ONCOIMMUNOLOGY 2022, VOL. 11, NO. 1, e2051845 (15 pages) https://doi.org/10.1080/2162402X.2022.2051845 ORIGINAL RESEARCH Mycobacterial surface characters remodeled by growth conditions drive different tumor-infiltrating cells and systemic IFN-γ/IL-17 release in bladder cancer treatment Sandra Guallar-Garrido a, Víctor Campo-Pérez a,b, Míriam Pérez-Trujillo c, Cecilia Cabrera d, Jordi Senserrich d , Alejandro Sánchez-Chardi e,f, Rosa Maria Rabanal g, Elisabet Gómez-Mora d, Estela Noguera-Ortega a*, Marina Luquin a, and Esther Julián a a Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain; bBacterial Infections and Antimicrobial Therapies group, Institute for Bioengineering of Catalonia (IBEC), Barcelona 08028, Spain; cServei de Ressonància Magnètica Nuclear i Departament de Química, Facultat de Ciències i Biociències, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain; dAIDS Research Institute IrsiCaixa, Institut de Recerca en Ciències de la Salut Germans Trias i Universitat Autònoma de Barcelona, Badalona, 08916, Spain; e Servei de Microscòpia, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain; fDepartament de Biologia Evolutiva, Ecologia i Universitat de Barcelona, Barcelona 08028, Spain; gUnitat de Patologia Murina i Universitat Autònoma de Barcelona, Bellaterra 08193, Spain ABSTRACT ARTICLE HISTORY The mechanism of action of intravesical Mycobacterium bovis BCG immunotherapy treatment for bladder Received 18 October 2021 cancer is not completely known, leading to misinterpretation of BCG-unresponsive patients, who have Revised 7 February 2022 scarce further therapeutic options. BCG is grown under diverse culture conditions worldwide, which can Accepted 7 March 2022 impact the antitumor effect of BCG strains and could be a key parameter of treatment success. Here, BCG KEYWORDS and the nonpathogenic Mycobacterium brumae were grown in four culture media currently used by Innate-lymphoid cells; innate research laboratories and BCG manufacturers: Sauton-A60, -G15 and -G60 and Middlebrook 7H10, and immune response; non- used as therapies in the orthotopic murine BC model. Our data reveal that each mycobacterium requires muscle invasive; BCG; lipid; specific culture conditions to induce an effective antitumor response. since higher survival rates of tumor- tumor microenvironment 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 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-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. CONTACT Esther Julián esther.julian@uab.cat Departament de Genètica i Departament de Genètica i, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain *Present address: Center for Cellular Immunotherapies and, Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Supplemental data for this article can be accessed on the publisher’s website. © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. e2051845-2 S. GUALLAR-GARRIDO ET AL. Background High-risk non-muscle invasive bladder cancer (NMIBC) is the of BC. We further analyzed the local and systemic immune most frequent malignant disease in the urothelial tract.1 The response and the lipidome of each mycobacterial strain, current treatment consists of surgical resection of the tumor enabling us to relate the antigenic profile to the antitumor followed by weekly intravesical instillations of Mycobacterium effect. bovis bacillus Calmette-Guérin (BCG), the tuberculosis vaccine. This treatment is efficacious in more than 70% of patients, pre­ venting recurrence and progression of the disease.2 Although BCG Material and methods treatment is generally well tolerated, both local and systemic Bacterial strains and cell lines complications have led to new research approaches as substitutes for BCG instillations.3 Among these alternatives, nonpathogenic M. brumae (ATCC 51384), and M. bovis BCG (Connaught mycobacteria such as Mycobacterium brumae have demonstrated strain) were grown in Middlebrook 7H10 agar (Difco antitumor effects. M. brumae inhibits bladder tumor cells prolif­ Laboratories, Surrey, UK) supplemented with 10% oleic- eration at the same time that triggers an antitumor immune albumin-dextrose-catalase enrichment medium for 1 and response both in ex vivo experiments and in the orthotopic murine 4 weeks, respectively. Mycobacteria grown in solid media model of the disease.4,5 M. brumae have also been demonstrated to were used to inoculate bottles with Middlebrook 7H9 med­ be safer than BCG in different animal models.6 Snapshots from in ium (Difco Laboratories) supplemented with 10 % albumin- vitro studies using bladder cancer (BC) cells, ex vivo studies using dextrose-catalase or different Sauton culture.11 L-asparagine immune cells, and studies of BC patients and orthotopic BC (A) or L-glutamate (G), and different glycerol concentra­ animal models have demonstrated that mycobacteria can interact tions (15 or 60 mL/litre) defines the different Sauton media with both BC cells and immune cells through different mechan­ used in this study (A60, G15 and G60). Macroscopic isms. The subsequent tumor infiltration by different immune cell appearance shown in (Supplementary Figure. 1). populations, ranging from macrophages and granulocytes to effec­ The mouse high-grade BC cell line MB49 were maintained tor T cells at the last instillations results in an effective antitumor in Dulbecco’s modified Eagle’s medium (DMEM) with stable response. The effect of mycobacteria-induced immunotherapy L-glutamine (Gibco BRL) and supplemented with 10% fetal translates successful treatment of a high percentage of NMIBC bovine serum (FBS, Lonza, Basel, Switzerland), containing patients. However, the effective and precise BCG-induced path­ 100 U/mL penicillin G (Laboratorios ERN, Barcelona, Spain) ways are still unknown, and they are needed to know whether and 100 g/mL streptomycin (Laboratorio Reig Jofré, Barcelona, other mycobacteria can play a major role as a therapeutic Spain), completed media. treatment. Many factors have been described as possible reasons for the failure of BCG treatment in almost one-third of treated patients, Orthotopic model of bladder cancer and intravesical for instance: the stage of the disease at the time of diagnosis,7 the treatment heterogenicity of the tumor,8 the genetic background and the Animal experiments were approved by the Animal Care immunological status of the patient,9,10 and