Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice

Abstract

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used.

Figure 1

Infiltrated immune cells into mycobacteria-treated and non-treated tumor bearing mice 29 days after tumor induction. Absolute number of T cells (CD3⁺, CD4⁺ and CD8⁺ subsets), NKT cells, NK cells and B cells that infiltrated the bladder. The different groups of animals are indicated using different colors on the horizontal axis of the graph: non-mycobacteria treated mice (No-bact) are represented by empty black symbols, BCG-treated mice by blue symbols and M. brumae-treated mice by red symbols. Dots represent animals treated with emulsion or emulsionated mycobacteria, triangles represent mice treated with PBS or mycobacteria in PBS. *p < 0.05; **p < 0.01.

Figure 2

Representative histological images of bladder sections stained with two immunological markers. Bladders from the OO-E No-bact group (top) and from the OO-E live M. brumae group (bottom). Sections were stained with a CD3 marker (left column) and CD20 marker (right column). Scale bar, 100 µm.

Figure 3

Systemic immune responses in tumor-bearing and healthy mice. Levels of anti-BCG (a) and anti-M. brumae (b) IgG antibodies present in sera from BCG-treated, M. brumae-treated and non-treated tumor-bearing mice, as well as in healthy animals. The presence of BCG antibodies in M. brumae-treated mouse serum is represented by empty blue symbols (b) and the presence of M. brumae antibodies in BCG-treated mouse serum by empty red symbols (a). For a and b, solid lines represent the mean of the OD (405 nm) values (two technical replicates) of the six mice from each group. *p < 0.05 compared to No-bact. ^&p < 0.05 compared to H. Splenocyte proliferation after ex vivo BCG (c) and M. brumae (d) restimulation. Proliferation is expressed relative to non-restimulated splenocytes of the same animal (proliferation of restimulated splenocytes/proliferation of non-restimulated splenocytes) after BCG and M. brumae stimulation. Proliferation of splenocytes from M. brumae-treated animals restimulated with BCG antigens is represented by empty red symbols (c) and proliferation of splenocytes from BCG-treated animals restimulated with M. brumae antigens is represented by empty blue symbols (d). For c and d, solid lines represent the mean of relative proliferation (three technical replicates) of 3–6 different spleens from each animal group. *p < 0.05 compared to No-bact. ^&p < 0.05 compared to H. For a, b, c, and d, dots represent animals treated with emulsified preparations and triangles represent animals treated with non-emulsified preparations. The different groups of animals are indicated by different colors on the horizontal axis of the graph as follows: empty grey triangles for the healthy (H) mice, empty black symbols for tumor-bearing mice receiving no mycobacteria treatment (No-bact), blue symbols for BCG-treated mice, and red symbols for M. brumae-treated mice.

Figure 4

Schedule of the animal experiments. Graphical representation of the schedule, in which tumor induction (day 0), mycobacteria treatments (days 1, 8, 15 and 22 after tumor induction) and sacrifice (day 29 after tumor induction) are indicated by arrows.