WITHDRAWN: Recombinant BCG overexpressing a STING agonist elicits trained immunity and improved antitumor efficacy in non-muscle invasive bladder cancer

The authors have withdrawn their manuscript owing to irregularities in the Source Data supporting some figures. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors.


INTRODUCTION:
Bacillus Calmette-Guérin (BCG)--the only FDA-approved bacterial agent for cancer immunotherapy--has been in use for the treatment of high-risk non-muscle invasive bladder cancer (NMIBC) as a first-line therapy since the late 1970s. More than one-third of patients with NMIBC will experience tumor recurrence after receiving BCG, and these patients have limited options other than removal of the bladder and creation of a urinary diversion -a major life-altering event. Thus, there is a significant unmet need for improved versions of BCG that provide superior response rates and prevent disease progression 1 . At the current time intravesical options for bladder preserving therapy remain limited.
Following bladder instillation, BCG induces a local inflammatory response accompanied by infiltration of granulocytes, macrophages, natural killer (NK) cells, dendritic cells (DCs), and CD4 + and CD8 + T cells, accompanied by release of pro-inflammatory cytokines including IL-6, TNF-a, and IFN-g [2][3][4][5][6][7] . However, the specific immune mechanisms leading to BCG-mediated tumor eradication as well as BCG-resistance are not well-understood. BCG has been found to impart potent heterologous protection against non-related viral and bacterial infections; it elicits this protection via an innate immune memory mechanism known as trained immunity [8][9][10][11] . Trained immunity is characterized by metabolic, epigenetic and transcriptional reprogramming of both myeloid and lymphoid lineages 12-15 but it has not been extensively studied as an antitumor mechanism during BCG immunotherapy for NMIBC.
Recent studies have implicated the cytosolically located, double strand DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signaling effector, stimulator of interferon genes (STING), in a key innate immune response pathway known as the cytosolic surveillance pathway (CSP), which responds to DNA and cyclic dinucleotides (CDN) aberrantly present in the cytosol [16][17][18][19] . CDNs can come from intracellular bacteria that synthesize and secrete them, or alternatively when DNA-activated cGAS catalyzes the formation of 2′3′-cGAMP, a CDN that is a potent STING agonist. Activation of STING leads TBK1 activation, which in turn stimulates canonical IRF3-driven Type I interferon and IKK-driven NF-κB-mediated innate immune responses, including elevated dendritic cell priming and recruitment of antigen-specific T-effector (Teff) cells [20][21][22][23] . Small molecule STING agonists elicit potent pro-inflammatory responses, and correspondingly have shown significant efficacy as cancer immunotherapies 24,25 .
BCG harbors an endogenous diadenylate synthase gene called disA, and the microbe naturally releases small amounts of the STING agonist c-di-AMP 18,26 . We hypothesized that this low-level engagement of STING may contribute to BCG's antitumor efficacy in NMIBC.
Consequently, sought to determine whether enhanced activation of STING may augment BCGmediated trained immunity in preclinical models of NMIBC. To address these questions, we tested a recombinant disA-overexpressing BCG strain called BCG-disA-OE for its antitumor potency and ability to elicit trained immunity in urothelial cancer models.

RESULTS
BCG-disA-OE shows improved efficacy against urothelial cancer in two animal models.