intolerance to BCG Committee at the Autonomous University of Barcelona and exe­ instillations.11,12 However, very few studies have investigated cuted according to. The orthotopic model of BC in mice was whether bacterial characteristics themselves influence the success performed following the schedule shown in Figure 1A.4 Briefly, 8 of treatment. BCG is an attenuated species derived originally from and 10 randomized C57Bl/6 female mice (6 to 8-week-old, Charles pathogenic Mycobacterium bovis. Its distribution and mainte­ River Laboratories, Barcelona, Spain) per group were used to study nance by subculturing in different laboratories worldwide created the triggered immune response and survival, respectively. genetically different substrains with different biochemical Animals were maintained in a supine position to per­ characteristics.13 The mycobacterial antigenic profile may be cru­ form the experiment. The bladder was emptied and chemi­ cial when mycobacteria are used for the treatment of NMIBC. cal lesions on the urothelium of the bladder were induced These features can differ not only since the existence of several by an intravesically instillation of 50 µL of Poly-L-Lysine BCG substrains but also because these strains are manufactured solution (PLL, Sigma) through a 24-gauge catheter for under different culture conditions that can affect the antigenic 15 min. After pouring out intravesical PLL, tumor was profile. We have recently shown in in vitro experiments that induced to randomized animals by intravesically instilling cytokine production triggered by BCG or M. brumae differs 100 µL of DMEM without FBS containing 105 MB49 cells. depending on the culture medium in which mycobacteria is Finally, the bladder was emptied, and animals recovered grown.11 The impact of culture conditions on BC treatment in from the anaesthesia. The day after tumour induction vivo is unknown, and the antigenic profile that is modified, if any, (day 1), mice were anaesthetized and treated with myco­ is also unidentified. bacteria grown in the different culture media. Accordingly, To address how BC immunotherapy treatment is influenced animals received 100 µL of PBS, 100 µL of PBS containing by mycobacteria culture conditions, we studied the antitumor 2 × 107 CFU of M. brumae, or 100 µL of PBS containing effect triggered by M. brumae and BCG grown in four different 2 × 106 CFU of M. bovis BCG grown on different culture culture media currently used by research laboratories or man­ media. Mycobacteria dose was previously optimized.4 ufacturers for BCG production in an orthotopic murine model Treatments were repeated weekly on day 8, 15 and 22, for ONCOIMMUNOLOGY e2051845-3 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. a total of 4 treatments. A negative (healthy mice) and a experiment, all animals were housed with a maximum of six positive (non-treated tumour-bearing mice) control group animals per cage with food and water ad libitum in an enriched were used in all the experiments. Healthy mice received the environment, in a Biosafety Level (BSL) 2 facility. Animal PLL instillation, but PBS was used instead of either BC behavior and well-being were evaluated daily to avoid unne­ tumour cells or mycobacteria treatments. Non-treated cessary suffering and animals were euthanized when it was tumour-bearing mice received PLL and BC tumour cells, required. To analyze the immune response, animals were sacri­ but PBS was used for the intravesical treatments. ficed at day 30, blood was collected by cardiac puncture and Mycobacteria suspension was prepared as previously spleens and bladders removed in aseptic conditions. In survival reported.11 Briefly, mycobacteria from pellicles were recovered analyses, surviving animals were sacrificed at day 60, and using a sterile Kölle handle, dried under the hood and transferred bladders and spleens were collected in aseptic conditions and to a sterile glass tube containing 3 mL of phosphate-buffered saline processed for histopathological evaluation and CFU counting.4 (PBS) and glass beads to disaggregate mycobacteria clumps. Then, For histopathologic analyses, bladders were collected and supernatant was adjusted to McFarland 1 and centrifuged at 1640 g immediately fixed in formaldehyde 4%. Then, hematoxylin- for 10 min. Pelleted bacteria were resuspended in PBS to achieve eosin staining was performed in 2–3 µm-sections of embedded the final concentration. Inoculums were corroborated by platting bladders in paraffin. Representative images were obtained from the solution in 7H10 Middlebrook plates. CFU/mL were obtained all conditions for histopathologic analyses. after incubating M. bovis BCG plates at 37°C for three weeks and M. brumae plates for one week. Restimulation and cytotoxic capacity of splenocytes All experiments were carried out under inhaled anaesthesia, 1.5–2% isoflurane in pure O2 and eyes protected with liquid pleens were disrupted with tweezers. Homogenized splenocytes ophthalmic gel. Tumour implantation was ensured from day 7 diluted in DMEM complete media were added on 96-well tissue to 10 due to the presence of blood in urine. During the culture plates (Thermo Scientific) containing 105 MB49 cells (25:1, e2051845-4 S. GUALLAR-GARRIDO ET AL. splenocyte:tumor cell). After incubation for 24 h at 37°C contain­ To analyze the total mycobacterial lipidic composition, cells ing 5% of CO2, wells were vigorously washed with warm PBS and recovered from four independent cultures of each strain in each MTT was performed to measure MB49 cells viability.4 Splenocytes culture medium were exposed to chloroform/methanol suspen­ adjusted to a final concentration of 3 × 106 cell/mL were also sions as described before.15 Lipidic extracts were analysed by thin- seeded to evaluate proliferation upon restimulation. Accordingly, layer chromatography (TLC) on silica gel-coated plates (G-60, 180 µL of splenocyte suspension was restimulated with 1 mg/mL of Merck, Germany) using chloroform/methanol (96:4, v/v) or PE heat-killed mycobacteria grown in each medium within 20 µL. (60–80°C b.p.)