The Problem
Malignant tumors are one of the leading causes of death worldwide. In the United States alone, there are 1.9 million new cancer cases projected to occur in 2022. [1] The pandemic of COVID-19 has put a tremendous burden on healthcare systems, resulting in delayed treatments and diagnoses.
Compared to recent years, colonoscopies in the United States dropped by 45%, while prostate biopsies and tomography scans decreased by 29 % and 10 % in 2020. [2] Patients in Hungary faced similar problems. The number of diagnoses decreased by 10-20% in 2020 and has not reached pre-pandemic as was reported in 2022. Performed procedures and therapies also declined: surgeries by 39.6% in lung, 12.0% in breast and 20.5% in colorectal cancer, or radiation therapy (22.3%, 15.8%, 28.3%) and the smallest for chemotherapy (4.3%, 4.1%, 3.8%). Mayer et al. reason these with extended time for examination, limited capacities, and restricted access to health care facilities. [3]
Delayed treatments and the backlog of procedures may cause a temporary decrease in cancer incidence, followed by a rise in more severe cases. [1] However, deciphering the results of these effects will take years; it demonstrates a clear need for solutions that deliver efficient treatment and accelerate diagnosis.
Project Overview
Team ELTE's project is titled NanoBlade, a novel bacterial-based tool for targeted tumor diagnostics and therapy.
Our Team has taken a theranostic approach to tackle cancer. The proposed NanoBlade is an engineered E. coli capable of detecting cancer cells and eliminating them locally upon blue light irradiation. Accordingly, it comprises two elements: a detecting device called Nanobody Display System and a drug delivery device, the BLADE Expression System, which secretes toxins into the tumor environment upon blue-light induction.
Nanobody Display System
Facultative anaerobic bacterial strains such as E. coli have an intrinsic ability to colonize and proliferate in the tumor microenvironment. [4] However, these features alone are insufficient to achieve adequate (colonization and) therapeutic effect. Therefore, targeting strategies should be implemented to tackle these obstacles. [5] For this aim, our Nanobody Display System introduces a tumor antigen-specific Nanobody to bacteria's outer membrane. Surface displaying is carried out with a bacterial adhesion factor fragment called Intimin.
These tumor-selective interactions can be visualized through fluorescence thanks to GFP co-expression. (Moreover, the construct contains two different epitope tags (such as E and Myc); therefore, the interaction can be verified via immunostaining) We hypothesized this could be used for determining the exact localization of tumors. We have targeted two different tumor-specific antigens. (1) Epidermal growth factor receptor (EGFR) that can be found in high copies on many types of cancer cells (ovarian, endometrial, breast, colorectal), (2) and carcinoembryonic antigen (CEA) that is associated with colorectal cancer progression.[6, 7]
We have proposed an advanced version of our tumor-targeting apparatus, the MiniNano System. This construct is more compact and integrates the tumor targeting and detection features into one component. It visualizes tumor tissues with a near-IR emitting fluorescence protein called miRFP670 nano, so it is more applicable since the light at this wavelength range penetrates tissues deeper. [8, 9] We also used a different surface displaying method. In this case, INPNC is a truncated N-C terminus version of Ice Nucleation Protein that can be used for surface display experiments. [10]
BLADE Expression System
Light can be exploited not only to visualize and investigate but also to manipulate biological systems. Many photosensitive proteins can be found in nature (photosensors, photoreceptors), which harbour important functions in the host organisms. These proteins can be implemented and genetically modified to control biological processes with light, which is the foundation of optogenetics. [11] Otherwise, light is harnessed widely in therapeutic applications (such as photodynamic therapies) because it is a precise and non-invasive tool and provides excellent spatio-temporal control with minimal perturbation. [8] We aimed to utilize these advantages and create a genetically encoded, light-inducible drug delivery system.