BCG-disA-OE is a genetically-engineered BCG strain in which disA is fused to a strong promoter, leading to a 300-fold overexpression of disA and a 15-fold increase in production of c-di-AMP (Fig. S1a) 18 . Compared with wild type BCG (BCG-WT), BCG-disA-OE elicits significantly increased STING activation in macrophages as measured by IRF3 induction (Fig.   S1b). To evaluate its NMIBC antitumor efficacy we tested BCG-disA-OE delivered intravesically in the rat N-methyl-N-nitrosourea (MNU) model of NMIBC [27][28][29] . In this model urothelial dysplasia develops at week 14 after the first intravesical instillation of MNU and by week 24 rats display a heterogeneous NIMBC including carcinoma-in-situ (CIS), papillary Ta, or higher-grade T1-T2 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human NMIBC (Fig. 1a). Rats treated with weekly intravesical BCG-disA-OE or BCG-5 WT from week 18-23 were sacrificed for bladder cytokine expression changes and histopathologic staging. As shown in Fig.1b, compared with BCG-WT, BCG-disA-OE elicited significantly increased levels of IFN-b, IFN-g, TNF-a, IL-1b, CXCL10, MCP-1, MIP-1a, and iNOS transcription while mRNA levels of the immunosuppressive cytokines IL-10 and TGF-b were reduced by both BCG strains (Fig. 1b). We confirmed these patterns of cytokine expression at the protein level using ELISA for TNF-a, IL-2, and IFN-g, and noted that intravesical BCG-disA-OE, strongly increased the levels of IFN-g in rat spleens while BCG-WT did not (Fig. S2a). Correspondingly, we found a significant decrease in highest pathology grade (Fig. 1c), tumor involvement index ( Fig. 1d) and highest tumor stage (Fig. 1e) in rats treated with BCG-disA-OE in comparison to untreated. By tumor involvement index BCG-disA-OE was statistically significantly superior to no treatment (p < 0.001) and to BCG-WT (p < 0.05), whereas BCG-WT showed only a trend towards improvement over no treatment. Importantly, the highest tumor stage observed in BCG-disA-OEtreated rats was CIS, whereas it was T1 in those receiving BCG-WT, and T2 in untreated rats, and 53.3% of BCG-disA-OE-treated rats were cancer free (p=0.009) compared with 31.2% of BCG-WT and 0% of the untreated rats (Fig. 1e). Immunohistochemical analyses revealed a significant reduction in Ki67 staining in BCG-disA-OE-treated MNU rat bladders when compared to untreated (p < 0.01) and BCG-WT (p < 0.05) suggesting reduced tumor proliferation (Fig. 1f).
CD68 staining of rat bladder showed significantly higher levels of macrophage recruitment, and a significant reduction in CD206 staining in the BCG-disA-OE-treated MNU-rats compared to those receiving BCG-WT (Fig. 1g). Among macrophages present, there was a trend toward elevation of the pro-inflammatory M1-like marker CD86 (Fig. 1g) and significant reduction in CD206 staining, an M2-like marker associated with resolution of inflammation, in the BCG-disA-OEtreated rats compared with untreated controls. These observations indicate that the enhanced induction of type I IFN and other proinflammatory signatures in bladders of tumor-bearing rats 6 treated with BCG-disA-OE correlated with the enhanced antitumor activity of the recombinant BCG strain.
To validate our findings in a second cancer model, we tested the efficacy of BCG-disA-OE against bladder cancer cells derived from mice in a syngeneic MB49 model of urothelial cancer. Following flank engraftment with MB49 tumor cells, mice received four intratumoral treatments over 9 days as shown in Fig. 1h. In this model we observed a significant reduction in MB49 tumor volume and tumor weight after intratumoral injection of BCG-disA-OE when compared with BCG-WT ( Fig. 1i and Fig. S2b). Histopathology demonstrated extensive necrosis and congestion in MB49 tumors treated with BCG-disA-OE when compared to BCG-WT and untreated (Fig. S2c). There were no significant changes in body weights of mice receiving BCG, however spleen weight of MB49 tumor mice significantly increased (Fig. S2d) after intratumoral BCG injection. We further characterized the impact of the treatments on macrophage polarization and recruitment of activated T cells in the tumor microenvironment (TME). As shown in Fig. 1j, compared with BCG-WT, BCG-disA-OE significantly reduced the abundance of immunosuppressive M2 macrophages when compared to untreated and BCG-WT and significantly (p < 0.01) increased proinflammatory M1 macrophages. Similarly, BCG-disA-OE recruited significantly more IFN-g-producing CD4 + T cells when compared to BCG-WT, and both BCG strains increased IFN-g-producing CD8 + T cells. While both BCG strains recruited more CD4 + and CD8 + cells to the tumors, BCG-disA-OE uniquely increased CD8 + T cells in the spleens of treated animals (Fig. S3a). BCG-disA-OE also significantly reduced tumor-associated Tregulatory (Treg) cells to a greater degree than BCG-WT in both tumor and spleen (Fig. S3b).
