BCG invokes superior STING‐mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer

Radiation and bacillus Calmette–Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS–STING pathway has been implicated in both BCG and radiation‐induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well‐established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING‐mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG‐treated non‐muscle‐invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING‐induced interferon and pro‐inflammatory molecules, intratumoral M1 macrophage and T‐cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non‐muscle‐invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG‐induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

[1]  W. Kassouf,et al.  Combined radiotherapy and immunotherapy in urothelial bladder cancer: harnessing the full potential of the anti-tumor immune response , 2020, World Journal of Urology.

[2]  Ee Sin Chen,et al.  cGAS-STING pathway in oncogenesis and cancer therapeutics , 2020, Oncotarget.

[3]  W. Bishai,et al.  WITHDRAWN: Recombinant BCG overexpressing a STING agonist elicits trained immunity and improved antitumor efficacy in non-muscle invasive bladder cancer , 2020, bioRxiv.

[4]  Laurentiu M. Pop,et al.  Type I Interferon Response in Radiation-Induced Anti-Tumor Immunity. , 2020, Seminars in radiation oncology.

[5]  C. Drake,et al.  Adaptive Immune Resistance to Intravesical BCG in Non–Muscle Invasive Bladder Cancer: Implications for Prospective BCG-Unresponsive Trials , 2019, Clinical Cancer Research.

[6]  A Study of Stimulator of Interferon Genes (STING) Agonist E7766 in Non-muscle Invasive Bladder Cancer (NMIBC) Including Participants Unresponsive to Bacillus Calmette-Guerin (BCG) Therapy, INPUT-102 , 2019, Case Medical Research.

[7]  C. Drake,et al.  Tumor immune microenvironment in non-muscle invasive urothelial carcinoma of bladder. , 2019, Human pathology.

[8]  Clemens Grassberger,et al.  Assessing the interactions between radiotherapy and antitumour immunity , 2019, Nature Reviews Clinical Oncology.

[9]  Bladder PREserVation by RadioTherapy and Immunotherapy in BCG Unresponsive Non-muscle Invasive Bladder Cancer , 2019, Case Medical Research.

[10]  W. Bishai,et al.  BCG overexpressing an endogenous STING agonist provides enhanced protection against pulmonary tuberculosis. , 2019, The Journal of infectious diseases.

[11]  Yuan Wu,et al.  Overexpression of Indoleamine 2,3-Dioxygenase 1 Promotes Epithelial-Mesenchymal Transition by Activation of the IL-6/STAT3/PD-L1 Pathway in Bladder Cancer , 2018, Translational oncology.

[12]  D. Siemens,et al.  Investigating the STING Pathway to Explain Mechanisms of BCG Failures in Non-Muscle Invasive Bladder Cancer: Prognostic and Therapeutic Implications , 2019 .

[13]  I. Lucca,et al.  Conventional and PD-L1-expressing Regulatory T Cells are Enriched During BCG Therapy and may Limit its Efficacy. , 2018, European urology.

[14]  J. Gong,et al.  Radiation therapy and PD-1/PD-L1 blockade: the clinical development of an evolving anticancer combination , 2018, Journal of Immunotherapy for Cancer.

[15]  C. Drake,et al.  TNFα and Radioresistant Stromal Cells Are Essential for Therapeutic Efficacy of Cyclic Dinucleotide STING Agonists in Nonimmunogenic Tumors , 2018, Cancer Immunology Research.

[16]  M. Babjuk,et al.  Grading of Urothelial Carcinoma and The New "World Health Organisation Classification of Tumours of the Urinary System and Male Genital Organs 2016". , 2018, European urology focus.

[17]  C. N. Coleman,et al.  DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity , 2017, Nature Communications.

[18]  C. Drake,et al.  Intravesical BCG Induces CD4+ T-Cell Expansion in an Immune Competent Model of Bladder Cancer , 2017, Cancer Immunology Research.

[19]  C. Drake,et al.  Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment , 2017, Cancer Immunology Research.

[20]  I. Mysorekar,et al.  Urothelial generation and regeneration in development, injury, and cancer , 2017, Developmental dynamics : an official publication of the American Association of Anatomists.

[21]  K. Ishibashi,et al.  Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site , 2017, Cancer Immunology, Immunotherapy.

