1 Scientific RepoRts | 6:27232 | DOI: 10.1038/srep27232 www.nature.com/scientificreports Mycobacteria emulsifed in olive oil-in-water trigger a robust immune response in bladder cancer treatment estela Noguera-Ortega 1 , Núria Blanco-Cabra 1 , Rosa Maria Rabanal 2 , Alejandro Sánchez-Chardi 3 , Mónica Roldán 3 , Sandra Guallar-Garrido 1 , Eduard Torrents 4 , Marina Luquin 1 & Esther Julián 1 The hydrophobic composition of mycobacterial cell walls leads to the formation of clumps when attempting to resuspend mycobacteria in aqueous solutions. Such aggregation may interfere in the mycobacteria-host cells interaction and, consequently, infuence their antitumor efect. To improve the immunotherapeutic activity of Mycobacterium brumae, we designed diferent emulsions and demonstrated their efcacy. The best formulation was initially selected based on homogeneity and stability. Both olive oil (OO)- and mineral oil-in-water emulsions better preserved the mycobacteria viability and provided higher disaggregation rates compared to the others. But, among both emulsions, the OO emulsion increased the mycobacteria capacity to induce cytokines’ production in bladder tumor cell cultures. The OO-mycobacteria emulsion properties: less hydrophobic, lower pH, more neutralized zeta potential, and increased afnity to fbronectin than non-emulsifed mycobacteria, indicated favorable conditions for reaching the bladder epithelium in vivo. Finally, intravesical OO-M. brumae-treated mice showed a signifcantly higher systemic immune response, together with a trend toward increased tumor-bearing mouse survival rates compared to the rest of the treated mice. The physicochemical characteristics and the induction of a robust immune response in vitro and in vivo highlight the potential of the OO emulsion as a good delivery vehicle for the mycobacterial treatment of bladder cancer. Intravesical instillation of Mycobacterium bovis bacillus Calmette-Guerin (BCG) is an efective treatment rou- tinely used for high-risk non-muscle-invasive bladder cancer (BC) and carcinoma in situ (CIS) patients 1 . Current evidence suggests that BCG therapy prevents recurrence and progression, which prolongs the patient’s survival 2,3 . Although the benefts of BCG immunotherapy are clear, the drawbacks should not be underestimated. Te major- ity of BCG-treated patients do not sufer severe adverse events, but approximately 5% of patients sufer side efects that are considered to be serious, and BCG infection cases have been detected 4 . As part of eforts to seek safer alternatives to BCG, we recently demonstrated the antitumor efcacy of the non-pathogenic Mycobacterium brumae. M. brumae was initially isolated from soil and water samples in Barcelona (Spain) 5,6 . No infection cases have been described until now either in humans or animals 7 . Similar to BCG, M. brumae inhibits BC cell growth in vitro and in vivo, which triggers an antitumor profle in the immune cells 8,9 . Te antitumor efect of mycobacteria, however, could be theoretically improved. Mycobacteria cells possess a high content of lipids in their cell walls (over 60% of their weight), which provides them with an elevated hydrophobic character that leads cells to form clumps of variable size and shape in aqueous solutions 10 . Tis tendency for clumping makes it difcult to obtain stable and homogeneous mycobacteria suspensions. In fact, the BCG aggregates in the usual aqueous solutions that are used for intravesical instillation in BC patients in a time-dependent manner, which afects the antitumor activity 11,12 . Terefore, for an adequate interaction between 1 Departament de Genètica i de Microbiologia, facultat de Biociències, Universitat Autònoma de Barcelona, Spain. 2 Unitat de Patologia Murina i comparada, Departament de Medicina i cirurgia Animals, facultat de Veterinària, Universitat Autònoma de Barcelona, Spain. 3 Servei de Microscopia, Universitat Autònoma de Barcelona, Spain. 4 Bacterial infections and Antimicrobial therapy group, institute for Bioengineering of catalonia (iBec), Spain. correspondence and requests for materials should be addressed to e.J. (email: esther.julian@uab.cat) received: 09 December 2015 Accepted: 17 May 2016 Published: 06 June 2016 OPEN