These results indicate that in this murine model of urothelial cancer, BCG-disA-OE has superior antitumor efficacy than BCG-WT, and its efficacy correlates with shift in polarization of macrophages to M1, increased activation of both CD4 + and CD8 + T cells, and a reduction of local intratumoral and systemic Treg cell populations.

BCG-disA-OE is a potent inducer of STING signaling and proinflammatory cytokine secretion by macrophages in vitro
BCG is known to be internalized by macrophages and other phagocytic cells 30 , and mycobacteria have been shown to release c-di-AMP into the cytosolic compartment where it activates the STING pathway ( Fig. 2a) 18,26 . To further investigate the mechanisms of antitumor activity of BCG-disA-OE, we evaluated its immunologic effects on human monocyte-derived macrophages (HMDMs), primary murine bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC) as well as macrophage cell line (J774.1). We found consistent induction of IRF3, IFN-β, IFN-g, TNF-a and IL-6 in all cell types in response to BCG-disA-OE that was significantly higher than that seen with BCG-WT-exposed cells (Fig. 2b-d and Fig S4a-c), and in human MDM and murine BMDM this difference was accentuated by IFN-g treatment (Fig. S4de). These differences were strictly STING-dependent as confirmed using BMDM from STING -/mice (Fig. 2c). Since STING activation also leads to upregulation of NF-kB via the TBK1 pathway, we found that expression of both TNF-a and IL-6 in the same panel of cells paralleled that of IFNb and was significantly higher following exposure to BCG-disA-OE compared with BCG-WT ( Fig.   2d and S4c). STING agonists are known to be potent inducers of several chemokines (CXCL9, CXCL10 [IP-10], CXCL22, and MCP-1) as well as iNOS 31,32 and consistent with this, IFN-gactivated BMDMs showed a more robust induction of these chemokines and iNOS when challenged with BCG-disA-OE strain than with BCG-WT (Fig. 2e) as we also observed in vivo in the MNU-rat model of NMIBC (Fig. 1b). We also assessed the cellular toxicity using annexin-PI staining and found that whereas late apoptotic cell death remained at baseline with BCG-disA-OE exposure in both BMDM and J774.1 macrophage, BCG-WT exposure elicited significantly higher levels of apoptotic cell death ( Fig. S5a-b) in the BMDM cells. These observations demonstrate the BCG-disA-OE elicits pro-inflammatory cytokine expression more potently than 8 BCG-WT in primary human MDM as well as murine primary macrophages and macrophage cell lines.

BCG-disA-OE is also a strong inducer of pro-inflammatory cytokines in urothelial carcinoma cells in vitro.
Because urothelial cells are capable of BCG phagocytosis and cytokine secretion [33][34][35] , we sought to determine if the STING agonist-overexpressing BCG strain elicits altered effects in urothelial carcinoma cells in vitro. Using monolayer cultures of 5637 (a human urothelial cancer cell line), we confirmed rapid BCG internalization of both BCG-WT and BCG-disA-OE as early as 30 mins after exposure (Fig. S6a) and documented that tumor cell survival was similar following BCG exposure with different MOI levels in three separate human urothelial cancer cell lines (Fig.   S6b). Next, we tested a panel of human and rodent urothelial carcinoma cell lines representing various tumor stages and observed that BCG-disA-OE elicited more potent gene and protein expression of the pro-inflammatory cytokines TNF-a, IL-6, IL1-b than BCG-WT in all the cell lines tested (Fig. 3a-d and Fig. S7a-b). In light of the fact that increased cytokine signaling from STING activation elicits increased autophagy 18,36,37 we evaluated the formation of LC3B puncta in human 5637 urothelial cancer cells exposed to BCG-disA-OE and BCG-WT. The majority of BCG-disA-OE bacilli co-localized with LC3B suggesting enhanced autophagic targeting by BCG-disA-OE, while similar co-localization was not observed with BCG-WT (Fig. 3e). These observations are consistent with the concept that urothelial cells contribute to immune activation following BCG exposure and demonstrate that they display increased pro-inflammatory signaling by BCG-disA-OE in agreement with enhanced efficacy we observed in vivo.
