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ERK translocation from cytosol to the ER

Supplementary Figures 1-11

ERK translocation from ER to the nucleus

Supplementary Figures S1-S4

Chemical Methods

Supplementary tables S1-8

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives.Significance:This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer.This article is highlighted in the In This Issue feature, p. 151

Supplementary Data from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Purpose:Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response.Experimental Design:We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.Results:[18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses.Conclusions:This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy.See related commentary by Weber et al., p. 5241

Supplementary Figure from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Purpose: Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response. Experimental Design: We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer. Results: [18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses. Conclusions: This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy. See related commentary by Weber et al., p. 5241

Two of the major obstacles that adoptive cell transfer immunotherapy needs to overcome to be successful in solid tumors are 1) the immunosuppressive tumor microenvironment (TME) and 2) the lack of robust biomarkers that allow for the identification of patients that would benefit from the therapy and for monitoring of the treatment response. Here we paired a CAR T cell therapy with a companion Positron Emission Tomography (PET) imaging approach that allows for serial, non-invasive, whole-body visualization of a biological target of interest -in this specific case, Fibroblast Activating Protein (FAP). FAP is a cell surface serine protease that is highly expressed by cancer associated fibroblasts in the TME that participate in the generation of the immunosuppressive stromagenic response in solid tumors. We designed a novel FAP CAR construct based on the scFv of the 4G5 antibody developed, in house, against canine FAP that cross-reacts against mouse and human FAP. In this study, we utilized the 18F-radiolabeled FAP inhibitor (FAPI), [18F]AlF-FAPI-74, to image FAP in two different mouse tumor models. First, the probe specificity was evaluated in the I45 human mesothelioma tumor model (a line which does not induce FAP+ fibroblasts). I45 WT and I45 cells transduced with human FAP were injected s.c. into the opposite flanks of a mouse and imaged following 2 weeks of tumor growth. We observed a 7.5-fold higher uptake of [18F]AlF-FAPI-74 in the I45 huFAP tumor compared to the WT, demonstrating the high specificity of the probe for FAP. Next, we used a more clinically-relevant A549 model where the tumor cells do not express FAP but induce a stromagenic response and drive the recruitment of FAP+ stromal cells in the TME. [18F]AlF-FAPI-74 PET/CT following 3 weeks of tumor growth showed a 6.5-fold increased radiotracer uptake in the tumor relative to the muscle at the baseline scan. Using this model, we evaluated the potential of our new CAR T cells to reduce tumor burden, as well as evaluating the potential of the [18F]AlF-FAPI-74 tracer as a tool to monitor the clearance of FAP-expressing cells in response to FAP CAR T cell therapy. Immediately after the baseline scan, we injected 5x106 FAP CAR T cells iv. At day 14 post-T cell injection, mice treated with FAP CAR T cells had significantly smaller tumors relative to the control group, which were treated with T cells that do not express the CAR, highlighting the therapeutic efficacy of the FAP CAR T cell therapy. Moreover, [18F]AlF-FAPI-74 PET/CT imaging showed no detectable tracer uptake in the tumors treated with FAP targeted CAR T. These findings were confirmed by immunofluorescence, indicating successful clearance of the FAP+ stroma by the injected FAP CAR T cells. In conclusion, the new 4G5 FAP CAR shows specific targeting toward mouse stroma infiltrating lung adenocarcinoma xenografts. PET imaging of FAP could be a highly useful approach to stratify patients prior to FAP CAR T therapy, as well as to monitor the pharmacodynamic response for FAP-targeted therapies. Citation Format: Estela Noguera-Ortega, Iris K Lee, Zebin Xiao, Leslie Todd, John Scholler, Decheng Song, Maria Liousia, Katheryn Lohith, Kexiang Xu, Kimberly J Eduards, Michael D Farwell, Carl H June, Steven M Albelda, Ellen Puré, Mark A Sellmyer. FAP CAR T cell therapy for solid tumors with PET imaging [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B19.

