM should begin to cluster
together, forming 3-dimensional mats. Upon transfer at Day 7, cells should form small, poorly-attached rosettes that grow into spheroids.
Developmentally reprogrammed cells should overexpress genes associated with normal, cancer and neural crest stem cells (Sox2, Oct3/4, Klf4, Sox9, ALCaM [CD166], β1-Integrin [CD29]).
Associated Publications
by Mannan Nouri, Josselin Caradec, Amy Anne Lubik, Na Li, Brett G. Hollier, Mandeep Takhar, Manuel Altimirano-Dimas, Mengqian Chen, Mani Roshan-Moniri, Miriam Butler, Melanie Lehman, Jennifer Bishop, Sarah Truong, Shih-Chieh Huang, Dawn Cochrane, Michael Cox
https://dx.doi.org/10.18632/oncotarget.14850
Supplementary Files
Supplement_0.Docx40.1KB Article file - Nouri et al. Prot. Exchange 2017
Protocol references
Nouri, M., Caradec, J., Lubik, A.A., Li, N., Hollier, B.G., Takhar, M., Altimirano-Dimas, M., Chen, M., Roshan-Moniri, M., Butler, M., et al. (2017). Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance. Oncotarget. DOI: 10.18632/oncotarget.14850.
Acknowledgements
See attached Article file