BCG-disA-OE-exposed macrophages are classically activated and inflammatory in nature.
The immunotherapeutic efficacy of BCG has been correlated with increased levels of inflammatory or M1-phenotype macrophages in NMIBC 38 .
Having observed an increased abundance of M1-like macrophages in MNU rat bladders following BCG-disA-OE instillation ( Fig.   1g-h) and higher levels of several M1-associated cytokines and chemokines in macrophages exposed to BCG-disA-OE ( Fig. 2d-e), we next measured the expression of immune markers delineating the M1 versus M2 macrophage phenotypes in the presence of BCG-WT or BCG-disA-OE in both murine and human primary macrophages. First, we focused on the MHC class IIexpressing CD45 + CD11b + F4/80 + murine BMDM population following in vitro BCG exposure ( Fig.   S8a-b). As may be seen in Fig. 4a and Fig. S9a-b, we observed a significant expansion of TNFa-expressing CD11b + F4/80 + murine BMDMs following exposure to BCG-disA-OE compared with BCG-WT. We next gated on cells expressing the M2 surface receptors CD206 + CD124 + (frequently reported as immunosuppressive of anti-tumor immunity) among CD45 + CD11b + F4/80 + macrophages and observed a reduction of this population with BCG-disA-OE relative to BCG-WT ( Fig. 4a and Fig. S9c-d). Within this immunosuppressive cell population, there was a high proportion of IL-10-expressing CD206 + CD124 + cells in BCG-WT-exposed macrophages, while IL-10-expressing cells were significantly reduced in response to BCG-disA-OE exposure ( Fig. 4a and Fig. S9e-f). These results demonstrate that compared with BCG-WT, BCG-disA-OE exposure elicits more extensive macrophage reprogramming with expansion of pro-inflammatory M1 macrophages displaying increased antigen presentation (MHC class II expression) and TNFa expression and contraction of immunosuppressive M2 macrophages expressing IL-10.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells known to foster immunosuppression within the TME 39,40 . Accordingly, we investigated the induction of monocytic-myeloid derived suppressor cells, M-MDSCs, (CD45 + Ly6C hi Ly6G -CD11b + F4/80 -) using primary murine BMDMs (Fig. S10). We observed a significant expansion of M-MDSCs following BCG-WT exposure, while this same population was significantly smaller following BCG-disA-OE exposure ( Fig. 4a and Fig. S11a-b). Moreover, the M-MDSCs elicited by BCG-WT exhibited higher IL-10 expression, whereas IL-10-expressing M-MDSCs were virtually absent after BCG-disA-OE exposure ( Fig. 4a and Fig. S11c-d). These observations suggest that BCG may contribute to an enhanced immunosuppressive TME (potentially contributing to BCG-unresponsiveness in some NMIBC patients) that may be overcome with the BCG strain overexpressing a STING agonist.
We next characterized the macrophage activation phenotypes in HMDMs isolated from several independent healthy human donors. Both the WT and BCG-disA-OE strains elicited increases in the population of classical macrophages (CD11b + CD14 + CD16 -), but these inductions were comparatively higher in response to BCG-disA-OE ( Fig. 4b and Fig. S12a-c).
