Collagenase Expression Augments the Interstitial Transport and Tumor Colonization Potential of Tumor-Targeting Salmonella Typhimurium
One of the principal impediments to the broad success of conventional chemotherapy is poor delivery to and transport within the tumor microenvironment (TME), caused by irregular and leaky vasculature, the lack of functional lymphatics, and underscored by the overproduction of extracellular matrix (ECM) proteins such as collagen. Co-administration of proteases that degrade the ECM has been shown to significantly enhance the transport of traditional macromolecule and nanoparticle-based chemotherapeutics in tumors. However, systemic administration causes collateral damage to normal tissue and has been suggested to increase the risk of tumor metastasis, necessitating targeted delivery. An innovative approach is to use engineered bacteria-based cancer therapy (BBCT), wherein microbes such as Salmonella Typhimurium, which preferentially replicate in the TME, are programmed to locally degrade the ECM. Not only would this target ECM degradation in tumors, but such an approach would augment the intrinsic benefits of BBCT over passive modalities such as active translocation and the ability to transport drug-loaded cargo. In this work, we engineered attenuated tumor-targeting S. Typhimurium VNP20009 to express and secrete a recombinant collagenase to locally degrade the ECM. We then developed a microfluidic assay to systematically characterize the engineered bacteria transport in a tissue-mimicking reductionist collagen model. Our platform supplies nutrients to support long-term growth and collagenase expression in time-lapse experiments while creating small, transient interstitial flow reminiscent of the TME. To eliminate the confounding effects of heterogeneity in population motility on transport, we performed experiments with non-motile mutants, finding that collagenase secretion increased the depth of penetration in collagen by 2.5-fold during 20 hr experiments. Using a diffusion-advection-reaction model of transport and growth, we show that diffusion plays little role in transport, but the average peak effective advection coefficient was significantly enhanced by more than 2-fold for the collagenase-secreting strain. We noted however that collagenase expression has deleterious effects on motility-driven transport due to a reduction in bacterial fitness and motility. To minimize this effect while maintaining a high level of collagenolytic activity, we modulated collagenase expression and studied bacteria transport in collagen and agar gels in the absence of flow. In agar, where collagenase had no effect, the highly motile and fast-growing control strain robustly outperformed the collagenase-expressing strain. However, engineered bacteria in collagen demonstrated a wide range of performance compared to the control based on their fitness and motility. We show that tempering the rate of collagenase expression enhanced the transport in and colonization of the collagen gel, allowing the engineered bacteria to outperform the control strain. In our current work, we are evaluating bacterial transport and colonization of ECM-rich tumor organoids and further optimizing collagenase expression to effectively modify the physical characteristics of the target tumor microenvironment. Overall, our results suggest that collagenase expression has the potential to robustly enhance bacterial transport when small pore size precludes motility, but precise engineering is required in less confined environments where fitness plays a role, both of which may be encountered in the notoriously heterogeneous tumor stroma. This research represents a substantial step forward in the rational engineering of BBCT, which today is increasingly being realized as a powerful treatment of cancer. BBCT has and continues to be proven in a plethora of in vitro and in vivo animal studies. The development of strains that have (1) been shown safe for human administration and (2) demonstrated a sufficient level of tumor colonization with minimal side effects in immunocompetent hosts are the primary hurdles for translation to the clinic. The first of these challenges has been repeatedly met; addressing bacterial transport limitations in desmoplastic tumors is a largely unexplored avenue and has the potential to surmount the second.
Collagenase Expression Augments the Interstitial Transport and Tumor Colonization Potential of Tumor-Targeting Salmonella Typhimurium
Category
Poster Presentation
Description
Session: 16-01-01 National Science Foundation Posters - On Demand
ASME Paper Number: IMECE2020-25299
Session Start Time: ,
Presenting Author: Eric Leaman
Presenting Author Bio: Eric Leaman is a Ph.D. candidate in the Department of Mechanical Engineering at Virginia Tech, where he studies ways to engineer microbial systems for cancer therapy using microfluidic tools and computational modeling in Prof. Bahareh Behkam's group. He received his B.S. in engineering from James Madison University in 2014 and his M.S. in mechanical engineering from Virginia Tech in 2016. Eric has been honored with several awards during his graduate studies, including two best paper awards, travel awards, and the Ellen E. Wade Graduate Studies Fellowship at Virginia Tech.
Authors: Eric Leaman Virginia Polytechnic Institute and State University
Bahareh Behkam Virginia Polytechnic Institute and State University