Resistance to BRAF and MEK inhibitors (BRAFi + MEKi) in BRAF-mutant tumors occurs through heterogeneous mechanisms, including ERK reactivation and autophagy. Little is known about the mechanisms by which ERK reactivation or autophagy is induced by BRAFi + MEKi. Here, we report that in BRAF-mutant melanoma cells, BRAFi + MEKi induced SEC61-dependent endoplasmic reticulum (ER) translocation of the MAPK pathway via GRP78 and KSR2. Inhibition of ER translocation prevented ERK reactivation and autophagy. Following ER translocation, ERK exited the ER and was rephosphorylated by PERK. Reactivated ERK phosphorylated ATF4, which activated cytoprotective autophagy. Upregulation of GRP78 and phosphorylation of ATF4 were detected in tumors of patients resistant to BRAFi + MEKi. ER translocation of the MAPK pathway was demonstrated in therapy-resistant patient-derived xenografts. Expression of a dominant-negative ATF4 mutant conferred sensitivity to BRAFi + MEKi in vivo. This mechanism reconciles two major targeted therapy resistance pathways and identifies druggable targets, whose inhibition would likely enhance the response to BRAFi + MEKi. Significance: ERK reactivation and autophagy are considered distinct resistance pathways to BRAF + MEK inhibition (BRAFi + MEKi) in BRAFV600E cancers. Here, we report BRAFi + MEKi–induced ER translocation of the MAPK pathway is necessary for ERK reactivation, which drives autophagy. The ER translocation mechanism is a major druggable driver of resistance to targeted therapy. This article is highlighted in the In This Issue feature, p. 305

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. Significance: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151

Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy for the treatment of hematological malignancies. However, in patients with solid tumors, objective responses to CAR-T cell therapy remain sporadic and transient. A major obstacle for CAR-T cells is the intrinsic ability of tumors to evade immune responses. Advanced solid tumors are largely composed of desmoplastic stroma and immunosuppressive modulators, and characterized by aberrant cell proliferation and vascularization, resulting in hypoxia and altered nutrient availability. To mount a curative response after infusion, CAR-T cells must infiltrate the tumor, recognize their cognate antigen and perform their effector function in this hostile tumor microenvironment, to then differentiate and persist as memory T cells that confer long-term protection. Fortunately, recent advances in synthetic biology provide a wide set of tools to genetically modify CAR-T cells to overcome some of these obstacles. In this review, we provide a comprehensive overview of the key tumor intrinsic mechanisms that prevent an effective CAR-T cell antitumor response and we discuss the most promising strategies to prevent tumor escape to CAR-T cell therapy.

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.

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used.

Summary: Autophagy has been identified as a potential therapeutic target in pancreatic ductal adenocarcinoma, one of the most lethal cancers, with few therapeutic options. Yang and colleagues successfully created a genetically engineered mouse model focused on the autophagy gene Atg4b that allows the study of therapeutic autophagy inhibition in fully formed tumors. Using this tool, they demonstrated that selective autophagy inhibition in either the tumor cells, normal host cells, or both suppresses tumor growth. Cancer Discov; 8(3); 266–8. ©2018 AACR See related article by Yang et al., p. 276.

New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers., Inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in murine melanoma models.

Background Bacillus Calmette-Guerin (BCG) prevents tumour recurrence and progression in non–muscle-invasive bladder cancer (BC). However, common adverse events occur, including BCG infections. Objective To find a mycobacterium with similar or superior antitumour activity to BCG but with greater safety. Design In vitro, ex vivo, and in vivo comparisons of the antitumour efficacy of nonpathogenic mycobacteria and BCG. Intervention The in vitro antitumour activity of a broad set of mycobacteria was studied in seven different BC cell lines. The most efficacious was selected and its ex vivo capacity to activate immune cells and its in vivo antitumour activity in an orthotopic murine model of BC were investigated. Outcome measurements and statistical analysis Growth inhibition of BC cells was the primary outcome measurement. Parametric and nonparametric tests were use to analyse the in vitro results, and a Kaplan-Meier test was applied to measure survival in mycobacteria-treated tumour-bearing mice. Results and limitations Mycobacterium brumae is superior to BCG in inhibiting low-grade BC cell growth, and has similar effects to BCG against high-grade cells. M. brumae triggers an indirect antitumour response by activating macrophages and the cytotoxic activity of peripheral blood cells against BC cells. Although no significant differences were observed between BCG and M. brumae treatments in mice, M. brumae treatment prolonged survival in comparison to BCG treatment in tumour-bearing mice. In contrast to BCG, M. brumae does not persist intracellularly or in tumour-bearing mice, so the risk of infection is lower. Conclusions Our preclinical data suggest that M. brumae represents a safe and efficacious candidate as a therapeutic agent for non–muscle-invasive BC. Patient summary We investigated the antitumour activity of nonpathogenic mycobacteria in in vitro and in vivo models of non–muscle-invasive bladder cancer. We found that Mycobacterium brumae effectively inhibits bladder cancer growth and helps the host immune system to eradicate cancer cells, and is a promising agent for antitumour immunotherapy.

