Mycobacteria emulsified in olive oil-in-water trigger a robust immune response in bladder cancer treatment

Abstract

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, influence their antitumor effect. To improve the immunotherapeutic activity of Mycobacterium brumae, we designed different emulsions and demonstrated their efficacy. 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 affinity to fibronectin than non-emulsified mycobacteria, indicated favorable conditions for reaching the bladder epithelium in vivo. Finally, intravesical OO-M. brumae-treated mice showed a significantly 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.

Figure 1. Macroscopic appearance of different OO-mycobacteria emulsions.

(a) W/O and (b) O/W emulsions obtained following the sonication protocol; (c) O/W emulsion following the rod sonication protocol. Right tubes (+) correspond to M. brumae preparations, and left tubes (–) are without mycobacteria. OO, olive oil; W/O, water-in-oil; O/W, oil-in-water.

Figure 2. Affinity of M. brumae for the different tested compounds and CFU counts in each emulsion.

(a) The hydrophobicity index was calculated from the means ± SEMs of the absorbance values from triplicate preparations of two different experiments. *p < 0.01 versus OO; ^&p < 0.01 versus MO (Mann-Whitney U test). (b) CFU of M. brumae in the different emulsions or in PBS-Tween (Non-E). Values are expressed as the mean ± SEM from bacterial culture triplicates of at least three independent experiments. *p < 0.05, versus OO-emulsified M. brumae; ^&p < 0.05 versus MO-emulsified M. brumae (Mann-Whitney U test). OO, olive-oil; SO, soybean oil; SE, squalene; MO, mineral oil; nH, n-hexadecane; Non-E, mycobacteria in PBS-Tween.

Figure 3. Viability and aggregation of M. brumae in O/W emulsions.

(a) Representative confocal capture images of M. brumae emulsified using different compounds, where live bacteria are shown in green (Syto^® 9 staining) and dead bacteria in red (propidium iodide staining). (b) A binary mask representing single mycobacteria cells (in blue), small clumps (in purple) and large clumps (in orange). (c) A binary mask representing only aggregates. The bars indicate 40 μm. (d) Mean percentage of live (green columns) and dead (red columns) bacteria present in the emulsion with respect to the total counts of green and red bacteria in “a” confocal images. (e) Means ± SEMs of the percentage of the area occupied by a single bacterium with respect to the total area (in blue) and the area occupied by bacteria forming each type of aggregate (small in purple, and large in orange) obtained from the “b” binary mask. *p < 0.01, differences in the percentage of single cells versus Non-E; ^&p < 0.001, differences in the percentage of single cells versus SE (Mann-Whitney U test). (f) Aggregate size determined from the “c” binary mask. The median size of the aggregates is indicated on top of each box. Error bars indicate range. *p < 0.01 versus Non-E; ^&p < 0.0001 versus SE (Mann-Whitney U test). Values in ‘d’, ‘e’ and ‘f’ were obtained by analyzing 20 fields per condition. OO, olive-oil; SO, soybean oil; SE, squalene; MO, mineral oil, Non-E, mycobacteria in PBS-tween; OO-E h-k, olive-oil emulsion of heat-killed M. brumae was used as a control.

Figure 4. Tumor growth inhibition and cytokine production triggered by emulsified or non-emulsified mycobacteria.

High grade human T24 (a) and murine MB49 (b), and human low grade 5637 (c) BC cells were treated with mycobacteria in OO emulsion (white columns), in MO emulsion (gray columns) or in culture media (Non-E, black columns). Emulsions without bacteria or cell culture media were added to parallel wells (No-bact). In (a–c) results from the MTT assay are shown as the percentage of proliferation in relation to the control cells (Non-E No-bact). Data are expressed as the means ± SEMs of the cell culture technical triplicates of three independent experiments. IL-6 (d–f) and IL-8/KC (g–i) production by infected and non-infected (No-bact) BC cells. Results are shown as the means ± SEMs of two technical replicates of the cell culture supernatants. *p < 0.05; **p < 0.01; ***p < 0.001; ^&p < 0.05; ^&&p < 0.01; ^&&&p < 0.001 versus the respective control No-bact; ^#p < 0.05; ^##p < 0.01 versus the respective BCG (Kruskal-Wallis H test followed by Mann-Whitney U test). OO, olive oil; MO, mineral oil; Non-E, mycobacteria in PBS-tween; No-Bact, preparations without mycobacteria.

