Genetic Design
Our engineered microbe incorporates four independent modules, which are designed to fullfill a series of functions from CRC targeting and ultrasonic reporting to preliminary treatment and finally, removal from the digestive tract.
Gene circuit overview diagram.
The CRC targeting module enables bacteria to specifically adhere to CRC lesions in the lumen. To this end, E.coli is designed to display a CRC antigen binding moiety on its surface, through which it can be tethered on the foci of CRC. For surface display vectors, we choose two engineered bacterial outer membrane proteins—OmpA (BBa_K1489002) and ice nucleation protein [1] Reference 1 [1]Bao, S., Yu, S., Guo, X., Zhang, F., Sun, Y., & Tan, L., et al. (2015). Construction of a cell-surface display system based on the n-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins. Journal of Applied Microbiology, 119(1), 236-244. , which are commonly used as carriers of protein/peptide to the outer surface of gram-negative bacteria.
As for the antigens to be targeted, there exists few choices. Despite the abundance of antigens we could think of regarding CRC, such as CEA and EGFR, the mucus layer in the gut prevents direct contact between cancer cells and microorganism. Therefore, a mucus related CRC antigen--Thomsen-Friedenreich (TF-antigen), is chosen as the target. TF-antigen is an epitope on mucin specifically secreted by most CRC cells, against which various targeting nanoprobes have been developed as optical imaging agents for early-stage CRC to assist colonoscopy [2] Reference 2 [2]Kumagai, H., Pham, W., Kataoka, M., Hiwatari, K., Mcbride, J., & Wilson, K. J., et al. (2013). Multifunctional nanobeacon for imaging thomsen-friedenreich antigen-associated colorectal cancer. International Journal of Cancer, 132(9), 2107-2117. [3] Reference 3 [3]Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., & Miyata, K., et al. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of thomsen-friedenreich associated colorectal cancer:. International Journal of Nanomedicine, 12, 1747-1755. .
For TF-antigen targeting, we find out two oligopeptides from a phage display library that bind specifically to TF-antigen [4] Reference 4 [4]Liu, R., Li, X., Xiao, W., & Lam, K. S. (2017). Tumor-targeting peptides from combinatorial libraries. Adv Drug Deliv Rev, 110-111, 13-37. . Moreover, these relatively hydrophilic peptides are supposed to inhibit non-specific interactions between bacteria and mucosa [3] Reference 3 [3]Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., & Miyata, K., et al. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of thomsen-friedenreich associated colorectal cancer:. International Journal of Nanomedicine, 12, 1747-1755. [5] Reference 5 [5]Lukic, J., Strahinic, I., Milenkovic, M., Nikolic, M., Tolinacki, M., & Kojic, M., et al. (2014). Aggregation factor as an inhibitor of bacterial binding to gut mucosa. Microbial Ecology, 68(3), 633-644. .
Bacterial adherence to TF-antigen via surface displayed peptides.
The ultrasonic reporting module contains a cluster of engineered gas vesicle proteins (ARG) that could assemble to form gas vesicles in the cytoplasm of E.coli, which are able to generate signals upon high-resolution ultrasonic inspection [6] Reference 6 [6]Bourdeau, R. W., Leegosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., & Nety, S. P., et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature,553(7686), 86. . The expression of these gas vesicles is controlled by T7 lac promoter. After introduction into the gut, these gas vesicles would indicate the location of our engineered bacteria via ultrasonic response.
Gas vesicles for acoustic response.
The suicide module is for removing the engineered bacteria from the digestive tract right after the examination, in a bid to eliminate the risk of, for example, interfering gut flora as much as possible. Considering that arabinose would serve as a safe and effective inducer of bacterial gene expression in the gut, we place the phage protein φX174E (BBa_K2500006), which triggers bacteria cell lysis under control of arabinose operon, to achieve an arabinose-governed bacterial cell death.
Cell lysis for removal and cargo release.
