Our long-term objective is to develop novel cancer therapies employing an attenuated strain of anaerobic bacteria called C. novyi-NT. When injected intravenously, C. novyi-NT spores are not toxic to healthy animals but can selectively germinate and spread within tumors, leading to substantial cures when combined with conventional therapies. Our IND (ref #BB-IND 12698) for C. novyi-NT single-agent therapy has recently been approved by the FDA and we are expecting to initiate a Phase I clinical trial within a month. As this clinical trial and the subsequent Phase II trial will involve C. novyi-NT, a "first-in-class" anticancer agent, sensitive surrogate makers are urgently needed to meet efficacy and safety concerns. Therefore, in the next funding cycle, our research effort will be focused on the development and validation of biomarkers that can predict therapeutic outcomes. The following specific aims reflect our short-term objectives. Aim 1. To develop circulating biomarkers for efficacy and toxicity associated with C. novyi-NT therapy in experimental systems: we will investigate three classes of biomarkers in preclinical studies in mice: (1) markers associated with host responses, (2) markers reflecting C. novyi-NT germination and invasion, and (3) markers indicating tumor response. Aim 2. To assess and develop circulating biomarkers in clinical trials: we will assess the biomarkers and assays developed in the animal models in Phase I and Phase II clinical trials, and validate them in subsequent larger clinical studies. Aim 3. To develop imaging modalities suitable for monitoring therapeutic responses: we have recently invented a method for imaging bacterial infection with radioactively labeled FIAU in animal models. In this aim, we will develop and implement this method to assess the correlation between C. novyi-NT colonization and treatment responses in both animal models and clinical trials. Aim 4. To investigate radiolabeled FIAU as a therapeutic agent: we will assess the possibility that [131IJFIAU can generate local radiation sufficiently potent to obliterate the well-oxygenated tumor rims resistant to C. novyi-NT. From the public health perspective, this new therapy we are developing utilizes a special strain of bacteria that only grows within tumors and kills cancer cells resistant to chemo and radiation therapies.