Conventionally, bladder tumors arising from the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) mouse model arise from a single clone. Urothelial carcinoma is often characterized by a “field effect” whereby normal appearing urothelium distinct from a bladder tumor shares similar underlying genetic susceptibility and exposure to environmental carcinogens to predispose tumorigenesis. In this study, the authors sought to transdifferentiate fibroblasts in vitro to basal urothelial stem cells and develop an in vivo engraftment model whereby at-risk urothelium could be “replaced” by engineered, transdifferentiated urothelial stem cells.
Transdifferentiation is the direct conversion of one differentiated cell type into another differentiated cell type without a pluripotent stem cell intermediate. This process is mediated by modulating expression of a host of transcription factors.
Urothelial stem cells give rise to all other cell types, including basal, intermediate, and umbrella cells, but do not maintain pluripotency. These cells strongly express KRT14 and are thought to be the origin site for bladder cancers. The investigators reviewed the published literature to identify a list of 37 candidate transcription factors known for their involvement in stimulating transdifferentiation. These were individually cloned into retroviral vectors and used to transduce fibroblasts. They were subsequently transplanted into mouse bladders for functional engraftment analysis.
The 37 transcription factor compliment did induce urothelial and basal marker expression, including Upk2, Upk3a, Krt5, and Krt14. The investigators then used a process of subtraction screening to identify the most influential 3-4 transcription factors involved in transdifferentiation to basal and suprabasal cells.
An injury-transplant model was utilized whereby the bladder urothelium was injured with imidazolium salts followed by instillation of transdifferentiated fibroblasts. They were able to achieve successful engraftment of KRT14 expressing stem cells.
The investigators then sought the characterize the pre-malignant phenotype in non-tumor urothelium adjacent to bladder tumors. They used single sell RNAseq and pseudotime analysis to generate a phylogeny of differentiation states in the bladder urothelium. Starting with healthy mouse bladders, they found that each time a BBN-treated cell was added to the compliment it tended to fall at the basal end of the spectrum. This concept of “basalization” is observed in normal urothelium from BBN-treated mice as well as in non-tumor urothelium from bladder cancer patients.
They then sought to reverse this basalization with pharmacologic intervention. Urothelium was characterized after treatment with BBN as well as BBN combined with erlotinib (EGFR inhibitor) and BBN combined with trametinib (MEK inhibitor). These combination therapies successfully decreased expression of basal markers.
Subsequent functional studies identified that indeed, erlotinib was able to decrease tumor burden and improve survival in mice treated with BBN.
The functional significance of EGFR inhibition with erlotinib in this basalization reversal phenomenon remains unclear. However, the authors provide compelling proof of concept that urothelial stem cells can be engineered with a minimal set of transcription factors. Basalization of the non-tumor urothelium appears to be modifiable and reversable with pharmacologic intervention. These results have significant implications in decreasing the risk of bladder cancer in high risk populations, decreasing recurrences in patients with know NMIBC, and even tissue engineering by engrafting scaffolds with engineered urothelial stem cells.
Presented by: Kris Prado, MD, a urologic oncologist at Stanford University
Written by: Patrick Hensley, MD, Urologic Oncologist at the University of Kentucky (@pjhensley11) with Ashish Kamat, MD, Urologic Oncologist at MD Anderson Cancer Center (@UroDocAsh) during the 2022 Bladder Cancer Advocacy Network Think Tank (#BCANTT22) Wednesday Aug 3 – Friday Aug 5, 2022
References:
- Merrell AJ, Stanger BZ. Adult cell plasticity in vivo: de-differentiation and transdifferentiation are back in style. Nat Rev Mol Cell Biol. 2016 Jul;17(7):413-25. doi: 10.1038/nrm.2016.24. Epub 2016 Mar 16. PMID: 26979497; PMCID: PMC5818993.
- Papafotiou G, Paraskevopoulou V, Vasilaki E, Kanaki Z, Paschalidis N, Klinakis A. KRT14 marks a subpopulation of bladder basal cells with pivotal role in regeneration and tumorigenesis. Nat Commun. 2016 Jun 20;7:11914. doi: 10.1038/ncomms11914. PMID: 27320313; PMCID: PMC4915139.