The centerpiece of our device is a blue-light inducible promoter system derived from the L-Arabinose operon, which is widely used for protein expression. The arabinose-binding domain of the transcription factor AraC is swapped to a blue-light sensitive Vivid (VVD) domain, which homodimerizes upon irradiation, triggering protein expression. This AraC-VVD fusion protein is called BLADE, abbreviating blue light-inducible AraC dimers in Escherichia coli. We have chosen BLADE over other light-responsive expression systems because it is a one-component system, which means only one protein is needed to achieve light-inducibility. Therefore, it does not burden the host organism as other, more complex systems, which was a key consideration for us. Nevertheless, blue light is not preferred in therapeutic applications; hence it exerts weak tissue penetration features, but in the literature, there are only one-component systems in this wavelength range. [12, 13]
For achieving the therapeutic effect of NanoBlade, we use BLADE to secrete a pore-forming toxin, Cytolysin A (ClyA), into the tumor environment to eliminate cancer cells locally. Cytolysin A is secreted autonomously by some bacterial strains of the Enterobacteriaceae family and thoroughly investigated its applicability in tumor therapy. [14]
To test our light-inducible system, we have developed an easy-to-use, easy-to-build irradiation setup, which can help others entering the field of optogenetics. (To learn more, please visit our Hardware page)
Project Inspiration
Our Team aimed to find a way to facilitate both tumor diagnostics and therapy to shorten the time it takes for patients to receive appropriate treatment. We have decided to work on a bacteria-based application since bacteria is one of the most versatile tool of synthetic biology and is highly preferred among living therapeutics. [14]
Now, we would like to briefly describe whose work inspired us to create NanoBlade. (To know what safety and socio-economical considerations shaped our project, please visit our Safety and Human Practices).
The Nanobody Display System is entirely adapted from the work of Lim and his colleagues. [15] Although the Intimin-based Nanobody surface display was developed by Carlos Piñero-Lambea and his co-workers, who showed the construct's usability in tumor-specific attachment, the interaction was detectable via immunostaining only. [16] Lim combined it with sfGFP co-expression, adding a fluorogenic feature to the system. Moreover, they demonstrated efficient, targeted drug delivery using salicylate hydroxylase to produce anticancer compounds. This aspect of their work inspired us to integrate this system into our project, and we converted it to Biobricks Parts.
During our design process, we explored synthetic biological approaches for optogenetic applications in bacteria. For this, we have heavily relied on two reviews by Florian Lindner & Andreas Diepold and Armin Baumschlager & Mustafa Khammash. [11, 12] We have decided to use BLADE (Romano et al., 2021), and we converted it to BioBricks Part. [17]
The work of Team UPENN 2012 was also an essential inspiration for us, as they pursued light-inducible targeted therapy too. In addition, we have integrated their surface display system into Mininano System, widening its functionalities. [18]
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[2] Englum, B. R.; Prasad, N. K.; Lake, R. E.; Mayorga-Carlin, M.; Turner, D. J.; Siddiqui, T.; Sorkin, J. D.; Lal, B. K. Impact of the COVID-19 Pandemic on Diagnosis of New Cancers: A National Multicenter Study of the Veterans Affairs Healthcare System. Cancer 2022, 128, 1048–1056. https://doi.org/10.1002/cncr.34011.
[3] Mayer, B.; Tóth, M.; Csanádi, M.; Zemplényi, A.; Fadgyas-Freyler, P.; Elek, P.; Szécsényi-Nagy, B. The Impact of the COVID-19 Pandemic on Cancer Care. Népegészségügy : A Népegészségügyi Képző- és Kutatóhelyek Országos Egyesületének Tudományos Folyóirata 2022, 99, 144–153. http://real.mtak.hu/146762/.
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[7] Kim, G.; Jung, E.-J.; Ryu, C.-G.; Hwang, D.-Y. Usefulness of Carcinoembryonic Antigen for Monitoring Tumor Progression during Palliative Chemotherapy in Metastatic Colorectal Cancer. Yonsei Med. J. 2013, 54, 116–122. https://doi.org/10.3349/ymj.2013.54.1.116.
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[10] Team UPenn 2012. https://2012.igem.org/Team:Penn.
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[12] Lindner, F.; Diepold, A. Optogenetics in Bacteria – Applications and Opportunities. FEMS Microbiol. Rev. 2021, 46. https://doi.org/10.1093/femsre/fuab055.
[13] Murase, K. Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins 2022, 14, 78. https://doi.org/10.3390/toxins14020078.
[14] Cubillos-Ruiz, A.; Guo, T.; Sokolovska, A.; Miller, P. F.; Collins, J. J.; Lu, T. K.; Lora, J. M. Engineering Living Therapeutics with Synthetic Biology. Nat. Rev. Drug Discov. 2021, 20, 941–960. https://doi.org/10.1038/s41573-021-00285-3.
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[17] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nat. Chem. Biol. 2021, 17, 817–827. https://doi.org/10.1038/s41589-021-00787-6.
[18] Magaraci, M. S.; Veerakumar, A.; Qiao, P.; Amurthur, A.; Lee, J. Y.; Miller, J. S.; Goulian, M.; Sarkar, C. A. Engineering Escherichia Coli for Light-Activated Cytolysis of Mammalian Cells. ACS Synth. Biol. 2014, 3, 944–948.https://doi.org/10.1021/sb400174s.