/diethyl ether (90:10, v/v). TLC were revealed by PBS was used as negative control, and 5 µg/mL of Concanavalin A using phosphomolybdic acid (VWR, USA; 10% in ethanol) fol­ (ConA, Sigma) as positive control. After 72 hours of incubation lowed by heating the plate at 120°C. One representative TLC for plates were centrifuged, supernatants collected and MTT assay each condition is shown in the Results section. performed in pelleted splenocytes. IFN-ɣ, Interleukin (IL)-4 and IL-17 cytokines were mea­ Lipid purification and analysis sured in supernatants by using commercially available ELISA tests (Mabtech AB, Nacka Strand, Sweden). Purification of spots 1, 2, 3 and 4 from two independent lipidic extracts was performed as previously described with slightly modifications.16 Briefly, lipid extract resuspended in chloro­ IgG antibody detection in sera form was added to a Silica Gel 60 (Merck, Germany) column stabilized with PE (60–80°C b.p). Increasing diethyl ether con­ Blood was centrifugated and sera frozen until use. To analyze centrations were used for spots elution. Fractions containing the presence of total IgG in sera, the bottom of 96-well plate the eluted spots were dried and used for nuclear magnetic wells was coated with 20 µg/mL of heat-killed-M. brumae or resonance (NMR) analyses. Purification of spots A, B and C heat-killed-BCG, grown in the different media. ELISA proce­ were done as 1, 2, 3 and 4 spots, yet using chloroform to dure was performed as described before.5 Blank wells consist stabilize the column and increasing methanol concentrations on sera without antigen and its absorbance was deducted to to elucidate the spots. wells with sera and antigen for each animal. For the NMR analysis each purified compound was dis­ solved in 600 µL of CDCl3 (99.80% D, Cortecnet, Voisins-le- Bretonneux, France). A Bruker Avance II 600 NMR spectro­ Detection of infiltrated immune cell subsets into the meter (Bruker Biospin, Rheinstetten, Germany) equipped with bladders a 5 mm TBI probe operating at a 1H and 13C NMR frequencies The immune infiltrate present into the bladder was analyzed by of 600.13 and 150.90 MHz, respectively, was used. All experi­ flow cytometry as previously described.5 Briefly, bladders were ments were performed at 298.0 K. 1D 1H NMR spectra were minced using a scalpel followed by digestion with RPMI medium acquired using a standard 90° pulse sequence, with an acquisi­ supplemented with 5% FBS containing 0.5 mg/mL collagenase II tion time of 1.71 s and a relaxation delay of 2 s. Data were (Sigma, Spain) and 1 U/mL DNAse I at 37°C for two-three collected into 32 K computer data points, with a spectral width successive 30 min cycles, with continuous shaking. The cell sus­ of 9590 Hz and as the sum of 1024 transients. In the case of pension obtained was filtered through a 40-μm disposable plastic quantitative 1D 1H NMR spectrum the relaxation delay was set strainer (Becton & Dickinson) and pelleted for staining. The cells to 15s. 2D NMR experiments 1H,1H-COSY (Correlation were labeled with the following antibodies: PerCP-CD45 APC- Spectroscopy) and 1H,13C-HSQC (Heteronuclear Single Cy7-CD3, FITC-CD4, Alexa 700-CD8, APC-CD62L and BV786- Quantum Coherence) were performed using standard Bruker CD127, PE-Dazzle-CD44 and FITC-CD45R/B220 (Biolegend). pulse sequences and acquired under routine conditions. All The lymphocyte gate was defined by morphological parameters, spectra were calibrated using the residual solvent signal and dead cells were excluded using a live/dead fixable Aqua Dead (CHCl3,δH, 7.26 and δC, 77.0 ppm). Multiplicity of peaks is Cell Stain kit (Invitrogen). Samples were acquired in a Fortessa abbreviated as s (singlet), d (doublet), t (triplet), dd (doublet of flow cytometer (Becton & Dickinson), and the data were analyzed doublets) and m (multiplet). Integration was performed with using FlowJo software (9.8 v; TreeStar, Portland, OR, USA). the global spectral deconvolution (GSD) application of Absolute cell numbers were obtained by using Perfect-Count MestreNova 8 (Mestrelab Research S.L.). Microspheres (Cytognos). Gating strategy is shown in Supplementary Figure 3. Ultrastructure of pellicles Grown pellicles were collected with nucleopore membrane Superficial and total lipid extraction of pellicles (Whatman® Nuclepore™) and placed in aluminum foil struc­ tures incorporated into 6-well plates (Nunc® Thermo After properly growth of four independent cultures of each Scientific™). Then, pellicles were fixed with osmium vapors by strain in each culture media, lipids from the surface of the adding 250 μL/well of 4% osmium tetraoxide (TAAB Lab., UK) mycobacteria pellicles were extracted with petroleum ether and maintained at 4°C overnight. Fixed biofilms were air dried, (PE) (40–60°C b.p.) for 5 min.14 PE extracts were dried placed on stubs and coated with Au-Pd (Emitech Au- under nitrogen stream and analyzed by thin layer chroma­ Metallizer). Pellicle ultrastructure was observed with a second­ tography (TLC) on silica gel-coated plates (G-60, Merck, ary electron detector in a SEM EVO® MA 10 (Zeiss, Germany). Oberkochen, Germany) operating at 15 kV. ONCOIMMUNOLOGY e2051845-5 Neutral red staining of mycobacteria the rest of the conditions (Figure 2A). Regarding cytokine production, a massive IFN-ɣ release triggered by BCG Neutral red staining was performed as previously reported with treatments compared to all M. brumae treatments (one few modifications.17 Briefly, mycobacteria pellicles were fil­ log inferior) was observed (Figure 2B), with M. bovis tered, completely dried under hood, and transferred to a screw- BCG-G15-treated mice having the highest IFN-ɣ produc­ cap tube containing 5 mL of 50% methanol in deionized water. tion. Splenocytes from M. brumae-A60-treated mice Then, cells were mixed and incubated for 1 h at 37°C. Washing exhibited more IFN-ɣ release than those form the other process was repeated twice. Finally, mycobacteria were mixed tested conditions, with a significant difference between M. with 2 mL of 0.002% neutral red (Merck, Germany) in barbital brumae-G60- and M. brumae-7H10-treated mice. In con­ buffer (1% sodium barbital in 5% NaCl [pH 9.8]) and incu­ trast to IFN-ɣ, IL-17 was overproduced in splenocytes bated at room temperature. Results were obtained 1 hour later. from M. brumae-treated mice compared to those from BCG-treated mice. Specifically, IL-17 levels in splenocyte Statistical analysis cultures from M. brumae-A60-treated mice were superior to those in splenocyte cultures from M. brumae-G15-, M. Graphpad Prism 6.0 software (San Diego, CA, USA) was used for brumae-G60- or M. brumae-7H10-treated mice (Figure statistical analyses. Log-rank (Mantel-Cox) test was used to eval­ 2B). Although no significantly different, splenocytes from uate differences between mice survival curses. Ex vivo experiments BCG-G15-treated mice showed slightly higher levels than were analyzed using ANOVA. Quantitative data are expressed as those from the other BCG-treated mice (Figure 2B). IL-4 the mean ± standard deviation (�x � SD). Immune infiltration data was not detected in any condition (data not shown). were analyzed using Mann–Whitney tests, and their correlations When splenocytes from A60-, G15- or G60-treated mice to IL-17 and IFN-ɣ were analyzed by Spearman tests. Statistical were restimulated with mycobacteria grown in 7H10, pro­ significance was assumed at p< 0.05. liferation ratios were lower than those obtained in spleno­ cytes restimulated with mycobacteria grown in the same Results medium used for intravesical treatment (Supplementary Figure. 