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Different systemic immune response triggered by mycobacteria. a) Percentage of proliferation of mycobacteria-restimulated splenocytes over non-stimulated splenocytes from mycobacteria-treated tumor-bearing mice; and b) IFN-ɣ and IL-17 cytokine detection in culture supernatants from restimulated splenocytes. Splenocytes were restimulated with heat-killed M. brumae (red) or BCG (blue) grown in the same medium used for intravesical treatment; c) MB49 cell inhibition triggered by splenocytes from M. brumae- (red), BCG- (blue), and non-treated (grey) tumor-bearing mice, and healthy (white) mice. Ratio 25:1 (splenocytes: MB49 cells). d) Anti-M. brumae (red) or anti-BCG (blue) IgG antibodies detected in sera from mycobacteria-treated mice. T is non-treated tumor-bearing mice; H is healthy mice. Data represents the mean and SD. *p< 0.05, #p< 0.05 with respect to tumor, and &p< 0.05 with respect to healthy mice (ANOVA test).
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The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can impact the antitumor effect of BCG strains and...
Citations
... Preclinical studies have shown that M. brumae inhibits bladder tumor cell proliferation being even more efficacious than BCG well-differentiated cells (low-grade tumors), and activated peripheral blood mononuclear cells triggering the production of cytokines and a cytotoxic profile against tumor cells (Noguera-Ortega et al., 2016a). In the orthotopic murine model of bladder cancer, the induction of local and systemic immune responses by M. brumae treatment leads to prolonging the survival of tumor-bearing mice with respect to non-treated tumor-bearing mice and by a similar ratio than BCG-treated mice (Noguera-Ortega et al., 2016a,b;Guallar-Garrido et al., 2022). ...
Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it’s in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M . brumae type strain ATCC 51384 T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M . brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M . tuberculosis T cell and B cell antigens in M . brumae genome is considerably lower than those antigens present in M . bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.
... However, our understanding of the BCG-induced immune landscape remains incomplete, mainly due to the difficulty associated with obtaining tissue samples from patients during treatment. Using animal models, it has been shown that CD4 + and CD8 + T-cells play a critical role in BCG-and M. brumaeinduced antitumor activity (10,(19)(20)(21), although comprehensive immune profiling has not been reported. ...
... Given the very few advances in therapeutic strategies for earlystage disease over the past two decades, there is a major unmet need for improved intravesical therapies for NMIBC. We very recently showed that the mycobacterial cell-surface is modified by culture conditions, which impacts antitumor immune activity (21,22). This is relevant since commercially available BCG sub-strains are currently cultured on different medium compositions, which is one of the plausible reasons for the different treatment outcomes observed in NMIBC patients after BCG treatment. ...
... Tumors were induced by initial intravesical administration of poly-L-lysine (Sigma-Aldrich) followed by MB49 cells instillation. 24 hours after tumor implantation, mice were intravesically treated with BCG or M. brumae grown in different conditions (21). Mycobacterial treatment was performed weekly for four weeks. ...
Intravesical BCG instillation after bladder tumor resection is the standard treatment for non-muscle invasive bladder cancer; however, it is not always effective and frequently has undesirable side effects. Therefore, new strategies that improve the clinical management of patients are urgently needed. This study aimed to comprehensively evaluate the bladder tumor immune microenvironment profile after intravesical treatment with a panel of mycobacteria with variation in their cell envelope composition and its impact on survival using an orthotopic murine model to identify more effective and safer therapeutic strategies. tumor-bearing mice were intravesically treated with a panel of BCG and M. brumae cultured under different conditions. Untreated tumor-bearing mice and healthy mice were also included as controls. After mycobacterial treatments, the infiltrating immune cell populations in the bladder were analysed by flow cytometry. We provide evidence that mycobacterial treatment triggered a strong immune infiltration into the bladder, with BCG inducing higher global absolute infiltration than M. brumae. The induced global immune microenvironment was strikingly different between the two mycobacterial species, affecting both innate and adaptive immunity. Compared with M. brumae, BCG treated mice exhibited a more robust infiltration of CD4⁺ and CD8⁺ T-cells skewed toward an effector memory phenotype, with higher frequencies of NKT cells, neutrophils/gMDSCs and monocytes, especially the inflammatory subset, and higher CD4⁺ TEM/CD4⁺ Treg and CD8⁺ TEM/CD4⁺ Treg ratios. Conversely, M. brumae treatment triggered higher proportions of total activated immune cells and activated CD4⁺ and CD8⁺ TEM cells and lower ratios of CD4⁺ TEM cells/CD4⁺ Tregs, CD8⁺ TEM cells/CD4⁺ Tregs and inflammatory/reparative monocytes. Notably, the mycobacterial cell envelope composition in M. brumae had a strong impact on the immune microenvironment, shaping the B and myeloid cell compartment and T-cell maturation profile and thus improving survival. Overall, we demonstrate that the bladder immune microenvironment induced by mycobacterial treatment is species specific and shaped by mycobacterial cell envelope composition. Therefore, the global bladder immune microenvironment can be remodelled, improving the quality of infiltrating immune cells, the balance between inflammatory and regulatory/suppressive responses and increasing survival.
... Thus, the same substrain grown under different conditions could trigger different immunomodulatory activities, which are relevant in vaccine efficacy, as demonstrated in a murine model by Venkataswamy and coworkers [21]. In the case of BC, recent studies in our laboratory demonstrated in vitro [9] and in vivo [22] different antitumour effects of BCG-Connaught grown on different media, which could also be related to the superficial characteristics expressed. ...
Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.