We considered antigen-presenting classically activated macrophages (CD14 + CD16 -HLA-DR + ) and their ability to produce TNF-a or IL-6 and found a significantly increased proportion of TNFa and IL6-producing HLA-DR + cells following exposure to BCG-disA-OE compared to BCG-WT ( Fig. 4b and Fig. S13a-b). We also investigated the immunosuppressive M2 surface markers, CD206 + and CD163 + , on transitional or intermediate macrophages (CD11b + CD14 + CD16 + ) and found a consistent decrease in them following BCG-disA-OE exposure ( Fig. 4c and Fig. S12b and Fig. 13b). Consistent with our findings in murine macrophages, the fraction of these immunosuppressive, intermediate macrophages expressing M2 surface markers and IL-10 was also significantly lower in response to exposure to BCG-disA-OE than with BCG-WT ( Fig. 4c and Macrophages harboring BCG-disA-OE are highly phagocytic. Macrophages exposed to STING agonists delivered by intratumoral injection have been reported to display increased phagocytosis and other markers of macrophage activation; they also recruit new macrophages which serve a phagocytic role in the TME [41][42][43][44] . Consistent with these observations we confirmed that HMDMs transfected with c-di-AMP showed increased phagocytosis and exhibited elongated dendrites compared to mock-transfected populations (Fig.   S14). We then evaluated the phagocytic properties of HMDMs following exposure to the different BCG strains and found significantly greater phagocytosis of IgG-opsonized FITC-latex beads by macrophages harboring BCG-disA-OE strains compared to those harboring BCG-WT (Fig. 4d).
In keeping with the previously established role of STING activation in augmenting autophagy 18,36,37 , we found that a majority of intracellular BCG-disA-OE bacilli were co-localized with LC3B in IFN-g-activated primary BMDMs ( Fig. 4e-f), similar to our findings with urothelial cancer cells (Fig. 3d), while autophagy induction in BCG-WT was significantly lower. We also found significantly greater co-localization of BCG-disA-OE bacilli with the autophagy adapter protein p62 compared to that observed with BCG-WT ( Fig. 4g-h). These results reveal BCG-disA-OE increases the levels of phagocytosis and autophagic processing within macrophages to a greater degree than BCG-WT, a phenomenon associated with enhanced peptide antigen presentation to MHC class-II molecules 45,46 .
In light of recent data showing BCG to be a potent inducer of long-lived innate immunitytermed trained immunity -through epigenetic modifications of key pro-inflammatory genes 10,11,13 we hypothesized that the addition of STING agonist overexpression to standard BCG might potentiate the epigenetic hallmarks of trained immunity in primary human monocytes. Having already established that BCG-disA-OE is a more potent inducer of macrophage TNF-a and IL-6 secretion than BCG-WT, we confirmed this in primary human monocytes from a group of 6 healthy human subjects (Fig. 5a). The ability of traditional BCG to elicit trained immunity has been correlated with changes in epigenetic marks that increase pro-inflammatory gene expression 12 .
Thus, we asked if the enhanced induction of TNF-a and IL-6 expression elicited by BCG-disA-OE compared with BCG-WT is epigenetically mediated. To this end, we evaluated the promoter regions of the TNF-a and IL-6 genes for durable, antigen-independent epigenetic changes using an assay 47 in which human monocytes exposed to BCG strains for 24 h were rested for five days prior to challenge with a heterologous PAMP (pathogen-associated molecular pattern), the TLR1/2 agonist Pam3CSK4 on day 6 ( Fig. 5b). Using chromatin immunoprecipitationpolymerase chain reaction (ChIP-PCR) assays, we quantified the activating histone methylation mark H3K4me3 present in the TNF-a and IL-6 promoters. We observed that exposure to BCG-disA-OE led to greater enrichment of this mark than BCG-WT even without the heterologous second stimulation (i.e., adding RPMI media alone at day 6). Upon re-stimulation with Pam3CSK4 at day 6, the abundance of the activating epigenetic mark was further increased by both BCG strains, but BCG-disA-OE-pretreatment yielded notably more enrichment than BCG-WT (Fig. 5c).
