Article Mycolicibacterium brumae is a Safe and Non-Toxic Immunomodulatory Agent for Cancer Treatment Marc Bach-Griera 1 , Víctor Campo-Pérez 1,2 , Sandra Barbosa 3,4 , Sara Traserra 3,4 , Sandra Guallar-Garrido 1 , Laura Moya-Andérico 2 , Paula Herrero-Abadía 1 , Marina Luquin 1 , Rosa Maria Rabanal 5 , Eduard Torrents 2,6 and Esther Julián 1, * 1 Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; marc.bach@uab.cat (M.B.-G.); victor.campo@uab.cat (V.C.-P.); sandra.guallar@uab.cat (S.G.-G.); paula.herrero@uab.cat (P.H.-A.); marina.luquin@uab.cat (M.L.) 2 Bacterial Infections and Antimicrobial Therapies group, Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain; lmoya@ibecbarcelona.eu (L.M.-A.); etorrents@ibecbarcelona.eu (E.T.) 3 Department of Cell Biology, Physiology and Immunology, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; sandra.barbosa@uab.cat (S.B.); sara.traserra@uab.cat (S.T.) 4 Integrated Services of Laboratory Animals, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 5 Unitat de Patologia Murina i Comparada, Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; rosa.rabanal@uab.cat 6 Microbiology Section, Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, 643 Diagonal Ave., 08028 Barcelona, Spain * Correspondence: esther.julian@uab.cat; Tel.: +34-93-581-4870  Received: 13 March 2020; Accepted: 22 April 2020; Published: 25 April 2020  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: bladder cancer; nontuberculous mycobacteria; BCG; safety; Galleria mellonella; mice 1. Introduction Bladder cancer (BC) currently represents the second most common malignancy of the urinary tract [1]. In 2018, approximately 549,393 newly diagnosed cases and 199,922 deaths from bladder cancer were reported worldwide [2]. Most BC cases are diagnosed when patients present with macroscopic Vaccines 2020, 8, 198; doi:10.3390/vaccines8020198 www.mdpi.com/journal/vaccines Vaccines 2020, 8, 198 2 of 18 hematuria, and confirmed after transurethral resection of bladder tumor (TURBT) [3]. In approximately 75% of BC patients, the disease is localized in the mucosa layer (Ta or carcinoma in situ [CIS]) or submucosa layer (T1) [1,4]. In these initial stages, known as non-muscle-invasive BC (NMIBC), intra-vesical/intra-bladder (IB) instillations of live Mycobacterium bovis Bacillus Calmette–Guérin (BCG) are still the gold standard treatment to avoid recurrence and progression of the disease [3,5]. BCG comprises a family of related sub-strains derived from a virulent strain of M. bovis, the causative agent of bovine tuberculosis. BCG was generated after eleven years of continuous culture passages, demonstrating, first in animal models and later in humans, in 1921, that it conferred protection against tuberculosis infection [6,7]. In the mid-1970s, Morales and his colleagues reported the efficacy of BCG in the control of recurrence and progression of NMIBC [8], and after several clinical trials, BCG was finally approved by the Food and Drug Administration (FDA) (in 1990) for the treatment of NMIBC [9–11]. The mechanism of action of BCG, as well as the best practice management, is still not completely understood. Nevertheless, the data support that the anti-tumor effects of BCG are due to an inhibition of tumor proliferation through infection of urothelial cells, a powerful local immune response involving the activity of neutrophils, macrophages, and dendritic cells, as well as the release of different cytokines such as Interleukin (IL)-1, IL-2, IL-6 and IL-8, Tumor Necrosis Factor (TNF)-α, and Interferon (IFN)-γ [1,12,13]. Despite its demonstrated efficacy, BCG treatment presents with local adverse effects in more than 50% of patients, systemic adverse effects in around 30% of patients, and 1% to 5% of patients suffer rare but severe complications such as pulmonary infections or sepsis [14–17]. In fact, after transurethral resection of the tumor, there must be a delay of around two weeks before patients can proceed to IB BCG instillations, in order to avoid the systemic dissemination of the bacilli through the wound. Nevertheless, the high number of instillations during the induction and maintenance treatment (up to three years) assume a risk of infection whenever traumatic instillation occurs. In order to avoid the possibility of BCG infection, different strategies involving non-pathogenic bacteria different from mycobacteria, or mycobacteria-derived agents, are currently under investigation [18–21]. The majority of these strategies consider the use of heat-killed or γ-irradiated mycobacteria, or fractions of mycobacteria cells. However, different studies have highlighted the importance of the use of live mycobacteria (at least at the first instillation) to achieve an adequate response against bladder tumors [22,23]. An interesting option to substituting BCG could be the use of non-infectious mycobacteria. Studies from our laboratory using this alternative demonstrated the anti-tumor and immunostimulatory capacity of Mycolicibacterium brumae (basonym Mycobacterium brumae) [Appendix A], a saprophytic mycobacterium with a rapid growth rate in vitro. M. brumae inhibits BC cell proliferation in vitro, triggers a cytotoxic profile in stimulated peripheral mononuclear blood cells against BC cells ex vivo, and increases survival of tumor-bearing mice by triggering both an IB and a systemic immune response that leads to tumor clearance [5,24,25]. Until now, no infections due to M. brumae in humans, other animals, or plants have been described. Both bladder tumor cells and macrophages can eliminate viable M. brumae in a few days after infection whereas BCG remains viable inside the cells [24,25]. Moreover, no colony forming units (CFU) have been isolated in tumor-bearing mice treated intravesically with M. brumae, suggesting the lack of pathogenicity of this species. However, no safety or toxicity studies have been undertaken. In this study, immunosuppressed and immunocompetent mice, after intravenous (IV) and IB administration, as well as the Galleria mellonella model, were used to assess the safety and potential toxicity of live and γ-irradiated M. brumae. 2. Materials and Methods 2.1. Bacterial Cultures M. bovis BCG Connaught (ATCC 35745) and M. brumae (ATCC 51384T ) were grown on Middlebrook 7H10 medium (Difco Laboratories, Detroit, Michigan) supplemented with 10% McFarland turbidity standard as previously described [26] in order to get the required concentration for each treatment. To confirm the dose of each experiment, representative bacterial suspensions of each treatment were serially diluted in PBS, and CFU were counted after plating on Middlebrook 7H10 medium as previously described [27]. Irradiation of M. brumae for intravenous infection and intravesical Vaccines 2020, 8,treatment 198 was performed at Aragogamma S.L. as previously described [5]. 3 of 18 2.2. Intravenous (IV) and Intravesical (IB) Mice Treatments Oleic-Acid-Dextrose-Catalase (OADC) enrichment for 3 or 1 week, respectively, at 37 °C. BCG and Animal experiments were performed according to procedures approved by the Ethics M. brumae cells were resuspended in sterile phosphate buffered saline (PBS) and adjusted to a 1.0 Committee on Animal Experiments (CEEA) of the Universitat Autònoma de Barcelona (UAB) and the McFarland turbidity standard as previously described [26] in order to get the required concentration Commission of Animal Experimentation of Generalitat de Catalunya (UAB procedure numbers: 3632 for each treatment. To confirm the dose of each experiment, representative bacterial suspensions of and 3633; and Generalitat de Catalunya procedure number: 9528). The minimum number of animals each treatment were serially diluted in PBS, and CFU were counted after plating on Middlebrook was used in compliance with current regulations. The welfare of the animals was considered in terms 7H10 medium as previously described [27]. Irradiation of M. brumae for intravenous infection and of number and extent of procedures to be performed. Before starting the experiment, mice were intravesical treatment was performed at Aragogamma S.L. as previously described [5]. randomized according to their weight. The animals were weighed and evaluated on a daily basis for theirIntravenous 2.2. general appearance, clinical (IB) (IV) and Intravesical signs, Miceand behavior, and were euthanized in order to avoid Treatments unnecessary suffering. Animal experiments were performed according to procedures approved by the Ethics Committee The IV infection was carried out in 8-week-old C.B-17/IcrHan®Hsd-Prkdcscid (severe combined on Animal Experiments (CEEA) of the Universitat Autònoma de Barcelona (UAB) and the Commission immunodeficient (SCID) mice) (Envigo) (8 female mice/group) [28,29]. Mice were IV treated via the of Animal Experimentation of Generalitat de Catalunya (UAB procedure numbers: 3632 and 3633; lateral tail vein with a dose of 2 × 106 