2A). These results indicate specificity in reactivity Tumor-bearing mouse survival rates depend on against mycobacterial antigens produced in each culture mycobacterial culture medium composition medium. Both the proliferation ratios and IL-17 production The antitumor effect of mycobacteria grown in different cul­ obtained in M. brumae-A60- and BCG-G15-treated mice ture media was studied in the orthotopic murine model of BC again showed the highest values (Supplementary Figure. (schedule shown in Figure 1A). Mycobacteria-treated tumor- 2A and 2B). bearing mice survived statistically longer than untreated mice, When cytotoxity was studied, splenocytes from M. brumae- with survival rates higher for M. brumae-treated mice than for A60-treated mice showed the highest growth inhibition of BCG-treated mice, in agreement with previous studies4 (Figure MB49 cells (reducing up to 30% of cell growth) compared to 1C-D). Notably, M. brumae grown in A60 culture medium (M. splenocytes from the rest of the groups (Figure 2C). Likewise, brumae-A60) prolonged survival in 100% of mice 40 days after splenocytes from the other groups of M. brumae- and BCG- tumor induction and in 89% of mice at the end of the experi­ treated mice had similar cytotoxic effects on MB49 cells ment, being superior to the rest of M. brumae treatments (Figure 2C). (Figure 1C and Supplementary Table 1 and Table 2), although No significant differences in anti-mycobacterial antibodies without statistical significance. For BCG treatments, 70% of in the serum were detected among treatments, but higher mice treated with BCG grown in G15 (BCG-G15) survived production of IgG was detected in M. brumae-treated mice until the end of the study, the most successful treatment than in BCG-treated mice when mycobacteria were grown in among all BCG treatment regimens (Figure 1D and Sauton media (Figure 2D), contrary to the results obtained Supplementary Table 1 and Table 2). Histological examination when mycobacteria were grown in 7H10 (Figure 2D and.[5]). of the urinary bladder showed mild to moderate lymphoplas­ When cross-reactivity was analyzed, IgG levels from serum of macytic infiltrates in the lamina propria, with lymphoid follicle M. brumae-7H10-treated mice were higher than those pro­ formation near the neck of the bladder, in most mycobacteria- duced against M. brumae grown in the culture medium used treated mice. The lymphocytic infiltrates were slightly more for intravesical treatment (Supplementary Figure. 2C). In all intense in BCG-treated mice (Figure 1E). cases, M. brumae-treated mice showed higher antibody levels Pathogenicity was not influenced by culture media, being than BCG-treated mice. BCG present in spleens from BCG-treated mice, and M. bru­ mae absent in M. brumae-treated mice, corroborating its non- Bladder immune cell infiltration differs between BCG and pathogenicity 4,6 (Figure 1B). M. brumae treatments All mycobacterial treatments induced robust infiltration of Culture medium composition influences the different populations of lymphocytes (CD45+, CD3+, CD4+ mycobacteria-triggered systemic immune response and CD8+ T cells, NK cells and B cells) into the bladder Restimulated splenocytes from M. brumae-A60- and BCG- compared to no treatment (Figure 3A). Statistical differences G15-treated mice showed the highest proliferation ratios, among groups are shown in Supplementary Table 3. When which were significantly higher than those observed for comparing both mycobacterial treatments, mice receiving BCG e2051845-6 S. GUALLAR-GARRIDO ET AL. 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). had significantly higher absolute immune infiltration of total by splenocytes (Figure 3C). Remarkably, these correlations CD45+ cells and T, NK and B cells than M. brumae-treated were observed when analyzing only M. brumae-treated animals (Figure 3A). Differences were also observed when mice, not BCG-treated mice. Both populations were comparing the effects of culturing each mycobacterial strain enriched in bladders from M. brumae-A60-treated tumor- in different media. Among all conditions, mycobacteria grown bearing mice in comparison to mice given any other treat­ in G15 induced the highest infiltration levels, while mycobac­ ment, although the differences only reached statistical sig­ teria grown in A60 induced the lowest infiltration levels. nificance for CD4+ TEM cells (Figure 3D). Interestingly, Statistical significance was only reached between animals trea­ although BCG treatment induced stronger tumor immune ted with BCG grown in G15 and A60 for the total numbers of cell infiltration than M. brumae treatment, the frequency of CD45+ lymphocytes and CD4+ T cells, with a trend for the ILCs was significantly higher in M. brumae-treated animals number of infiltrating CD3+ T cells (Figure 3A). In M. brumae- than in BCG-treated animals. In contrast, we did not treated mice, B cell numbers were also significantly increased observe differences in the proportion of CD4+ TEM cells in G15 culture conditions compared to the other culture con­ between BCG- and M. brumae-treated mice when the ditions, and a trend toward differences in NK cell infiltration results for each mycobacteria strain, regardless of culture was also found when comparing G15 and A60 media (Figure medium, were pooled together (Figure 3D). 