The therapeutic module aims for a preliminary bio-treatment of CRC after its diagnosis. This module is to be introduced into another strain, which is employed for treatment only, together with the CRC targeting module and suicide module. As a minor part of our project, the therapeutic module mainly consists of a well established anticancer agent—a peptide derived from azurin (BBa_K2500002), used by ETH Zurich 2017. In order to produce the agent in a CRC specific manner, a nitric oxide responsive promoter (pYeaR/pNorV) manipulates its expression, since nitric oxide is an unstable molecule overproduced by CRC cells and polyps, with its concentration highest around these lesions [7] Reference 7 [7]Cianchi, F., Cortesini, C., Fantappiè, Messerini, L., Schiavone, N., & Vannacci, A., et al. (2003). Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis. American Journal of Pathology, 162(3), 793-801. [8] Reference 8 [8]Keum, D. H., Jung, H. S., Wang, T., Shin, M. H., Kim, Y. E., & Kim, K. H., et al. (2015). Microneedle Biosensor for Real-Time Electrical Detection of Nitric Oxide for In Situ Cancer Diagnosis During Endomicroscopy. Advanced Healthcare Materials, 1153-1158. . The targeting module would work in synergy, and the suicide module releases the agent from the cells.
Application Design
Ultrasonic imaging: the key to successful cancer imaging using our engineered bacteria is the sufficiency of maximal attachment to the tumor and minimal attachment to normal mucus of our bacteria. Therefore, we incorporate the delivery of our bacteria into the conventional bowel preparation protocols for colonoscopy and ultrasonic imaging of the colon, which our HP team learned from interview with experts.
The bowel preparation procedure is roughly summarized as follows:
laxative –clear liquid –enema
Among which clear liquid, typically 500-1000ml, orally introduced 4 hours before ultrasonography, is most suitable for carrying our engineered bacteria in terms of sufficient quantity, time for adhesion, and upcoming enema which could wash away non-specifically attached bacteria.
Bacteria should be induced with 1uM lactose for at least 22h before mixed into 500-1000ml clear liquid, with final concentration around 107cell/ml. After ultrasonography, the bacteria could be removed by orally administered arabinose.
The imaging procedure can be adapted from our protocols. In general, ultrasound plays two roles: imaging and collapsing. As Bourdeau et. al. [6] Reference 6 [6]Bourdeau, R. W., Leegosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., & Nety, S. P., et al. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature,553(7686), 86. reported, the gas vesicles produced by the engineered microbe can be collapsed with acoustic pressure that exceeds certain intensity, therefore enabling distinguishing target signal with background. The strategy is collecting imaging graphs right before and after introducing high-power ultrasound to collapse target gas vesicles in areas of interest, and the subtraction between two images would indicate the target signal.
Treatment: after diagnosis of CRC, capsules containing the treatment strain could be administered. Likewise, arabinose should be taken subsequently to release the agent.
Reference
[1] Bao, S., Yu, S., Guo, X., Zhang, F., Sun, Y., Tan, L., ... & Ding, C. (2015). Construction of a cell‐surface display system based on the N‐terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins. Journal of applied microbiology, 119(1), 236-244.
[2] Kumagai, H., Pham, W., Kataoka, M., Hiwatari, K. I., McBride, J., Wilson, K. J., ... & Gore, J. C. (2013). Multifunctional nanobeacon for imaging Thomsen‐Friedenreich antigen‐associated colorectal cancer. International journal of cancer, 132(9), 2107-2117.
[3] Nakase, H., Sakuma, S., Fukuchi, T., Yoshino, T., Mohri, K., Miyata, K., ... & Tobita, E. (2017). Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer. International journal of nanomedicine, 12, 1747.
[4] Liu, R., Li, X., Xiao, W., & Lam, K. S. (2017). Tumor-targeting peptides from combinatorial libraries. Advanced drug delivery reviews, 110, 13-37.
[5] Lukic, J., Strahinic, I., Milenkovic, M., Nikolic, M., Tolinacki, M., Kojic, M., & Begovic, J. (2014). Aggregation factor as an inhibitor of bacterial binding to gut mucosa. Microbial ecology, 68(3), 633-644.
[6] Bourdeau, R. W., Lee-Gosselin, A., Lakshmanan, A., Farhadi, A., Kumar, S. R., Nety, S. P., & Shapiro, M. G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature, 553(7686), 86.
[7] Cianchi, F., Cortesini, C., Fantappiè, O., Messerini, L., Schiavone, N., Vannacci, A., ... & Perna, F. (2003). Inducible nitric oxide synthase expression in human colorectal cancer: correlation with tumor angiogenesis. The American journal of pathology, 162(3), 793-801.
[8] Keum, D. H., Jung, H. S., Wang, T., Shin, M. H., Kim, Y. E., Kim, K. H., ... & Hahn, S. K. (2015). Microneedle Biosensor for Real‐Time Electrical Detection of Nitric Oxide for In Situ Cancer Diagnosis During Endomicroscopy. Advanced healthcare materials, 4(8), 1153-1158.
