9a, d, e), thus confirming the importance and generalization of PGE2/COX2 signalling during development of chemoresistance, while other metabolites of the arachidonic acid pathway may also be involved. repopulation can be abrogated by a PGE2-neutralizing antibody and celecoxib drug-mediated blockade of PGE2 signalling. administration of the cyclooxygenase-2 (COX2) inhibitor celecoxib effectively abolishes a PGE2- and COX2-mediated wound response gene signature, and attenuates progressive manifestation of chemoresistance in xenograft tumours, including primary xenografts derived from a patient who was resistant to chemotherapy. Collectively, these findings uncover a new underlying mechanism that models the progressive development of clinical chemoresistance, and implicate an adjunctive therapy to enhance chemotherapeutic response of bladder urothelial carcinomas by abrogating early tumour repopulation. Cytotoxic chemotherapy remains the standard of care for many advanced carcinomas. Although chemotherapy is effective in debulking tumour mass, certain Mercaptopurine patients show initial response but progressively become unresponsive after multiple treatments. Chemotherapy is administered in cycles to induce fractionated killing of unsynchronized proliferating cancer cells, and treatments are spaced out to allow recovery of normal tissues between cycles8. However, repopulation of residual surviving cancer cells also occurs, which is an undesirable phenomenon that limits chemotherapeutic response in subsequent cycles8. Recent studies demonstrated that CSCs have a survival Mercaptopurine advantage in response to chemotherapy1C3. Here we investigate the unexplored concept that CSCs may actively proliferate in response to chemotherapy-induced damages, similar to how tissue resident stem cells mobilize to wound sites during tissue repair4C7,9. Bladder urothelial carcinomas contain cells that span various cellular differentiation stages10C15, cytokeratin 14 (CK14) marks the most primitive (or least differentiated) cells11,13 and patients with abundant CK14 staining correlate with poor survival11,13. Here, comparative analysis of matching pre- and post-chemotherapy patient tissues IKBA revealed one group with CK14 staining enrichment/persistence (Fig. 1a and Extended Data Fig. 1aCc) and another group with no CK14 staining after chemotherapy (Fig. 1a and Extended Data Fig. 1a, b, d). KaplanCMeier analysis revealed patients with CK14+ cancer cell Mercaptopurine enrichment/persistence showed worse survival (Fig. 1a), justifying further need to investigate their chemotherapeutic response. Using the standard chemotherapy regimen for advanced bladder urothelial carcinomas (that is, gemcitabine and cisplatin (GC)), one chemotherapy cycle effectively reduced the growth rate of all xenograft tumours in comparison to controls (Fig. 1b and Extended Data Fig. 2a), while leading to a generalized enrichment of CK14+ cancer cells (1.7C4.3-fold) (Fig. 1c, d and Extended Data Fig. 2b, c). This enrichment is unexpectedly contributed by proliferation marked by mitosis phaseprotein phosphohistone H3 (Extended Data Fig. 2d, e; white arrows). In addition to the conventional thinking that chemotherapy selects for chemoresistant cancer cells, this active proliferative response may represent a new mechanism contributing to repopulation of residual tumours. To investigate this phenomenon further, we constructed a lentiviral reporter to Mercaptopurine enable prospective isolation of CK14+ cells by fluorescence activated cell sorting (FACS), as CK14 is an intracellular protein that would not allow for cell surface antibody labelling. We sub-cloned a previously validated gene promoter region of human (ref. 16) into a promoterless lentiviral vector carrying a tdTomato (hK14. tdTomato) red fluorescent protein (Extended Data Fig. 3a). With this reporter stably transduced into urothelial carcinoma cells (Fig. 1e and Extended Data Fig. 3bCd), we could readily detect a tdTomato+ (Tm+) subpopulation that exclusively expressed CK14 at the protein (Fig. 1f; white arrows) and messenger RNA (Fig. 1g; (Extended Data Fig. 3e) and tumorigenic cells when engrafted (Extended Data Fig. 3f), demonstrating recognized functional criteria for CSCs thus. To judge their chemotherapeutic response, we purified Tm+ Tm and CK14+? CK14? cancers cells and examined their comparative cell viability after GC chemotherapy (Fig. expanded and 1h Data Fig. 4). Tm+ CK14+cancers cells survived chemotherapy-induced apoptosis much better than Tm significantly? CK14? cells beginning at time 3 (Fig. Mercaptopurine 1h and Prolonged Data Fig. 4). Concurrent cell routine analyses revealed an urgent proliferative response of.