3A). Remarkably, the amount of IFN-γ secreted by splenocytes from treated mice restimulated with mycobacteria showed strong correlations with the frequencies of CD3+, CD4+, and Culture medium composition modifies the mycobacterial CD8+ T cells and CD4+ effector memory T (TEM) cells within cell-surface lipidomic profile bladder tumors (Figure 3B). As M. brumae treatment induced high IL-17 production The ultrastructural appearance of the outermost layer deci­ by splenocytes upon restimulation (Figure 2B), we sought phered a thin layer covering all bacilli, although growing in to identify whether there is a link between systemic the different culture media (Figure 4A and 5A). However, immune response and local immune infiltration. while M. brumae appears as a prearranged mass in which Frequencies of innate lymphoid cells (ILCs, defined as individual cells can be clearly observed, BCG cultures showed CD3−CD127+ cells) and CD4+ TEM cells in the bladder an extracellular matrix covering the cells, as previously robustly correlated with the levels of this cytokine produced reported.11 ONCOIMMUNOLOGY e2051845-7 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 (TEM) 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+ TEM 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+ TEM 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. Then, the lipidic composition of the superficial layer of showed a slightly different H1a and H1b signal pattern mycobacteria was obtained by a short extraction with PE compared to that shown before.18 These differences could and analyzed by TLC and NMR spectroscopy. Eluting the be due to a change in the solvent used and/or to differences TLC products with chloroform/methanol (96:4, v/v), spot A in the global structure of the molecule (since previous was only detected in M. brumae-A60 and M. brumae-G60 GroMM molecule analysed contains a different mycolate). (Figure 4B). The 1H NMR spectrum of spot A was in However, in the present study, α-GroMM also showed two accordance with that of glycerol monomycolate (GroMM) different H1b signals, indicated as H1b (dd at 4.242 ppm) structures,18 with the mycolate unit being an α-mycolic acid and H1b’ (dd at 4.228 ppm), which are partially overlapped (Figure 4D). Confirmation of this molecule was achieved by (Supplementary Figure 4 and Supplementary Table 4). the performance of 1D and 2D NMR experiments (1D 1H, Additionally, the relative integration of H1a and (H1b 1 H-1H COSY and 1H-13C HSQC), which allowed complete +H1b’) signals was 1.00 to 1.03. This evidence suggested 1 H and 13C NMR characterization of the molecule the presence of a mixture of the R and S diastereoisomers (Supplementary Table 4). Resonances of the glycerol mono­ Eluting the TLC products with PE/diethyl ether (90:10, substituted unit were identified as H1a and H1b at 4.28 and v/v), spots 1, 2, 3, and 4 were observed in M. brumae 4.24 ppm, respectively, H2 at 3.96 ppm, and H3a and H3b grown in all cultures except in the A60 culture. According resonated at 3.75 and 3.63 ppm, respectively. The typical to NMR analyses, spot 1 was identified as having molecular peaks of α-mycolates were identified and were in accor­ structures containing long aliphatic chains and aromatic dance with published studies.19 Among them, olefinic pro­ groups (Supplementary Figure 5), spot 2 was identified as tons of trans double bonds (multiplets at 5.33 and 5.24 having molecules containing long chains with cis and trans ppm from H10 and H11, respectively) and cis double insaturations (Supplementary Figure 6), and spots 3 and 4 bonds (multiplet at 5.35 ppm from H9) were shown. were identified as acylglycerides (AGs). Nevertheless, all Finally, peak integrations were in accordance with the culture conditions permitted the accumulation of AG in structure of α-GroMM. To determine the specific diaster­ deeper layers of the cell wall or the cytoplasm since a eoisomers of α-GroMM of spot A, a more detailed analysis total lipidic extraction of M. brumae clearly showed a of H1a and H1b was carried out. The 1D 1H spectrum spot corresponding to AG (Figure 4C), even in the case e2051845-8 S. GUALLAR-GARRIDO ET AL. 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. of M. brumae-A60, in which AG was absent on the outer­ Moreover, less PGL and almost untraceable phthiocerol most cell surface. Similar lipidic profiles in G15-, G60- or dimycocerosate (PDIM) levels were detected on the surface 7H9-M. brumae were observed (Figure 4B). of BCG-G15 pellicles in comparison with the other BCG Notable differences were observed among lipid extracts conditions (Figure 5B). The analysis of the total lipid from BCG pellicles grown in different culture media (Figure extract corroborated a diminished production of PGL, 5B and 5C). In the A60 and G60 cultures, two spots (B and PDIM and GroMM by BCG-G15 cultures (Figure 5C). C) were highly expressed, while both spots were less pro­ Finally, the physicochemical properties of mycobacteria duced by BCG grown in medium with a low glycerol con­ were studied by neutral red staining of cells. BCG-A60, centration (G15). Remarkably, only spot C was detected in BCG-G60 and BCG-7H10 cells showed a distinctive strong BCG-7H9 (Figure 5B). Spot B and spot C of BCG were pink color but not the intense deep red color produced by identified and fully characterized using 1D and 2D NMR Mycobacterium tuberculosis pathogenic strains.21 This result spectroscopy. Spot B corresponded to phenol glycolipid indicates the presence of mycobacterial compounds with (PGL) (Supplementary Figure 7 and Supplementary Table reducing power, suggesting different physicochemical char­ 5),20 whereas spot C corresponded to a GroMM structure acters of the cell wall in all M. brumae conditions and formed mainly by keto-mycolates (k-GroMM, Figure 5D).18 BCG-G15 cells (Figures 4F and 5E). For spot C, the NMR resonances of the monosubstituted glycerol unit of k-GroMM coincided with those described Discussion before for α-GroMM and typical peaks of the keto group and cis cyclopropane appeared (Figure 5D, Supplementary Our results indicate that mycobacteria grown in different cul­ Figure 8 and Table 4). The integration of signals in a ture media trigger different antitumor activities in an orthoto­ quantitative 1D 1H NMR spectrum showed that the pic murine model of BC. Furthermore, each mycobacterial GroMM molecular structure in spot C was mainly formed strain requires specific culture conditions to induce an effective by keto-mycolates