mycobacteria CFU of live M. brumae, γ-irradiated M. brumae, or and Generalitat de Catalunya procedure number: 9528). The minimum number of animals was used in live BCG in 0.1 mL of PBS, or 0.1 mL of PBS as a control. At 15 weeks, the surviving mice were compliance with current regulations. The welfare of the animals was considered in terms of number sacrificed by exsanguination under terminal inhalation anesthesia for a complete systematic necropsy and extent of procedures to be performed. Before starting the experiment, mice were randomized and histopathology assessment. Collected blood was processed for biochemistry and hematology according to their weight. The animals were weighed and evaluated on a daily basis for their general parameters, and the main mycobacteria-target organs (liver, lung and spleen) were removed for appearance, clinical signs, and behavior, and were euthanized in order to avoid unnecessary suffering. quantitation of CFU and/or histopathology analysis (schedule is shown in Figure 1A). The IV infection was carried out in 8-week-old C.B-17/IcrHan®Hsd-Prkdcscid (severe combined The evaluation of safety and toxicity of M. brumae after repeated IB administrations was immunodeficient (SCID) mice) (Envigo) (8 female mice/group) [28,29]. Mice were IV treated via the performed in 8-week-old BALB/c OlaHsd (Envigo) mice (8 female mice/group) [25,30]. A dose of 2 × lateral tail vein with a dose of 2 × 106 mycobacteria CFU of live M. brumae, γ-irradiated M. brumae, 106 CFU/mouse (low dose) or 2 × 109 CFU (high dose) of live or γ-irradiated M. brumae in 0.1 mL PBS, or live BCG in 0.1 mL of PBS, or 0.1 mL of PBS as a control. At 15 weeks, the surviving mice were or PBS alone for control group, was administered through IB instillations (dwell time 60 minutes) [5]. sacrificed by exsanguination under terminal inhalation anesthesia for a complete systematic necropsy Animals were treated weekly for 6 weeks (Figure 1B), and were euthanized two weeks after the last and histopathology assessment. Collected blood was processed for biochemistry and hematology treatment. A complete systematic necropsy was undertaken. Lung, spleen, liver, and kidney were parameters, and the main mycobacteria-target organs (liver, lung and spleen) were removed for aseptically removed to quantify CFU. Urinary bladders were also preserved for histopathology quantitation of CFU and/or histopathology analysis (schedule is shown in Figure 1A). analysis. Figure 1.1.Schematic Figure Schematicschedule scheduleof of (A)(A) intravenous intravenous infection infection in severe in severe combined combined immunodeficient immunodeficient (SCID) (SCID)(B) mice, mice, (B) intravesical intravesical treatments treatments in BALB/c, in BALB/c, and (C)and (C) intrahemacoelic intrahemacoelic infection infection in G. mellonella in G. mellonella larvae. larvae. Abbreviations: Abbreviations: PBS, phosphate PBS, phosphate bufferedbuffered saline; saline; BCG, BCG, Bacillus Bacillus Calmette–Guérin. Calmette–Guérin. The 2.3. G. evaluation mellonella Model and toxicity of M. brumae after repeated IB administrations was of safety Infection performed in 8-week-old BALB/c OlaHsd (Envigo) mice (8 female mice/group) [25,30]. A dose of 2 ×G.10mellonella larvae(low 6 CFU/mouse were reared dose) or at 2 ×3410ºC9 and CFUfed an artificial (high dose) ofdiet liveconsisting of cornM. or γ-irradiated flour, wheat brumae in flour, powdered milk, cereals, brewer’s yeast, honey, and glycerol. Larvae were infected 0.1 mL PBS, or PBS alone for control group, was administered through IB instillations (dwell time through an intrahemacoelic 60 minutes) [5]. injection Animals with wereatreated Hamilton 22-gauge weekly for 6 syringe using three weeks (Figure 1B), different and wereconcentrations euthanized twoof M. brumae and BCG corresponding to 104, 105, and 106 CFU/larva in 0.01 mL of PBS prepared as weeks after the last treatment. A complete systematic necropsy was undertaken. Lung, spleen, liver, described and kidneyabove. were Fifteen larvae aseptically were used removed for eachCFU. to quantify group, and thebladders Urinary experiment werewas alsoperformed preservedfour for histopathology analysis. 