Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells

Abstract

Major clinical issues in bladder cancer include the identification of prediction markers and novel therapeutic targets for invasive bladder cancer. In the current study, we describe the isolation and characterization of a tumor-initiating cell (T-IC) subpopulation in primary human bladder cancer, based on the expression of markers similar to that of normal bladder basal cells (Lineage-CD44(+)CK5(+)CK20(-)). The bladder T-IC subpopulation was defined functionally by its enriched ability to induce xenograft tumors in vivo that recapitulated the heterogeneity of the original tumor. Further, molecular analysis of more than 300 bladder cancer specimens revealed heterogeneity among activated oncogenic pathways in T-IC (e.g., 80% Gli1, 45% Stat3, 10% Bmi-1, and 5% beta-catenin). Despite this molecular heterogeneity, we identified a unique bladder T-IC gene signature by gene chip analysis. This T-IC gene signature, which effectively distinguishes muscle-invasive bladder cancer with worse clinical prognosis from non-muscle-invasive (superficial) cancer, has significant clinical value. It also can predict the progression of a subset of recurring non-muscle-invasive cancers. Finally, we found that CD47, a protein that provides an inhibitory signal for macrophage phagocytosis, is highly expressed in bladder T-ICs compared with the rest of the tumor. Blockade of CD47 by a mAb resulted in macrophage engulfment of bladder cancer cells in vitro. In summary, we have identified a T-IC subpopulation with potential prognostic and therapeutic value for invasive bladder cancer.

Fig. 1.

Bladder tumor-initiating cells possess cell properties in common with bladder basal cells. (A) FACS (Becton Dickinson) analysis of CD44 expression in BC-1 and serially derived xenograft tumors. Infiltrating mouse cells positive for CD45 (hematopoietic cell marker), CD31 (endothelial cell marker), and H2Kd (mouse MHC class I) are indicated in blue boxes. Patient CD44⁺ tumor cells (black box) can form xenografts comprised of both CD44⁺ (black box) and CD44⁻ (red box) tumor cells, whereas patient CD44^- tumor cells form xenograft tumors comprised primarily of CD44⁻ tumor cells (red box). (B and C) Immunofluorescence analysis of CD44 (red) and CK5/CK20 (green) in a representative bladder xenograft section. (D) Heatmap summarizing the relative expression of CD44, CK5, and CK20 by immunohistochemistry in bladder cancer tissue array containing ≈300 specimens. Arrow indicates a subgroup of bladder cancers expressing all 3 markers (CD44, CK5, and CK20). (E) Representative serial sections of bladder cancers showing the relative distribution of CD44, cytokeratin 5 (basal cell marker), and cytokeratin 20 (differentiated cell marker).

Fig. 2.

Molecular heterogeneity: activation of diverse self-renewal proteins in bladder cancer. (A–D) Immunofluorescence staining of ß-catenin, followed by analysis with confocal microscopy. White arrowheads indicate cells with nuclear localization of ß-catenin. (E) Real-time PCR analysis of Gli1 mRNA level in fractionated tumor cells from BC-1, BC-8, and 2 representative xenografted tumors derived from BC-8. (F) Heatmap summarizing the expression level of CD44 and the activated forms of several self-renewal proteins. (G) Representative immunohistochemical staining of bladder cancers with positive CD44 expression and nuclear localization of β-catenin, Stat3, and Bmi-1 in serial sections. (H and I) Immunohistochemical staining of Oct-4 (H) and Nanog (I) in seminomas as positive control and staining in normal testis as negative control.

Fig. 3.

Bladder T-IC gene signature predicts the progression of non-muscle-invasive to muscle-invasive bladder cancers. (A) Schematic diagram depicting the clinical progression of the 2 major subtypes of bladder cancers. Thirty percent of cases begin as carcinoma in situ/dysplasia that progresses to muscle-invasive bladder caner. Seventy percent of bladder cancers are non-muscle-invasive, and 15% of these can progress to invasive cancer. (B and C) Unsupervised hierarchical clustering of bladder T-IC signature genes from bladder cancer specimens obtained from 2 separate sets of published microarray data. Red indicates an activated bladder T-IC gene signature, and green indicates a repressed bladder T-IC gene signature. Approximately 97% (B) and 87% (C) of non-muscle-invasive bladder cancer clustered into the subgroup with the repressed bladder T-IC gene signature. (D) Unsupervised hierarchical clustering of bladder T-IC signature genes from published gene-expression data of non-muscle-invasive bladder cancer samples with long-term clinical follow-up. (E) Kaplan-Meier analysis of disease progression in patients who had bladder cancer categorized by an activated (n = 5) or repressed (n = 9) T-IC gene signature as shown in D.

Fig. 4.

CD47 as a putative therapeutic target for eradicating bladder tumor-initiating cells. (A) Relative CD47 cell-surface protein expression on bulk bladder cancer cells in CD44⁺ and CD44⁻ subpopulations from human bladder cancer samples quantified by FACS (Becton-Dickinson). Mean fluorescence intensity was determined for each specimen. All samples except BC-8* were primary tumors human cells; BC-8* was sorted from a xenograft. (B) H&E staining of a xenografted bladder cancer (BC-6) at an early stage underneath the mouse skin of RAG2⁻/γc⁻ mice. Islands of tumor cells (TC) grow around blood vessels (BV). (C) Immunofluorescence staining showing the relative distribution of CD44 (red) and CD47 (green) in xenografted tumor cells derived from BC-6; yellow color indicates co-localization of CD44 and CD47. (D) CFSE-labeled bladder cancer cells (from BC-1) were incubated with mouse bone marrow-derived macrophages in the presence of IgG1 isotype control, anti-HLA IgG1, or anti-CD47 IgG1 mAb. After 2 hours, the presence of fluorescently labeled bladder cancer cells within macrophages (arrowheads) was observed by immunofluorescence microscopy. (E) Phagocytosis of lineage-depleted human bladder cancer cells by mouse macrophages for each antibody condition per sample is shown. The phagocytic index for each antibody condition was determined by calculating the number of phagocytosed cells per 100 macrophages. Horizontal bars indicate the mean value for bladder cancer samples. (F) Phagocytosis of the same human bladder cancer samples by human macrophages for each antibody condition per sample is shown. Horizontal bars indicate the mean value for bladder cancer samples.