in form of a mixture of R and S diaster­ antitumor response since higher survival rates of tumor- eoisomers (Supplementary Figure 4). bearing mice were achieved using BCG-G15 and M. brumae- ONCOIMMUNOLOGY e2051845-9 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. A60 than the rest of the treatments. The inhibition of bladder directly inhibiting BC cells22 and inducing higher infiltration of tumor growth was accompanied by local and systemic immune neutrophils into the bladder after BCG instillation.23 In M. bru­ responses that also differed between the two species. mae-treated mice but not in BCG-treated mice the production of Outstandingly, M. brumae-A60 was the most efficacious IL-17 correlates positively with the presence of CD127+ ILCs in the among all tested treatments in terms of mouse survival and bladder, whose infiltration is significantly increased, especially in cytotoxic activity of splenocytes against tumor cells. Therefore, M. brumae-A60-treated mice. comparing M. brumae-A60 with BCG treatments provides the ILCs include cytotoxic natural killer (NK) cells and cyto­ possibility to unravel traits of the mechanisms involved in the kine-producing ILCs (helper-like ILC types 1, 2 and 3), which antitumor activity of mycobacteria in vivo. are considered the innate homologs of Th1, Th2 and Th17 After the evaluation of the systemic immune activation, we responses 24,25 and display features of mature effector lympho­ observed that M. brumae-A60 and BCG-G15 triggered the highest cytes. Considering the positive correlation between IL-17 pro­ IL-17 and IFN-γ production, respectively, which was significantly duction and the presence of ILCs, the presence of type 3 ILCs, different from that triggered by the rest of the treatments. The role which produce IL-17 after stimulation, is plausible in M. bru­ of the cytokine IL-17 in cancer immunotherapy is under discus­ mae-treated mice. The roles of these subsets in shaping anti­ sion, as its function can vary depending on the type of cancer. In tumor immunity are the focus of intense study.26–29 In the case BC, high levels of IL-17 in BCG-treated patients have been related of BC, Chevalier et al. analyzed the immune cell distribution in to high survival rates. IL-17 upregulates IL-8 and IL-6 expression, the urine of NMIBC patients during BCG treatment, and ILC2s e2051845-10 S. GUALLAR-GARRIDO ET AL. were found to be the most abundant innate lymphoid cell A60 was unique and showed only GroMM, while BCG-G15 subpopulation present in patients with BCG treatment showed less PDIM, PGL and GroMM located outside of the cell failure.30 The same authors demonstrated that CCR4 may be than the rest of the BCG cultures (Figures 4 and 5). The involved in the recruitment of ILCs in the bladder during BCG presence/absence of each of these lipids might be a determi­ therapy.30 Thus, ILCs are stimulated after mycobacterial treat­ nant of the triggered immune response. ment, and each type of ILC could play a distinct role in BC The first observation was the positive correlation between outcomes. Interestingly, transcriptome cross-species analysis GroMM presence in the outermost layer and a high glycerol showed similarities between mouse ILC1 and ex-ILC3 subsets concentration in the culture medium, as previously described.39 and human CD56bright NK cells,31 which have a relevant role in GroMM constitutes glycerol and a monomycolate acid39,40 and, BC patients treated with BCG.32 Hence, understanding how like other mycolic acid (MA)-derived molecules (such as TDM and which of these subsets are regulated after M. brumae or or TMM), has been related to the cell hydrophobicity,41 persis­ BCG instillation in BC might yield significant insights into the tence and virulence of pathogenic mycobacteria species,42–44 mechanisms of mycobacteria-based therapy. resistance to therapeutic agents45 and immune system interac­ CD4 TEM cells are another immune cell population able to tion. M. brumae presented only α-GroMM, while GroMM produce IL-17, which positively correlates with M. brumae- exposed on BCG corresponded to only keto-mycolic acid, treated mice, in contrast to the negative correlation found in although Connaught BCG contains α- and ƙ-mycolates. The BCG-treated mice. In the case of BCG, CD4 TEM cells were presence of only ƙ-mycolic acid can be explained since its only positively correlated with IFN-ɣ production. In the case of conformation is more stable than that acquired by α-mycolic M. brumae-A60, the IL-17 response was accompanied by sig­ acid.46 GroMM is an immunostimulatory molecule that can be nificant production of IFN-ɣ compared to the rest of the M. recognized by the human innate immune receptor macrophage- brumae treatments. The role of mycobacteria-triggered IFN-ɣ inducible C-type lectin (Mincle), which leads to a proinflamma­ production via the Th1 response has been widely described in tory response including synthesis of NO, modulation of Toll- the resolution of BC.13,30,33 Early works showed the relevance like receptor 2-mediated cytokine responses and release of of the recruitment of CD4 and CD8 T cells to resolve bladder proinflammatory cytokines,47–49 independent of the α- or ƙ- tumors in animal models, and even recombinant BCG expres­ mycolic acid content.50 Although the R-isomer from BCG has sing Th1 cytokines and combinations of BCG therapy plus been described as more reactive than the S-isomer,51 a mixture cytokine addition are currently under study to overcome of both isomers was found in our mycobacterial extracts. BCG unresponsiveness in BC patients.34,35 Remarkably, BCG- Moreover, although initial reports found that GroMM does G15 treatment triggered the highest systemic IFN-ɣ produc­ not bind to the mouse Mincle receptor, in contrast to human tion and massive bladder infiltration of immune cells, mainly Mincle, recent research and preliminary results from our CD3+ (CD4+ and CD8+) T cells, observed by both flow cyto­ laboratory indicate that GroMM can bind to the mouse metry and histological analysis. Overall, systemic IL-17 and Mincle receptor at higher concentrations,52 explaining the IFN-ɣ production together with immune cell infiltration into observed immune response triggered by M. brumae-A60. the bladder indicates that while BCG-G15 antitumor activity Moreover, M. brumae-A60 was specifically devoid of AGs on could be based on massive