Figure 5. BCG and M. brumae survival inside T24, MB49 and 5637 BC cells.

Colony-forming units (CFUs) from cell lysates at different time-points after infection are represented (h, hours). Values represent the means ± SEMs of three serial dilutions of triplicate culture wells of M. brumae infected T24 cells (a), MB49 cells (b) and 5637 cells (c), and BCG-infected T24 cells (a). Data are representative of one out of three (for T24) and two (for MB49 and 5637) independent experiments. BCG counts were estimated from the results obtained in the 96-well plate 7H10-based spot assay from dilutions of infected MB49 and 5637 BC cells. ^& < 0.001 (Friedman test); *p < 0.05; **p < 0.01 versus their respective M. brumae. (Kruskal-Wallis H test followed by Mann-Whitney U test). Representative pictures of the wells in which grown BCG from MB49 (d) cultures and 5637 (e) can be observed. OO-E, olive oil emulsion; MO-E, mineral oil emulsion; Non-E, non-emulsified preparation.

Figure 6. Characterization of OO-M. brumae emulsion.

(a) Adhesion of M. brumae to BSA-coated (control), fibronectin-coated and uncoated (hydrophobicity assay) polystyrene plates. The bars represent means ± SEMs of absorbance values of triplicate wells from three independent experiments. ^&p < 0.05 versus respective BSA-coated wells; *p < 0.05; **p < 0.001 (Mann-Whitney U test). (b) Zeta potential of mycobacteria preparations. The results are expressed as the mean ± SEM of 20 measurements of each triplicate sample from two independent experiments. ^#p < 0.01 versus OO-E BCG; *p < 0.001 versus Non-E (Mann-Whitney U test). (c) Light microscopy (LM): trypan blue and fluorescence staining in upper images and separated canals in bottom images, and Field Emission Scanning Electron Microscopy (FESEM) images of OO emulsions with (M. brumae OO-E) or without (No-bact OO-E) M. brumae. Bars: 10 μm in images of LM; in FESEM: 5 μm, in the general views (2 large images), and 1 μm, in images of the details. OO, olive oil; Non-E, mycobacteria in PBS-tween; No-b, No-bact, preparation without bacteria; BSA, Bovine Serum Albumina; Fibron, fibronectin; Hydrofob, Hydrofobicity assay.

Figure 7. In vivo treatment with OO-emulsified mycobacteria.

(a) Schematic schedule of the induction of the tumor and treatments. C57BL/6 mice (n = 6) received four weekly intravesical instillations (indicated by arrows) of only the vehicle: emulsion alone (No-bact OO-E) or PBS (No-bact Non-E) or mycobacteria in emulsion (OO-E) or in PBS (Non-E), after tumor implantation. (b) On the right, macroscopic pictures taken from representative bladders of tumor-bearing mice after receiving the different treatments. Each mark is 1 mm. On the left, histological images (hematoxylin-eosin staining) of bladder sections from the same bladders. Scale bar, 1 mm. (c) Kaplan-Meier curve of survival in tumor-bearing mice after treatments. ^&p < 0.05 versus respective no mycobacteria groups (Log-Rank Mantel-Cox tests). (d) Colony-forming units (CFU) of splenocytes cultures from treated mice. The means of three replicates of the viable bacteria from each spleen is represented as a dot; the line identifies the mean of the animal group. ^#p < 0.05 versus respective BCG groups (Mann-Whitney U test). (e) Production of IFN-γ by mycobacteria-stimulated splenocytes from treated mice measured 72 hours after stimulation. Dots represent the means of two technical replicates per spleen; the line identifies the mean of the animal group. ^&p < 0.05 versus PBS group; *p < 0.05; **p < 0.01 (Mann-Whitney U test).