Mycolicibacterium brumae Is a Safe and Non-Toxic Immunomodulatory Agent for Cancer Treatment

Abstract

Intravesical Mycobacterium bovis Bacillus Calmette-Guérin (BCG) immunotherapy remains the gold-standard treatment for non-muscle-invasive bladder cancer patients, even though half of the patients develop adverse events to this therapy. On exploring BCG-alternative therapies, Mycolicibacterium brumae, a nontuberculous mycobacterium, has shown outstanding anti-tumor and immunomodulatory capabilities. As no infections due to M. brumae in humans, animals, or plants have been described, the safety and/or toxicity of this mycobacterium have not been previously addressed. In the present study, an analysis was made of M. brumae- and BCG-intravenously-infected severe combined immunodeficient (SCID) mice, M. brumae-intravesically-treated BALB/c mice, and intrahemacoelic-infected-Galleria mellonella larvae. Organs from infected mice and the hemolymph from larvae were processed to count bacterial burden. Blood samples from mice were also taken, and a wide range of hematological and biochemical parameters were analyzed. Finally, histopathological alterations in mouse tissues were evaluated. Our results demonstrate the safety and non-toxic profile of M. brumae. Differences were observed in the biochemical, hematological and histopathological analysis between M. brumae and BCG-infected mice, as well as survival curves rates and colony forming units (CFU) counts in both animal models. M. brumae constitutes a safe therapeutic biological agent, overcoming the safety and toxicity disadvantages presented by BCG in both mice and G. mellonella animal models.

Keywords: BCG; Galleria mellonella; bladder cancer; mice; nontuberculous mycobacteria; safety.

Figure 1

Schematic schedule of (A) intravenous infection in severe combined immunodeficient (SCID) mice, (B) intravesical treatments in BALB/c, and (C) intrahemacoelic infection in G. mellonella larvae. Abbreviations: PBS, phosphate buffered saline; BCG, Bacillus Calmette–Guérin.

Figure 2

Kaplan–Meier survival curves after intravenous (IV)-infection and intravesical (IB)-inoculation in mice and intrahemacoelic infection in G. mellonella. (A) SCID mice (n = 8/group) received a single intravenous infection of live (red) or (purple) γ-irradiated M. brumae or Bacillus Calmette–Guérin (BCG) (blue), or PBS (black). BCG-infected mice survived for over 48 days, whereas the rest of mice groups survived until the end of the study. **** p < 0.0001 (Mantel–Cox test). (B) Balb/C mice (n = 8/group) received IB instillations with low (purple) or high doses (violet) of live γ-irradiated; low (red) or high doses (orange) of live M. brumae; or PBS (green). All animals survived until the end of the experiment; (C) G. mellonella larvae (n = 60/group) were infected with 1 × 10⁴; 1 × 10⁵ or 1 × 10⁶ CFU/larvae of M. brumae or BCG, or PBS as control *** p < 0.0005; **** p < 0.0001 (Mantel–Cox test).

Figure 3

Colony forming units (CFU) counts obtained from (A) lungs, (B) spleens, and (C) livers from mycobacteria-infected SCID mice. No CFUs were detected in the organs of M. brumae-infected mice, while CFU counts were significantly elevated in the lung, spleen and liver tissues of BCG-infected mice. (D) Bacterial burden recovered in hemolymph samples from G. mellonella larvae. While no CFU counts were observed in M. brumae-infected larvae, high CFU counts were observed in BCG-infected larvae. Data are presented as the mean values ± SD of the mycobacteria CFU counts. ** p < 0.01, **** p < 0.0001 (Kruskal–Wallis H test).

Figure 4

Histopathology analysis of the different organs from IV-infected mice. (A) Hematoxylin and eosin staining of lungs, spleens and livers from PBS, γ-irradiated-M. brumae, M. brumae and BCG- IV infected mice. Only BCG-infected mice showed lesions consisting of granulomas with inflammatory component (*, black asterisks). The lesions were massive, showing in some organs a coalescent pattern (note lung and spleen sections). (B) A score (0 to 4) in accordance with the degree of the histological findings. Only BCG-infected mice exhibited systemic injuries of maximum gravity. Statistically significant differences were seen in 11 out of 16 organs between BCG-infected mice and the other infected mice. * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001 (Kruskal–Wallis H test).

Figure 5

Histology score in bladders from IB-treated mice. A score (0 to 4) in accordance with the degree of inflammatory cells observed during the histopathology analysis of the bladders.