infiltration together with high IFN-ɣ the cell surface, although the ultrastructural analysis and total release (high systemic and local inflammation), M. brumae- lipidic profile of M. brumae showed the presence of AGs. AG A60 could be more effective in inducing the infiltration of accumulation could occur as peripheral deposits associated with selected immune cells, mainly IL-17 producers, which could the cell envelope and in ILIs,53 both in M. tuberculosis and create an adequate niche to help the host subvert immunosup­ nontuberculous mycobacteria.54,55 In M. tuberculosis, produced pressive tumor actions.33,36 AGs are either transported towards the outer cell wall through Reasonably, the first interaction between intravesically the cytosol or accumulate in the cytosol as ILIs. Overexpression instilled mycobacteria and BC or innate immune host cells of an efflux pump and the membrane lipoprotein LprG/Rv1410 could be the determinant for proper antitumor action. Since is responsible for the release of AG into culture medium,56,57 mycobacteria are characterized by a rich lipid fraction on the enabling the resistance of mycobacteria in the host immune cell surface,37,38 changes in the mycobacterial lipid content environment. The function of surface-exposed AG on nontu­ after growth in each culture medium may be involved in the berculous mycobacteria, such as M. brumae, with host interac­ different antitumor effects observed. tions is still unknown. Experiments with Mycobacterium M. brumae and BCG share the structural skeleton common abscessus indicated that the presence of AG in the cell wall is to all mycobacteria, composed of arabinogalactan, peptidogly­ not associated with excess AG storage in ILIs.58 Nonetheless, it can and mycolic acids, and the presence of some noncovalent could also be hypothesized that AG absence on M. brumae-A60 linked lipids such as PIM, TDM, TMM and TAG that were could favor the interaction of host receptors with PAMPs. present in the cell wall under all culture conditions (Figures 4 Further studies are needed to elucidate the unknown functions and 5). However, the outermost layer is enriched in specific of AG. lipids depending on the culture medium in which they are In BCG-G15, the diminished amounts of PDIM and PGL grown. Here, after a short exposition to an apolar solvent that compared to the amounts found in the rest of the BCG cultures permits the extraction of the outermost lipid molecules,14 a could be a determinant of its efficacy. PDIM and PGL are reduced variety of lipids was strikingly observed in the outer­ present in only a selected group of mycobacteria, such as M. most layer of M. brumae-A60 and BCG-G15 compared to the tuberculosis, Mycobacterium marinum, and some strains of rest of the cultures. Among all M. brumae cultures, M. brumae- BCG.59,60 PDIM is required for M. tuberculosis and M. ONCOIMMUNOLOGY e2051845-11 marinum virulence in mouse and zebrafish models, could be of interest to recover cultured mycobacteria at differ­ respectively,45,61–65 mycobacterial persistence in host tissues ent time points to unravel the role of each lipid profile and mediated by inhibiting phagosome maturation66 and disease decipher the convenient antigenic profile for enhancing a dissemination.67 PDIM interacts with host membranes to desired immunostimulatory activity. Moreover, we have stu­ remodel lipid organization,68–70 interferes with IFN-ɣ- died the BCG Connaught substrain, but the influence of the independent host defense,71 and masks mycobacterial culture medium used for manufacturing other BCG substrains PAMPs, affecting innate immune signaling pathways.72 devoid of PDIM and PGL glycolipids in clinical use is Infection with PDIM-mutant strains induced dendritic cells unknown. Similar to BCG Connaught, other BCG substrains and macrophages to secrete more tumor necrosis factor showed a diminished lipidic content on the outermost cell (TNF)-alpha and interleukin (IL)-6 than infection with the surface when culture in G15 medium.79 Accordingly, another wild-type strain.73 Likewise, PGL has been associated with the interesting point will also be to study different BCG substrains M. tuberculosis hypervirulent phenotype, inhibiting host pro­ to investigate whether these lipids or other types of molecules duction of proinflammatory cytokines such as TNF-α, IL-6 and are specifically responsible for the immune response. To IL-1β.74–76 Moreover, both compounds also play a role in cell understand how mycobacteria helps the immune system wall permeability since mutants lacking PDIM and PGL are could be relevant for the selection of other bacterial species to more sensitive to various hydrophobic and hydrophilic treat patients following the current immunotherapy regimen, drugs.77 In the case of BCG, the presence of PDIM and/or or in combination with new immunotherapy agents such as PGL varies among BCG substrains,78 yet few works associate checkpoint inhibitors.83 their presence with a specific role when using BCG as an Our study had some limitations. First, only female mice immunomodulatory agent. In the context of tuberculosis pro­ were used. Because anatomic characteristics make mycobacter­ tection, PDIM present on the BCG Pasteur substrain cell wall is ial treatment modeling in male mice impractical, differences in needed for complete protection against M. tuberculosis chal­ sex could influence the response to treatment, modifying the lenge in mouse models,76 although a PDIM/PGL-mutant strain results obtained here.84 The MB49 cell line used to induce did not alter the release of IFN-ɣ by CD4 and CD8 T cells tumor in mice generates aggressive bladder tumors compare compared to the wt strain. In the context of BC, the roles of to those treated with mycobacteria in BC patients. Moreover, these antigens are known. Our work demonstrates that the the different immune systems between mice and humans also antitumor effect is enhanced when the amount of these com­ limited our conclusions. The fact that mice are devoid of group pounds is diminished in the BCG cell wall. Although a direct 1 CD1 molecules prevented us from determining whether sur­ relationship between PDIM/PGL presence in BCG substrains face-exposed mycolate-derived antigens have a role in trigger­ and BC growth inhibition in vitro has not been observed,78 the ing the adaptative immune response. After demonstrating