PROJECT SUMMARY/ABSTRACT Cancer is the second leading cause of death globally and many available treatments lack the safety and effica- cy sought by oncologists and patients alike. Major efforts have been made to use living organisms, such as bacteria, as vehicles to produce and deliver site-specific therapeutic payloads, however they are mostly cen- tered on Gram-negative bacteria. In this proposal, we aim to expand the list of candidates available for probi- otic cancer therapy by exploring the potential for species of Bacillus to colonize tumor microenvironments. Many Bacillus species are generally regarded as safe (GRAS) and as Gram-positives are capable of high pro- tein product secretion. Therefore, we will also aim to expand the genetic toolbox for model organism, Bacillus subtilis, in order to develop Gram-positive production hubs of anticancer therapies. Using the bacteria-in- spheroid coculture (BSCC) model, we can rapidly screen for optimal isolates and recombinant protein products and use in vivo mouse models for follow up experiments exploring only the high preforming candidate strains. With in vitro and in vivo approaches, we aim to develop novel, safe, and effective alternatives to treat tumor growth and increase the quality of cancer therapy.