Purpose To determine the security and toxicities of sequential MMC + BCG in individuals with non-muscle invasive bladder malignancy (NMIBC) and explore evidence for potentiation of BCG activity by MMC. and fatigue were common. Eleven (91.7%) individuals were free of disease at a mean (range) follow-up of 21.4 (8.4-27.0) weeks. Median Ercalcidiol post-treatment urine concentrations of IL-2 IL-8 IL-10 and TNF-α improved on the 6-week treatment period. A larger increase in post-treatment urinary IL-8 during the 6-week period was observed in individuals receiving MMC + BCG compared to individuals receiving BCG monotherapy. In mice intravesical MMC + BCG skewed tumor-associated macrophages (TAMs) towards a beneficial M1 phenotype. Conclusions Instillation of sequential MMC + BCG is definitely safe and tolerable up to 40 mg MMC plus full strength BCG. This approach could provide improved antitumor activity over BCG monotherapy by augmenting beneficial M1 TAMs. obvious tumor or erythematous lesion). Response assessment relied on cystoscopic evaluation and biopsies when performed. Adverse events were graded according to the National Tumor Institute common toxicity criteria version 4.0. was defined as the event of culture-proven cystitis (not BCG-related). Irritative bladder symptoms with bad urine culture were classified as non-infectious cystitis (BCG-related cystitis). Treatment related grade 3 or 4 4 systemic toxicities or grade 4 local bladder toxicities defined a DLT. No evidence of disease (NED) was defined as absence of visual tumor seen on cystoscopy and cytology. Urinary Cytokine Measurement Urine was collected from each patient immediately prior to therapy instillation and 4-6 hours following therapy (1st voided urine specimen following evacuation of BCG) on weeks 1 4 and 6. Urine was also collected on a prospective control human population of individuals receiving BCG monotherapy (n=5). Samples were filter-centrifuged having a 0.45 μm membrane pore size filter (Corning? Costar? Spin-X? Centrifuge Tube Filter Corning NY) for 10 minutes at 800 revolutions per minute to remove debris. Supernatants were stored at -80° Ercalcidiol C until assayed for cytokines using customized MilliplexTM packages (Millipore St. Charles MO) according to the manufacturer’s instructions. Data were collected and analyzed using Luminex software (Luminex Corporation Austin TX). Samples were run in duplicate analyzed and standard curves were generated using Bio-Plex Manager v5.0 software (Bio-Rad Laboratories Hercules CA). Screening for normality of data distribution was performed using the Shapiro-Wilk normality test. Statistical analysis was performed with Prism 6 (GraphPad Software Inc. La Jolla CA) and Stata 10.0 (StataCorp LP College Train station TX). Data were analyzed by a one-way analysis of variance (ANOVA). When the variance was not equivalent a non-parametric test for tendency was performed across the organizations. Murine studies Orthotopic MB49 Ercalcidiol tumors were founded in syngeneic C57BL/6 female mice aged 6-12 weeks (Jackson Laboratory) as explained (8 14 Briefly poly-L-lysine (0.1 mg/ml) (Sigma-Aldrich) was instilled into mouse bladders PRL for 20 minutes prior to instillation of 1 1 × 105 MB49 cells in 50 ml PBS for 1 hour MMC (0.25 mg in 50 μl sterile water) was instilled into the bladder for 20 minutes. BCG (3 × 106 CFUs in 50 μl sterile water) was instilled for 1 hour. In mice receiving MMC + BCG a 10 minute washout was performed with sterile water after MMC and before BCG. Ercalcidiol Mouse bladders were surgically harvested under sterile conditions 6 hours after the 4th weekly instillation minced with 5 ml of collagenase (Sigma-Aldrich St. Louis MO) in RPMI medium (2 mg/ml) and incubated at 37° C for Ercalcidiol 1 hour. Bladder cells was then repeatedly pipetted through a 1 ml tip filtered through a 100 μm cell strainer (BD Falcon) and then reconstituted in 14 ml of RPMI. Our Institutional Animal Care and Use Committee authorized all animal studies. Circulation cytometry We isolated and stained cells as previously explained (15) using LSR II and FACSAriaII circulation cytometers and FACSDiva software (BD Biosciences San Jose CA). Antibodies for circulation cytometry: antibodies Ercalcidiol against CD3 (clone 17A2) and CD11b (clone M1/70) were purchased from eBioscience (San Diego CA); antibodies against CD45 (clone 30-F11) were purchased from BD Bioscience (San Jose CA); antibodies against CD11c (clone N418) were purchased from Biolegend (San Diego CA); antibodies against Gr-1 (clone.