Similarly, we investigated the chromatin repression mark H3K9me3 at the same two promoters and found that, while both BCG strains led to reduced levels of H3K9me3 (which were further accentuated by addition of Pam3CSK4), the degree of reduction mediated by BCG-disA-OE was consistently greater than that mediated by BCG-WT, both upon initial exposure and after rest and re-stimulation ( Fig. 5d). Simultaneous measurement of TNF-a and IL-6 in BCG-trained culture supernatant following non-specific stimulation by Pam3CSK4 revealed that BCG-disA-OE-trained macrophages produced significantly higher levels of these pro-inflammatory cytokines than did those trained with WT-BCG ( Fig. 5e-f). Next, an LC-MS based investigation of the immunometabolic state of primary human and murine macrophages exposed to BCG-disA-OE suggested increased intracellular glucose and lactate as compared to BCG-WT-exposed macrophages ( Fig. 5g-i), suggesting an enhanced glycolytic state in macrophages infected with BCG-disA-OE. In the same screen, there was a trend towards increased levels of intracellular, tryptophan and significantly lower levels of immunosuppressive kynurenine ( Fig. 5g-i) suggesting 13 more extensive metabolic reprograming in the BCG-disA-OE-infected macrophages compared with those infected with WT-BCG. In addition, modulation of several key metabolites such as itaconate and UDP-N-acetyl-glucosamine (UDP-GlcNac) suggest extensive and stronger metabolic shift in BCG-disA-OE macrophages. Together, these results reveal that overexpression of a STING agonist by BCG potentiates the degree of epigenetic reprogramming normally conferred by BCG-WT and that in turn leads to increased proinflammatory cytokine responses and immunometabolic changes associated with improved antitumor immunity in the TME 48-50 .

BCG-disA-OE is less pathogenic than BCG-WT in two mouse models.
To assess the pathogenicity of the two BCG strains, we used an immunocompetent BALB/c mouse model of aerosol exposure and measured the lung bacillary burden after four weeks when adaptive immune responses are maximal (Fig. 6a). While the day 1 implantation of the two BCG strains was equivalent, we observed that BCG-disA-OE proliferated in murine lungs to a significantly lower degree than BCG-WT by a margin of 0.43 log10 colony forming units ( Fig.   6b and Fig. S15a). As we observed in earlier in cell-based models, pro-inflammatory cytokine levels in both lungs and spleens were significantly higher in BCG-disA-OE-exposed mice than those receiving BCG-WT ( Fig. 6c-d and Fig. S15b). We also tested the two strains in immunocompromised SCID mice, again using a low dose aerosol exposure model ( Fig. 6e) and observed a statistically significant survival prolongation with BCG-disA-OE compared to BCG-WT ( Fig. 6f and Fig. S15c). Thus, despite eliciting more profound inflammatory signatures in numerous model systems, BCG-disA-OE is less pathogenic than BCG-WT in these two murine model systems.

DISCUSSION
Bladder cancer ranks as the ninth most common cancer worldwide, and approximately 75% of patients diagnosed with bladder cancer present initially with NMIBC 51 . For over 40 years, transurethral resection of the bladder mass followed by BCG adjuvant immunotherapy has been the standard of care for high-risk NMIBC. Although BCG has been shown to reduce tumor recurrence and progression, ~30-50% of patients will recur after BCG, placing them at a high risk of recurrence and progression to MIBC 52,53 . This high risk for recurrence combined with the lack of effective alternative or salvage therapies, and a worldwide shortage of BCG has generated an unmet need for improved versions of BCG 1,2 . The economic burden and inability to develop effective bladder-sparing approaches in BCG-unresponsive and relapsing populations prompted us to investigate alternative mechanism-based therapies exploiting the STING-signaling pathway 54 .
A primary goal of this study was to determine whether the antitumor efficacy of BCG in urothelial cancer models could be enhanced by engineering excess production of the STING we found that BCG-disA-OE was less pathogenic than BCG-WT in two mouse models, suggesting that BCG-disA-OE may be safer than BCG-WT and that STING agonist overexpression may not result in an undesirable adverse event profile.
