Urease-powered nanobots for radionuclide bladder cancer therapy.

Bladder cancer treatment via intravesical drug administration achieves reasonable survival rates but suffers from low therapeutic efficacy. To address the latter, self-propelled nanoparticles or nanobots have been proposed, taking advantage of their enhanced diffusion and mixing capabilities in urine when compared with conventional drugs or passive nanoparticles. However, the translational capabilities of nanobots in treating bladder cancer are underexplored. Here, we tested radiolabelled mesoporous silica-based urease-powered nanobots in an orthotopic mouse model of bladder cancer. In vivo and ex vivo results demonstrated enhanced nanobot accumulation at the tumour site, with an eightfold increase revealed by positron emission tomography in vivo. Label-free optical contrast based on polarization-dependent scattered light-sheet microscopy of cleared bladders confirmed tumour penetration by nanobots ex vivo. Treating tumour-bearing mice with intravesically administered radio-iodinated nanobots for radionuclide therapy resulted in a tumour size reduction of about 90%, positioning nanobots as efficient delivery nanosystems for bladder cancer therapy.

The High Plasticity of Nonpathogenic Mycobacterium brumae Induces Rapid Changes in Its Lipid Profile during Pellicle Maturation: The Potential of This Bacterium as a Versatile Cell Factory for Lipid Compounds of Therapeutic Interest.

The immunomodulatory potential of mycobacteria to be used for therapeutic purposes varies by species and culture conditions and is closely related to mycobacterial lipid composition. Although the lipids present in the mycobacterial cell wall are relevant, lipids are mainly stored in intracellular lipid inclusions (ILIs), which have emerged as a crucial structure in understanding mycobacteria-host interaction. Little is known about ILI ultrastructure, production, and composition in nonpathogenic species. In this study, we compared the lipid profiles of the nonpathogenic immunomodulatory agent Mycobacterium brumae during pellicle maturation under different culture conditions with qualitative and quantitative approaches by using high-resolution imaging and biochemical and composition analyses to understand ILI dynamics. The results showed wax esters, mainly in early stages of development, and acylglycerols in mature ILI composition, revealing changes in dynamics, amount, and morphometry, depending on pellicle maturation and the culture media used. Low-glycerol cultures induced ILIs with lower molecular weights which were smaller in size in comparison with the ILIs produced in glycerol-enriched media. The data also indicate the simple metabolic plasticity of lipid synthesis in M. brumae, as well as its high versatility in generating different lipid profiles. These findings provide an interesting way to enhance the production of key lipid structures via the simple modulation of cell culture conditions.

Remodeling the bladder tumor immune microenvironment by mycobacterial species with changes in their cell envelope composition.

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.

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

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-É£, and bladder infiltration of selected immune cells such as ILCs and CD4TEM. 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-É£ 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.

Swarming behavior and in vivo monitoring of enzymatic nanomotors within the bladder.

Enzyme-powered nanomotors are an exciting technology for biomedical applications due to their ability to navigate within biological environments using endogenous fuels. However, limited studies into their collective behavior and demonstrations of tracking enzyme nanomotors in vivo have hindered progress toward their clinical translation. Here, we report the swarming behavior of urease-powered nanomotors and its tracking using positron emission tomography (PET), both in vitro and in vivo. For that, mesoporous silica nanoparticles containing urease enzymes and gold nanoparticles were used as nanomotors. To image them, nanomotors were radiolabeled with either 124I on gold nanoparticles or 18F-labeled prosthetic group to urease. In vitro experiments showed enhanced fluid mixing and collective migration of nanomotors, demonstrating higher capability to swim across complex paths inside microfabricated phantoms, compared with inactive nanomotors. In vivo intravenous administration in mice confirmed their biocompatibility at the administered dose and the suitability of PET to quantitatively track nanomotors in vivo. Furthermore, nanomotors were administered directly into the bladder of mice by intravesical injection. When injected with the fuel, urea, a homogeneous distribution was observed even after the entrance of fresh urine. By contrast, control experiments using nonmotile nanomotors (i.e., without fuel or without urease) resulted in sustained phase separation, indicating that the nanomotors' self-propulsion promotes convection and mixing in living reservoirs. Active collective dynamics, together with the medical imaging tracking, constitute a key milestone and a step forward in the field of biomedical nanorobotics, paving the way toward their use in theranostic applications.

Analysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography.

