Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-γ/IL-17 release in bladder cancer treatment

Abstract

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 could be a key parameter of treatment success. Here, BCG and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor-bearing mice were achieved using M. brumae-A60 and BCG-G15 than the rest of the treatments. M. brumae-A60 was the most efficacious among all tested treatments in terms of mouse survival, cytotoxic activity of splenocytes against tumor cells, higher systemic production of IL-17 and IFN-ɣ, and bladder infiltration of selected immune cells such as ILCs and CD4_TEM. BCG-G15 triggered an antitumor activity based on a massive infiltration of immune cells, mainly CD3⁺ (CD4⁺ and CD8⁺) T cells, together with high systemic IFN-ɣ release. Finally, a reduced variety of lipids was strikingly observed in the outermost layer of M. brumae-A60 and BCG-G15 compared to the rest of the cultures, suggesting an influence on the antitumor immune response triggered. These findings contribute to understand how mycobacteria create an adequate niche to help the host subvert immunosuppressive tumor actions.

Keywords: BCG; Innate-lymphoid cells; innate immune response; lipid; non-muscle invasive; tumor microenvironment.

Graphical abstract

Figure 1.

Culture media composition affects the antitumor capacity of mycobacteria. a) Schematic schedule of in vivo experiments. Mycobacteria (M. brumae and M. bovis BCG) pellicles grown in different culture media or PBS were used as treatments for 4 weeks, after tumor induction in female C57Bl/6 mice. In a first set of experiments, survival rates were evaluated 60 days after tumor induction (N = 10). In a second set of experiments the triggered immune response in tumor-bearing mice was studied 30 days after tumor induction(N = 8).b) Colony-forming units (CFU) of spleens from M. brumae- (red) and M. bovis BCG-(blue) treated mice. Mycobacteria counts from each spleen is represented by a dot; the line identifies the median for each group. *p< 0.05 Kruskal–Wallis test, Dunn’s multiple comparison test. c–d) Kaplan–Meier analysis of healthy and tumor-bearing mice survival after PBS or M. brumae or M. bovis BCG treatments. *p< 0.05 versus PBS group, log-Rank (Mantel-Cox) test. e) Representative histological images (hematoxylin-eosin staining) of bladder sections from non-treated (PBS), M. brumae-, and M. bovis BCG-treated tumor-bearing mice. Scale refers to 1 mm. A60 is L-asparagine plus 60 mL of glycerol per L of medium; G15 is L-glutamate plus 15 mL/L of glycerol; G60 is L-glutamate plus 60 mL/L of glycerol; 7H10 is Middlebrook 7H10 medium.

Figure 2.

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).

Figure 3.

Mycobacteria systemic effects are linked to local bladder tumor immune features. a) Immune infiltration in the bladder of healthy mice (H), untreated tumor-bearing mice (T) and mycobacteria-treated tumor-bearing mice, either BCG (B) or M. brumae (M) grown in different media. Absolute numbers of infiltrating CD45⁺, CD3⁺, CD4⁺ and CD8⁺ T cells (top graph) and B and NK cells (bottom graph) are given. b) Positive correlation between CD3⁺, CD4⁺, CD8⁺ T and CD4⁺ effector memory (T_EM) cell frequencies and the levels of IFN-ɣ produced by re-stimulated splenocytes. Each dot represents one mouse (M. brumae in red; BCG in blue; healthy in light grey; untreated tumor in dark grey). Pearson r and p values are shown. c) Secretion of IL-17 by re-stimulated splenocytes significantly correlates to the frequency of CD4⁺ T_EM cells and innate lymphoid cells (ILCs) in M. brumae-treated mice (M. brumae in red; BCG in blue). Pearson r and p values are indicated. d) Frequency of CD4⁺ T_EM cells and ILCs infiltrating bladder tissue from healthy or tumor-bearing mice, treated with BCG or M. brumae grown in different culture media. Mean ± SD values are given, with dots representing individual mice. **p< 0.01.

Figure 4.

Superficial lipid analyses of M. brumae. a) SEM representative micrographs of M. brumae grown in the four culture media, bars size are 40 µm; b and c) Thin layer chromatografies (TLC) corresponding to the superficial lipids (b) and total lipids (c) of M. brumae pellicles grown on different culture media. The volume of lipid extracts applied to TLC plates was equal in all culture conditions and derived from the same amount of lipid extractions in the surface of the pellicles. The results are one representative experiment out of at least four biological replicates. TLC plates were eluted in chloroform-methanol at 96:4 (v/v), or PE 60–80°C -diethyl ether at 90:10 (v/v). TLC plates were revealed with 10% phosphomolybdic acid in ethanol; d) ¹H NMR spectra of purified α-GroMM. CDCl₃ was used as solvent and spectra were acquired at a magnetic field of 600 MHz and at a temperature of 298.0 K; e) summarized tables of the superficial lipids obtained from M. brumae pellicles; f) images corresponding to the neutral red staining in M. brumae cells. α-GroMM, alpha-glycerol-monomycolates; AG, acylglycerol.

Figure 5.

Superficial lipid analyses of M. bovis BCG. a) SEM representative micrographs of M. bovis BCG grown in the four media, bars size are 40 µm; b and c) Thin layer chromatografies (TLC) corresponding to the superficial lipids (b) and total lipids (c) of BCG pellicles. The volume of lipid extracts applied to TLC plates was equal in all culture conditions and derived from the same amount of lipid extractions in the surface of the pellicles. The results are one representative experiment out of at least four biological replicates. TLC analyses were eluted in chloroform-methanol at 96:4 (v/v), or PE 60–80°C -diethyl ether at 90:10 (v/v). TLC plates were revealed with 10% phosphomolybdic acid in ethanol; d) ¹H NMR spectra of purified k-GroMM. CDCl₃ was used as solvent and spectra were acquired at a magnetic field of 600 MHz and at a temperature of 298.0 K; e) images corresponding to the neutral red staining in BCG cells; (f) summarized tables of the superficial lipids obtained from BCG pellicles. k-GroMM, keto-glycerol-monomycolates; PGL, phenolic glycolipid; AG, acylglycerol, PDIM, phthiocerol dimycocerosates.