2.3. G. mellonella Infection Model G. mellonella larvae were reared at 34 ºC and fed an artificial diet consisting of corn flour, wheat flour, powdered milk, cereals, brewer’s yeast, honey, and glycerol. Larvae were infected through an Vaccines 2020, 8, 198 4 of 18 intrahemacoelic injection with a Hamilton 22-gauge syringe using three different concentrations of M. brumae and BCG corresponding to 104 , 105 , and 106 CFU/larva in 0.01 mL of PBS prepared as described above. Fifteen larvae were used for each group, and the experiment was performed four times (n = 60). A group of fifteen larvae injected with PBS was also included in each experiment as a control group (Figure 1C). 2.4. Mycobacteria CFU Counting in Different Organs from Mice and G. mellonella Hemolymph Lungs, spleens, and livers from IV-treated mice were weighed and imaged after collection. A portion of spleen, liver, and upper right lung lobe from each mouse was then weighed and processed for CFU quantification. Furthermore, kidneys from mice subjected to IB instillations were also analyzed, since in the context of IB instillations, kidneys are another critical organ due to its direct contact to the bladder lumen. Lungs and livers were resuspended in 1mL of PBS and disrupted using glass homogenizers, while spleens were resuspended in 5 mL of Roswell Park Memorial Institute medium (RPMI, Lonza, Switzerland), and disrupted using forceps and by passing through a needle. Afterwards, serial dilutions from the homogenates were cultured on 7H10 agar plates. Plates were incubated at 37 ◦ C for 1 week or 3 weeks for M. brumae or BCG, respectively, in order to count CFU. In addition, hemolymph from surviving mycobacteria-treated larvae at 144 h post-infection was collected and cultivated on 7H10 agar plates in order to count CFU. Data reported represent the mean ± SD of CFU/g for each organ and CFU/mL from hemolymph of larvae. 2.5. Determination of Biochemical and Hematological Parameters Blood samples were obtained through a cardiac puncture from IV-infected and IB-inoculated mice. Blood was divided into two different tubes: an EDTA-containing tube for hematology analysis and a lithium heparin-containing tube for the biochemical analysis. Plasma was obtained from the blood of lithium heparin tubes by centrifugation (15 min, 3500 rpm/1200× g, 4 ◦ C, Heraeus Megafuge 11R) and stored at −80 ◦ C until biochemical analysis. A broad range of biochemical and hematology parameters were analyzed using Olympus AU400 chemistry and ADVIA 120 (Siemens®) hematology analyzers, respectively. Biochemistry analysis included the quantification of alanine transaminase (ALT), aspartate transaminase (AST), creatinine, iron, alkaline phosphatase, glucose, potassium, total proteins, sodium, urea, and albumin. For the hematology analysis, whole blood was immediately processed for the white blood cell counts (WBCB), red blood cell counts (RBC), hematocrit, platelets, total neutrophils, total lymphocytes, total basophils, total eosinophils, and total monocytes. 2.6. Histopathological Analysis in Mouse Organs At necropsy, the representative tissues of all systems from IV-infected mice and urinary bladders from IB-inoculated mice were immediately fixed in 4% formaldehyde and embedded in paraffin blocks. Hematoxylin and eosin staining was performed on 2–3 µm sections of all specimens. Histopathology assessment included the analysis of stomach, duodenum, pancreas, jejunum, ileum, cecum, colon, lymph nodes, liver, spleen, ovary, uterus, vagina, kidneys, adrenal glands, urinary bladder, salivary glands, submandibular lymph nodes, esophagus, trachea, lung, heart, thymus, brain, sciatic nerve, skin, muscle, aorta, and bone marrow in IV-infected mice, and the urinary bladders of IB-inoculated mice. Representative images were taken from all processed specimens. Histological damage was scored as follows: 0, no apparent lesions; 1, very slight lesions; 2, mild lesions; 3, moderate lesions; 4, intense lesions. 2.7. Statistical Analysis The statistical significance of CFU counts and histopathology analysis (organs from IV-infected mice and urinary bladders from IB-inoculated mice) was assessed using the non-parametric one-way analysis of variance by ranks Kruskal–Wallis H test. Mean values ± standard deviation (SD) of weight gain and weight loss were compared between different treatment groups using two-way ANOVA. Vaccines 2020, 8, 198 5 of 18 Body weight, weight of organs, hematology, and biochemistry results were expressed as mean ± SD Vaccines and were 2020,compared 8, x 5 of 18 using one-way ANOVA. Mouse and G. mellonella survival was analyzed using Log–Rank (Mantel–Cox) test and plotted as Kaplan–Meier curves. Significance was considered as