How urinary bladder tumors grow and expand: The bladder as a model for the study of clonal organization and field cancerization
Final Report Abstract
In the performed project we wanted to investigate the spatial composition of urinary bladder mucosa (urothelium) out of stem/progenitor cells and their descendants in normal and malignant conditions. We used tissue areas deficient for the mitochondrially-encoded enzyme cytochrome c oxidase (CCO), detected by dual colour enzyme histochemistry. To show that these patches represent clonal proliferations originating from a common stem/progenitor cell, small CCO-proficient and -deficient tissue areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) was sequenced to identify mtDNA mutations. We found CCO-deficient epithelial cell patches of varying number, location and size. Each cell area within a CCO-deficient patch contained an identical mtDNA mutation, indicating the patch was a clonal unit. The presence of one or more patches (max. 26) confirmed that the bladder mucosa surface is built by several stem/progenitor cells. Different locations of the patches show that there is no specific stem cell site in the bladder. The two-dimensional length of these negative patches reached from 2-3 cells (about 30 μm) up to 4.7 mm, indicating that the patches can expand horizontally and suggesting that one stem cell clone can be replaced by another. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a (unipotential) stem cell that actively replenishes the urothelium during ageing. The CCO-deficient patches always comprised the basal epithelial layer, but not necessarily reached the epithelial surface, which indirectly proves a basal location of the stem cells. Probably due to the fast growth of urothelial neoplasias we were not able to detect slowly developing CCO-deficiency in premalignant conditions and cancer in an appropriate time frame. Therefore we extended the project to prostatic epithelium which is known to have slowly growing premalignant prostatic intraepithelial neoplasia (PIN) and cancer. Entire CCO-deficient prostatic acini, and part-deficient prostatic acini were found. Deficient patches spanned either basal or luminal cells, but sometimes also both epithelial cell types in normal, hyperplastic or atrophic epithelium and PIN. These results show that all human prostatic epithelial conditions contain stem cell-derived clonal units that actively maintain the epithelium. CCO-deficient patches in benign epithelium contained basal, luminal and endocrine cells, demonstrating multilineage differentiation and therefore the presence of a multipotent stem cell. Most deficient areas included the basal compartment indicating again the basal layer as the location of the stem cell. Additionally, clonal patches comprising both PIN and invasive cancer were observed, supporting PIN as the pre-invasive lesion for prostate cancer. Taken together, the method of CCO-deficiency based lineage tracing in bladder and prostatic epithelium has revealed indirect evidence for the location and spread of tissue specific stem/progenitor cells. Further mathematical modelling is now necessary in order to calculate clone growth rates, which will be of clinical relevance for tumour development and detection time, as well as clinical surveillance intervals in bladder cancer patients.
Publications
- The human urothelium consists of multiple clonal units, each maintained by a stem cell. J Path April 2011 May 30
Gaisa NT, Graham TA, McDonald SAC, Cañadillas-Lopez S, Poulsom R, Heidenreich A, Jakse G, Tadrous PJ, Knuechel R, Wright NA
(See online at https://doi.org/10.1002/path.2945)
