Clostridium sporogenes-mediated intratumoral production of cytokines, combined with radiotherapy, reduces tumor growth and prolongs survival

Abstract Cancer immunotherapy transformed cancer treatment, as reflected by the continued development of immunotherapeutics for multiple cancer types. Despite successes, adverse effects due to systemic exposure remain a major challenge, potentially tackled using tumor-targeted strategies. Most solid cancers contain necrosis, associated with poor prognosis. This can be exploited by anaerobic bacteria, which selectively colonize necrotic regions, thereby eliciting anticancer effects. We “armed” Clostridium sporogenes-nontoxic (NT) to express immunotherapeutic genes serving as intratumoral Trojan horse. Genes encoding murine interleukin-2 (mIL-2) or Granulocyte-Macrophage Colony-Stimulating factor (mGM-CSF) were chromosomally inserted. Genetic insertions did not affect germination, growth, or colonization properties of clostridial strains. ELISA results showed successful secretion of mIL-2 and mGM-CSF up to ~3400 and ~560 pg/ml, respectively. Secreted mIL-2 increased T-cell proliferation 6-fold, while secreted mGM-CSF increased NF-κB expression ~2-fold compared to control. In vivo combined with radiotherapy (RT), significant tumor growth inhibition was observed with C. sporogenes-NT (p<0.01) +RT (p<0.01), C. sporogenes-NT-mIL-2 (p<0.0001) +RT (p<0.0001), and C. sporogenes-NT-mGM-CSF +RT (p<0.01). Treatment with clostridial strains significantly prolonged survival (p<0.001) up to tumor cure upon C. sporogenes-NT-mIL-2 +RT treatment, without causing toxicity. Our data reports successful secretion of active immunotherapy by C. sporogenes-mIL-2 and C. sporogenes-NT-mGM-CSF, resulting in prolonged survival in vivo .