Adoptive natural killer (NK) cell immunotherapy using primary NK cells has been challenging, to date. The use of an ”off-the -shelf” NK cell line where multiple genetic changes can be introduced has advantages. In this study, we explored the use of the NK-92MI cell line expressing a chimeric antigen receptor (CAR) targeting human mesothelin, the current target in our human CAR-T cell therapy trials. The NK-92MI cell line (which makes its own IL-2) was purchased from ATCC and transduced with a lentivirus encoding GFP (vector control) or an anti-human mesothelin scFv-41BB-CD3 ζ second-generation CAR (MesoCAR). Addition of IL-21 enhanced transduction. The transduced cells (MesoCar-NK-92MI) were sorted by flow cytometry to achieve an expression level of >98%. The NK markers NKp30, NKp44, NKG2D, 2B4, and CD56 were highly expressed on the Car-NK-92MI cell line (from 75% to 99.9%). To test the antitumor effects in vitro, three types of human tumor lines transduced to express human mesothelin were tested. Lung cancer (A549Meso), mesothelioma (EMMeso), and head and neck cancer (SSC15Meso) cells were exposed to vector control NK-92MI or MesoCar-NK-92MI cells for 22 hours at varying E:T ratios. The supernatants were collected for detection of cytokines and granzyme B. We also determined the killing function of MesoCar-NK-92MI cells after irradiation of the cells at 10 Gy. To test in vivo activity, NSG mice were injected in the flank with 5 million EMMeso cells. After tumors were established, each mouse was intravenously injected with 10 million vector control NK-92MI cells or MesoCar-NK-92MI cells with a boost injection of 5 million effectors on days 4, 8, and 11. Tumor volumes were assessed over time and mice were sacrificed on day 18. In vitro killing results showed augmented antitumor effect in MesoCar-NK-92MI compared to vector control NK-92MI. Irradiated MesoCar-NK-92MI cells still possessed good antitumor activity with only 32% loss of activity on average. Increased cytokine and granzyme B secretion was also seen with the MesoCar-NK-92MI cell line (with or without irradiation) compared to NK-92MI cells. In animal studies, irradiated MesoCar-NK-92MI cells significantly inhibited tumor growth by an average of 59% with no obvious toxicity or tumor growth in other organs. The non-CAR-expressing NK-92MI cells had significantly less antitumor activity. In conclusion, use of an irradiated NK-92MI cell line expressing a CAR targeting human mesothelin could become a safe and possible “off-the-shelf” adoptive cell therapy that could be used alone or in combination with CAR-T cells or other treatments. Citation Format: Jing Sun, Patrick Woodruff, Soyeon Kim, Estela Noguera-Ortega, Evguenia Arguiri, Maria Liousia, Steven Maceko, Edmund Moon, Steven Albelda. Augmented antitumor activity of NK-92 MI cells expressing a chimeric antigen receptor targeting human mesothelin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A67.