[22]  Yuanyuan Ruan,et al.  Decreased expression of STING predicts poor prognosis in patients with gastric cancer , 2017, Scientific Reports.

[23]  G. Schäfer,et al.  Tumor-infiltrating immune cell subpopulations influence the oncologic outcome after intravesical Bacillus Calmette-Guérin therapy in bladder cancer , 2016, Oncotarget.

[24]  J. Taube,et al.  The ratio of CD8 to Treg tumor-infiltrating lymphocytes is associated with response to cisplatin-based neoadjuvant chemotherapy in patients with muscle invasive urothelial carcinoma of the bladder , 2016, Oncoimmunology.

[25]  Miao-Fen Chen,et al.  The role of PD-L1 in the radiation response and clinical outcome for bladder cancer , 2016, Scientific Reports.

[26]  F. Grillo,et al.  Residual tumor micro-foci and overwhelming regulatory T lymphocyte infiltration are the causes of bladder cancer recurrence , 2016, Oncotarget.

[27]  G. Barber,et al.  Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis. , 2016, Cell reports.

[28]  Xinchen Sun,et al.  Effects of radiation on T regulatory cells in normal states and cancer: mechanisms and clinical implications. , 2015, American journal of cancer research.

[29]  George E. Katibah,et al.  Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. , 2015, Cell reports.

[30]  W. Bishai,et al.  A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis , 2015, Nature Medicine.

[31]  Peng Huang,et al.  Efficacy of intravesical Bacillus Calmette-Guérin therapy against tumor immune escape in an orthotopic model of bladder cancer , 2014, Experimental and therapeutic medicine.

[32]  E. Janssen,et al.  STING-Mediated DNA Sensing Promotes Antitumor and Autoimmune Responses to Dying Cells , 2014, The Journal of Immunology.

[33]  Ying Wang,et al.  STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. , 2014, Immunity.

[34]  I. Stratford,et al.  Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. , 2014, Cancer research.

[35]  M. Glickman,et al.  The mechanism of action of BCG therapy for bladder cancer—a current perspective , 2014, Nature Reviews Urology.

[36]  Reid F Thompson,et al.  Radiotherapy and the tumor microenvironment: mutual influence and clinical implications. , 2014, Advances in experimental medicine and biology.

[37]  D. Getnet,et al.  Combined treatment effects of radiation and immunotherapy: studies in an autochthonous prostate cancer model. , 2013, International journal of radiation oncology, biology, physics.

[38]  D. Quail,et al.  Microenvironmental regulation of tumor progression and metastasis , 2014 .

[39]  R. Xavier,et al.  Long-Lasting Effects of BCG Vaccination on Both Heterologous Th1/Th17 Responses and Innate Trained Immunity , 2013, Journal of Innate Immunity.

[40]  M. Albert,et al.  From infection to immunotherapy: host immune responses to bacteria at the bladder mucosa , 2013, Mucosal Immunology.

[41]  P. Darcy,et al.  Enhancing immunotherapy using chemotherapy and radiation to modify the tumor microenvironment , 2013, Oncoimmunology.

[42]  T. Hagemann,et al.  The tumor microenvironment at a glance , 2012, Journal of Cell Science.

[43]  R. Weichselbaum,et al.  The efficacy of radiotherapy relies upon induction of type i interferon-dependent innate and adaptive immunity. , 2011, Cancer research.

[44]  A. Seth,et al.  A disproportion of TH1/TH2 cytokines with predominance of TH2, in urothelial carcinoma of bladder. , 2011, Urologic oncology.

[45]  D. Zamboni,et al.  A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells , 2010, PloS one.

[46]  E. Shinya,et al.  A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth , 2009, Cancer Immunology, Immunotherapy.

[47]  M. Stöckle,et al.  Neutrophil granulocytes are required for effective Bacillus Calmette-Guérin immunotherapy of bladder cancer and orchestrate local immune responses. , 2006, Cancer research.

[48]  K. Naito,et al.  Urinary interleukin-2 may predict clinical outcome of intravesical bacillus Calmette-Guérin immunotherapy for carcinoma in situ of the bladder , 2003, Cancer Immunology, Immunotherapy.

[49]  T. Ratliff,et al.  NK cells are essential for effective BCG immunotherapy , 2001, International journal of cancer.

[50]  W. Catalona,et al.  T-cell subsets required for intravesical BCG immunotherapy for bladder cancer. , 1993, The Journal of urology.