ABSTRACT
Hypoxia is one of the main features of solid tumors including breast cancer and has been shown to
correlate with a poor prognosis. Although many chemotherapeutic agents such as paclitaxel and
doxorubicin are significantly effective in a normoxia environment, they are less effective in hypoxic tumor
regions due to poor infusion, hypoxia, and acidity. Given its significant impact on treatment and tumor
progression, the development of a new approach to specifically target the hypoxic regions of tumors is
clinically needed. To address the challenges (unmet medical needs), our objective for this proposal is to
develop a new probiotic-assisted approach as bacteria preferentially migrate and accumulate in the hypoxic
region of the tumor. We developed our original hypoxia-inducible expression system and have optimized its
use in E. coli G3/10 cells, which have been used as a probiotic in humans. We demonstrated that the G3/10
cells preferentially localized the hypoxic regions of xenografted breast tumors in mice. With the new tools of
the hypoxia-inducible expression system in G3/10 cells, we will deliver cancer-killing cupredoxin proteins,
azurin or rusticyanin, in the hypoxic region of triple-negative breast cancer as they have limited treatment
options and a worse prognosis. We hypothesize that the development of a new bacteria-assisted approach
to express cancer-killing proteins under the hypoxia-inducible promoter will provide functional tools for
specifically targeting hypoxic tumors. To test our hypothesis and improve current therapy, we designed
experiments based on our “bi-directional” strategy; a combination of the G3/10-based therapy with standard
chemotherapeutic agents (paclitaxel and doxorubicin), so that the chemotherapy suppresses tumors in the
outer periphery regions, perfused tumor regions, and our bacterial approach kills tumor in the hypoxic inner
regions. Our preliminary data strongly support this concept. Considering the NIH/NCI’s focusing areas in
FOA PAR-22-085 (Microbial-based Cancer Imaging and Therapy - Bugs as Drugs), we specifically formed a
multidisciplinary team that integrates expertise in basic, translational, and clinical breast cancer biology,
microbiology, molecular biology, immunology, pathology, and biostatistics. With the five years of preclinical
research proposed in this application, we will discern the value of engineered G3/10 cells as effective
bacteria-assisted functional tools. Results from the proposed studies will potentially provide new and
effective treatment strategies that specifically target the hypoxic tumor microenvironment.