Mycobacterium bovis bacillus Calmette-Guérin is the most effective treatment for high risk noninvasive bladder cancer. Although bacillus Calmette-Guérin immunotherapy clearly decreases recurrence and progression rates, side effects are common and infection with the bacillus has been described. For these reasons it is necessary to find safer alternatives to the live bacillus. We explored the possibility of using killed but metabolically active bacillus Calmette-Guérin.T24, J82 and RT4 bladder tumor cell lines were cultured with live and irradiation or heat treated bacillus Calmette-Guérin Connaught. We measured the inhibition of cell proliferation and the production of cytokines in cell culture supernatants. Peripheral mononuclear blood cells were also infected and the production of different cytokines in cell culture supernatants was analyzed. Peripheral blood mononuclear cell and cell culture supernatants activated by mycobacteria were then cultured with T24 cells to analyze whether they showed cytotoxic activity.Compared to the other bacillus Calmette-Guérin treatments, γ irradiated bacillus Calmette-Guérin showed activity similar to that of the live bacillus for inhibiting tumor growth and inducing cytokine production. Irradiated bacillus Calmette-Guérin showed metabolic activity and, thus, was considered killed but metabolically active. This is the treatment that most accurately preserved the mycobacterial structure. Killed but metabolically active bacillus Calmette-Guérin induced cytokine production by infected peripheral mononuclear blood cells. Mycobacteria activated peripheral blood mononuclear cell and cell supernatants showed cytotoxic activity against tumor cells, retaining the antitumor capacity of the live bacillus.Our results suggest that killed but metabolically active bacillus Calmette-Guérin could be considered a safer immunotherapy alternative to treatment with the live bacillus.

The rough morphotypes of non-tuberculous mycobacteria have been associated with the most severe illnesses in humans. This idea is consistent with the fact that Mycobacterium tuberculosis presents a stable rough morphotype. Unlike smooth morphotypes, the bacilli of rough morphotypes grow close together, leaving no spaces among them and forming large aggregates (clumps). Currently, the initial interaction of macrophages with clumps remains unclear. Thus, we infected J774 macrophages with bacterial suspensions of rough morphotypes of Mycobacterium abscessus containing clumps and suspensions of smooth morphotypes, primarily containing isolated bacilli. Using confocal laser scanning microscopy and electron microscopy, we observed clumps of at least 5 rough-morphotype bacilli inside the phagocytic vesicles of macrophages at 3 hours post-infection. These clumps grew within the phagocytic vesicles, killing 100% of the macrophages at 72 hours post-infection, whereas the proliferation of macrophages infected with smooth morphotypes remained unaltered at 96 hours post-infection. Thus, macrophages phagocytose large clumps, exceeding the bactericidal capacities of these cells. Furthermore, proinflammatory cytokines and granuloma-like structures were only produced by macrophages infected with rough morphotypes. Thus, the present study provides a foundation for further studies that consider mycobacterial clumps as virulence factors.

γ Irradiated Mycobacterium bovis bacillus Calmette-Guérin has shown in vitro and ex vivo antitumor activity. However, to our knowledge the potential antitumor capacity has not been demonstrated in vivo. We studied the in vivo potential of γ irradiated bacillus Calmette-Guérin and γ irradiated M. brumae, a saprophytic mycobacterium that was recently described as an immunotherapeutic agent.The antitumor capacity of γ irradiated M. brumae was first investigated by analyzing the in vitro inhibition of bladder tumor cell proliferation and the ex vivo cytotoxic effect of M. brumae activated peripheral blood cells. The effect of γ irradiated M. brumae or bacillus Calmette-Guérin intravesical treatment was then compared to treatment with live mycobacteria in the orthotopic murine model of bladder cancer.Nonviable M. brumae showed a capacity to inhibit in vitro bladder cancer cell lines similar to that of live mycobacteria. However, its capacity to induce cytokine production was decreased compared to that of live M. brumae. γ Irradiated M. brumae could activate immune cells to inhibit tumor cell growth, although to a lesser extent than live mycobacteria. Finally, intravesical treatment with γ irradiated M. brumae or bacillus Calmette-Guérin significantly increased survival with respect to that of nontreated tumor bearing mice. Both γ irradiated mycobacteria showed lower survival rates than those of live mycobacteria but the minor efficacy of γ irradiated vs live mycobacteria was only significant for bacillus Calmette-Guérin.Our results show that although γ irradiated mycobacteria is less efficacious than live mycobacteria, it induces an antitumor effect in vivo, avoiding the possibility of further mycobacterial infections.