BCG vaccination for tuberculosis has long been known to confer heterologous crossprotective effects against antigenically unrelated viral and bacterial infections 10,11,60 . This phenomenon has recently been shown to be mediated by an innate immune mechanism called trained immunity in which epigenetic modifications following BCG exposure confer an elevated set-point of transcriptional activation in genes governing pro-inflammatory responses including cytokine genes, immunometabolism and cell polarization 12,61-64 . Trained myeloid cells demonstrate elevated responses to subsequent challenge by unrelated antigens or pathogens, and the effect is long-lasting since transcriptional changes are induced by BCG in hematopoietic stem cells and myeloid progenitor cells 11 . These same trained immunity changes elicited by BCG may underlie the immunotherapeutic effects of BCG in cancer prevention [65][66][67][68] . Therefore, another goal of this study was to evaluate whether or not the salutary effects of BCG in bladder cancer therapy are mediated through a trained immunity mechanism, and how overexpression of a STING agonist may modulate BCG-mediated trained immunity.
Indeed, we observed that BCG-disA-OE potentiated trained immune responses to a greater degree than BCG-WT in multiple in vivo and in vitro analyses. We observed elevated levels of pro-inflammatory cytokines and chemokines in bladders from tumor-bearing animals treated with BCG-disA-OE compared to BCG-WT. Since non-immune cells have also been shown to possess immunological memory 69 , we considered the possibility that this cytokine response may have originated from myeloid cells in the TME and/or the tumor cells themselves. Indeed, we found that compared with BCG-WT, BCG-disA-OE elicited more potent cytokine responses in both primary macrophages and urothelial cancer cells representing various tumor stages. We found dramatically reduced induction of these cytokines in BMDMs from STING -/mice, indicating for the first time that that this trained immunity is a downstream consequence of STING activation.
In addition, we found robust induction of several chemokines as has been observed in other studies with stimulation using exogenous STING agonists 22,32 .
Enhanced trained immune responses leading to reacquisition of pro-inflammatory traits of macrophages, also called repolarization, has been shown to correlate with increased survival in tumor-bearing mice and in cancer patients 44,70 . Consistent with this we observed that, compared with BCG-WT, BCG-disA-OE led to greater increases in M1 macrophages in vivo in two separate in vivo models of urothelial cancer, and we noted the same pattern in vitro with both mouse and human primary macrophages. STING activation in host cells is known to augment autophagy, and polymorphisms in autophagy-related genes correlate with progression and recurrence of bladder cancer following BCG treatment 71 . Consistent with this, we observed that BCG-disA-OEtreated myeloid cells also exhibited enhanced phagocytosis and autophagy to a greater degree than BCG-WT. This finding raises the intriguing possibility that tumor-associated macrophages that take up BCG or BCG-disA-OE may also augment adaptive immunity through enhanced antigen presentation of tumor-associated peptides to the lymphocytes. In summary, our findings reveal a significant enhancement of BCG's antitumor activity with a reengineered BCG that overexpresses a STING agonist. Importantly, STING pathway activation potentiates the expression of pro-inflammatory cytokines and chemokines and induces macrophage polarization towards an M1 phenotype. We also found that BCG-disA-OE elicits epigenetic changes that govern cytokine expression which are known to be mechanistically linked to immunometabolic and cell polarization changes. These observations reveal that boosting STING agonism improves the ability of BCG to elicit trained immunity and also its immunotherapeutic benefit against NMIBC. This association strongly suggests that a fundamental mechanism of BCG's efficacy in urothelial cancer is through trained immunity.   The following antibodies were used to stain myeloid and lymphoid cells:      confocal microscope and Fiji software processing. Quantification was by mean fluorescence intensity. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by 2-tailed Student's t-test. Data

Methods: Online Methods
shown are for BCG-Tice; similar findings were observed for BCG-Pasteur as shown in Fig. S9 and S11.  Cytokines and chemokine levels were measured by ELISA after 24 hr exposures at a MOI of 20:1.