Mycobacteria species can differ from one another in the rate of growth, presence of pigmentation, the colony morphology displayed on solid media, as well as other phenotypic characteristics. However, they all have in common the most relevant character of mycobacteria: its unique and highly hydrophobic cell wall. Mycobacteria species contain a membrane-covalent linked complex that includes arabinogalactan, peptidoglycan, and long-chains of mycolic acids with types that differ between mycobacteria species. Additionally, mycobacteria can also produce lipids that are located, non-covalently linked, on their cell surfaces, such as phthiocerol dimycocerosates (PDIM), phenolic glycolipids (PGL), glycopeptidolipids (GPL), acyltrehaloses (AT), or phosphatidil-inositol mannosides (PIM), among others. Some of them are considered virulence factors in pathogenic mycobacteria, or critical antigenic lipids in host-mycobacteria interaction. For these reasons, there is a significant interest in the study of mycobacterial lipids due to their application in several fields, from understanding their role in the pathogenicity of mycobacteria infections, to a possible implication as immunomodulatory agents for the treatment of infectious diseases and other pathologies such as cancer. Here, a simple approach to extract and analyze the total lipid content and the mycolic acid composition of mycobacteria cells grown in a solid medium using mixtures of organic solvents is presented. Once the lipid extracts are obtained, thin-layer chromatography (TLC) is performed to monitor the extracted compounds. The example experiment is performed with four different mycobacteria: the environmental fast-growing Mycolicibacterium brumae and Mycolicibacterium fortuitum, the attenuated slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG), and the opportunistic pathogen fast-growing Mycobacterium abscessus, demonstrating that methods shown in the present protocol can be used to a wide range of mycobacteria.

BCG Substrains Change Their Outermost Surface as a Function of Growth Media.

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.

Mycobacteria-Based Vaccines as Immunotherapy for Non-urological Cancers.

The arsenal against different types of cancers has increased impressively in the last decade. The detailed knowledge of the tumor microenvironment enables it to be manipulated in order to help the immune system fight against tumor cells by using specific checkpoint inhibitors, cell-based treatments, targeted antibodies, and immune stimulants. In fact, it is widely known that the first immunotherapeutic tools as immune stimulants for cancer treatment were bacteria and still are; specifically, the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) continues to be the treatment of choice for preventing cancer recurrence and progression in non-invasive bladder cancer. BCG and also other mycobacteria or their components are currently under study for the immunotherapeutic treatment of different malignancies. This review focuses on the preclinical and clinical assays using mycobacteria to treat non-urological cancers, providing a wide knowledge of the beneficial applications of these microorganisms to manipulate the tumor microenvironment aiming at tumor clearance.

Each Mycobacterium Requires a Specific Culture Medium Composition for Triggering an Optimized Immunomodulatory and Antitumoral Effect.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the first treatment option for non-muscle-invasive bladder cancer (BC) patients. In research laboratories, M. bovis BCG is mainly grown in commercially available media supplemented with animal-derived agents that favor its growth, while biomass production for patient treatment is performed in Sauton medium which lacks animal-derived components. However, there is not a standardized formulation of Sauton medium, which could affect mycobacterial characteristics. Here, the impact of culture composition on the immunomodulatory and antitumor capacity of M. bovis BCG and Mycolicibacterium brumae, recently described as efficacious for BC treatment, has been addressed. Both mycobacteria grown in Middlebrook and different Sauton formulations, differing in the source of nitrogen and amount of carbon source, were studied. Our results indicate the relevance of culture medium composition on the antitumor effect triggered by mycobacteria, indicating that the most productive culture medium is not necessarily the formulation that provides the most favorable immunomodulatory profile and the highest capacity to inhibit BC cell growth. Strikingly, each mycobacterial species requires a specific culture medium composition to provide the best profile as an immunotherapeutic agent for BC treatment. Our results highlight the relevance of meticulousness in mycobacteria production, providing insight into the application of these bacteria in BC research.

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

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.

Bacillus Calmette-Guérin (BCG) Therapy for Bladder Cancer: An Update.

Physicians treating patients affected by nonmuscle-invasive bladder cancer (NMIBC) have been in shock during the last six years since manufacturing restrictions on the production of the first-option medicine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), have resulted in worldwide shortages. This shortage of BCG has led to a rethinking of the established treatment guidelines for the rationing of the administration of BCG. Some possible schedule modifications consist of a decrease in the length of maintenance treatment, a reduction in the dose of BCG in intravesical instillations or the use of different BCG substrains. All these strategies have been considered valuable in times of BCG shortage. In addition, the lack of availability of BCG has also led to the general recognition of the need to find new treatment options for these patients so that they are not dependent on a single treatment. Few alternatives are committed to definitively replacing BCG intravesical instillations, but several options are being evaluated to improve its efficacy or to combine it with other chemotherapeutic or immunotherapeutic options that can also improve its effect. In this article, we review the current state of the treatment with BCG in terms of all of the aforementioned aspects.

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

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.