here presence of PDIM/PGL could modify the interactions of the surface exposure of some mycolic acid-derived antigens, PAMPs with immune cells or even alter cell permeability, further work with humanized mouse models with those recep­ allowing the release of other antigens with immunomodulatory tors will shed light on these newly generated questions.85 effects. Overall, we have demonstrated that the mycobacterial lipi­ Moreover, the profile of the outermost lipidome can influ­ dome depends on the culture conditions and that these profiles ence the antitumor effect not only via a direct interaction with impact the triggered immune response, both local and sys­ immune receptors but also by altering the physicochemical temic, when using mycobacteria for BC treatment, suggesting properties of the mycobacteria that modify the host- that the mycobacterial lipidome could be one of the factors mycobacteria interaction. Our results show that BCG-G15, in influencing the success of the antitumor effect. Based on the contrast to the rest of the BCG cultures, was not able to take the data described here, we can conclude that the outcome of neutral red stain in its non-ionic hydrophobic form due to a NMIBC depends not only on patient and/or tumor character­ non-adherence on mycobacteria surface and/or the lack of istics but also on mycobacterial characteristics that can be compounds with reducing power on the cells. The variation modulated as a function of the growth conditions. This evi­ on the hydrophobicity and/or charge on the cell surface of dence strengthens the understanding of the mechanism of mycobacteria cells,79 could be an advantage when interacting action of mycobacteria in cancer treatment, providing new with BC cells. In contrast to normal bladder cells, tumor cells insights to improve this approach. contain a diminished amount of sulfated glycosaminoglycan (GAGs), which alter the physicochemical characters of the cells.80 Thus, BCG-G15 could be properly attached to tumor Conclusions cells. Additionally, it is plausible that the exposure of fibronec­ tin-binding proteins involved in the interactions between M. Growth conditions can impact the antitumor effect of BCG brumae or BCG and tumor cells 81 could be altered as a strains and could be a key parameter of treatment success. function of the mycobacterial surface lipidome. Tumor-bearing mouse survival, the splenocyte antitumor In our experiments, mycobacteria were meticulously har­ effect, and immune cell infiltration into tumors in mycobac­ vested at the same time from all mycobacterial cultures to teria-treated mice differed as a function of the culture condi­ assess the influence of the culture conditions on the mycobac­ tions for each mycobacterial strain. Our data reveal that the terial antitumor effect. However, the lipidic profile is built in a mycobacterial surface is modified by culture conditions, sug­ time-dependent manner as a function of the environmental gesting an influence on the antitumor immune response trig­ and nutritional statuses of the cells.82 In view of our results, it gered. These findings contribute to understanding the e2051845-12 S. GUALLAR-GARRIDO ET AL. antitumor mechanism of mycobacteria, allowing improved Availability of data and materials management of BC patients. All data generated or analysed during this study are included in this published article [and its supplementary information files]. List of abbreviations Disclosure statement BC, Bladder Cancer BCG, Mycobacterium bovis bacillus Calmette-Guérin No potential conflict of interest was reported by the author(s). BSA, Bovine Serum Albumin CDCl3, Deuterate chloroform CFU, Colony Forming Units Funding CO2, Carbon dioxide ConA, Concanavalin A This work was funded by the Spanish Ministry of Science, Innovation and DMEM, Dulbecco’s modified Eagle’s medium Universities grant RTI2018-098777-B-I00 (EJ) and FEDER Funds (EJ), ELISA, Enzyme-linked Immunosorbent Assay Generalitat of Catalunya grant 2017SGR-229 (EJ, ML), Generalitat de FBS, Fetal Bovine Serum Catalunya PhD contracts FI (SG-G, VC-P). GAG, Glycosaminoglycans GroMM, Glycerol Monomycolate 1 H NMR, Proton Nuclear Magnetic Resonance ORCID 1 1 H, H-COSY, Correlation Spectroscopy 1 13 H, C-HSQC, Heteronuclear Single Quantum Coherence Sandra Guallar-Garrido http://orcid.org/0000-0002-9666-5757 Ig, Immunoglobulin Víctor Campo-Pérez http://orcid.org/0000-0003-3161-9984 IL, Interleukin Míriam Pérez-Trujillo http://orcid.org/0000-0002-6919-7417 ILC, Innate Lymphoid Cells Cecilia Cabrera http://orcid.org/0000-0002-9941-6828 IFN-ɣ, Interferon-γ Jordi Senserrich http://orcid.org/0000-0002-8021-1718 M. brumae, Mycobacterium brumae Alejandro Sánchez-Chardi http://orcid.org/0000-0002-8789-1883 MTT, Thiazolyl blue tetrazolium bromide Rosa Maria Rabanal http://orcid.org/0000-0002-9521-791X NF-κβ, Nuclear Factor – κβ Elisabet Gómez-Mora http://orcid.org/0000-0002-9779-5940 NK, Natural Killer Estela Noguera-Ortega http://orcid.org/0000-0001-6507-0673 NMIBC, Non-Muscle Invasive Bladder Cancer Marina Luquin http://orcid.org/0000-0002-6015-3282 NMR, Nuclear Magnetic Resonance Esther Julián http://orcid.org/0000-0002-6558-3978 PBS, Phosphate Buffered Saline PDIM, Phtiocerol dimycocerosates PGL, Phenol Glycolipid Authors’ contributions PE, Petroleum Ether OD, Optical Density Conceptualization, S.G-G. and E.J.; Formal Analysis, S.G-G., V.C.P., M.P. RPMI 1640, Roswell Park Memorial Institute 1640 T., C.C., J.S.; Investigation, S.G-G., V.C.P., M.P.T., C.C., J.S., A.S.Ch., R.R., SD, Standard Deviation E.G-M., E.N-O., M.L. and E.J.; Data Curation, S.G-G.; Writing –Original SEM, Scanning Electron microscopy Draft Preparation, S.G-G., C.C., M.L., and E.J.; Writing –Review & Editing TEM, Effector Memory CD4+ T cells Preparation, all authors; Visualization Preparation, all authors; TLC, Thin-layer chromatography Supervision, Project Administration and Funding Acquisition EJ. TNF-α, Tumour Necrosis Factor-α References Acknowledgments 1. Babjuk M, Burger M, Zigeuner R, Shariat SF, van Rhijn BWG, Compérat E, Hernández V, Kaasinen E, Palou J, Rouprêt M, et al. We are grateful to the technical staff from the Estabulari UAB for their EAU guidelines on non-muscle-invasive urothelial carcinoma of outstanding animal management. We thank the IGTP Cytometry Core the bladder: update 2013. 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