Introduction

In Hungary, 70,000 patients are diagnosed with malignant tumors every year [1]. Cancer is the leading cause of death in almost every part of the world. Doctors and researchers are constantly fighting the disease and trying to stay one step ahead. Our team's goal was nothing less than to join the fight with a new perspective, armed with cutting-edge tools of synthetic biology. That's why we created the NanoBlade system, which approaches the problem from both a diagnostic and a therapeutic aspect. NanoBlade is a bacterial-based tumor therapy.

Bacteria-based therapies have been known in the scientific world for decades, but in recent years (especially after the 2000s), the number of publications on the subject has increased significantly. It is due to the fact that technologies are constantly evolving, and our knowledge is expanding. Scientists have recognized that bacteria-based therapies have many advantages that can help overcome the limitations of conventional treatments. Cancer, for example, is precisely one of the challenges facing science that requires a multipronged approach to treat it effectively [3]. However, traditional techniques such as chemotherapy or radiotherapy have three very important shortcomings: incomplete tumor targeting, inadequate tissue penetration, and limited toxicity. Bacteria-based therapies can address all three problems, as they can penetrate tissues and specifically colonize tumors, where they can induce cytotoxicity [4]. Another great advantage of bacteria is that they can be easily genetically manipulated, allowing for the simultaneous and precise combination of several useful functions. It may include immunotoxin transport or local production [5].

Bacteria are versatile and can be used in many different ways in cancer therapy. Currently, a number of studies have been carried out using different strains, such as Salmonella, Escherichia, Clostridium, and Bifidobacterium, some of which have been tested in mammalian and human trials. Although most experimental results have reported a reduction in tumor size and increased survival, each strain of bacteria has advantages and disadvantages, so the choice of which one to use needs careful consideration [4] [5].

There are still several obstacles to the use of bacteria-based therapies, such as the genetic instability of the organisms. Our team has already tried to take this into our project design, which is why we have improved the originally multi-component Nanobody Display System to a single component (for details, visit Here). Another big problem currently with these therapeutic approaches is that the bacteria themselves can be toxic, causing significant side effects. Therefore, researchers are experimenting with strains that contain weakened or modified bacteria to reduce the risk of this problem [3].

At present, bacteria-based therapies have their limitations, but it is likely that in the future, many such therapies will emerge, both for diagnostic and therapeutic purposes. These systems may enhance the efficacy of current therapies, but they can also be used on their own. Therefore, the potential of this field should not be overlooked. The results achieved so far with bacteria-based therapies are encouraging, but they are few and far between, and it will take time before they become widespread.

Use of the product

System operation and end users

The bacteria in the NanoBlade system are able to attach to the antigens of cancer cells and colonize them. Within the organism, they can be identified by their fluorescence properties and secrete an immunotoxin upon blue light induction that kills cancer cells (Figure 2.). The system we have designed has several advantages that make it a very effective tool in the fight against cancer. It has the benefit of being able to bind specifically to the supracellular molecules of cancer cells, thus ensuring precise targeting of the tumor in the body. This property also provides that the cells cannot interact with other cells in the body. Moreover, blue light induction allows us to control the toxin production of our bacteria in space and time. The production of Cytolisin-A, which is a highly effective ”hole-punching” protein, can eliminate cells with excellent efficiency.

As our product has both a diagnostic and a therapeutic element, the number of end users could be widespread, from skin cancer to colon cancer patients. Nevertheless, according to Dr. Imre Kacskovics (DVM., Ph.D., D.SC.), our system can be really effective where it is not essentially necessary for the bacteria to penetrate the mucosal membrane. Thus, the primary target of our therapy can be colon cancer, where the implementation of the illumination system is also relatively simple. Our system could be suitable for the confirmation of suspected cancer cases, as fluorescent cells with GFP may be able to detect cancerous tissues and take action if needed. Furthermore, the product can not only detect tumors but can also kill them locally by light induction. Thus, it can be usable in patients with a later, more severe stage of cancer. Our solution may benefit even more patients with metastatic cancer, as our bacteria could reach sporadic cancer tissues. The result is a simple, fast, and highly effective system.

With our final product, we want to help doctors in the fight against cancer as much as possible, so we envision a wide range of medical applications. First of all, the type of cancer to be cured affects the applicability of the elements of our system and the methods of its implementation. However, the complexity of the illumination system requires medical expertise, so it can be used in hospitals and clinics for most types of cancer.

The delivery of a medicinal product to the body is always a key issue because the choice of this has a major impact on the acceptability and actual use of the medicine. Since bacterial-based therapies are not yet a standard treatment, the question of how to administer these products is relatively open. However, the results of several cancer-related studies are known, in which the products have been administered in different ways. For example, the intratumoral approach is one known solution that has been tested and reported to have an anti-tumor effect. The advantage of this method is that it can easily avoid the systemic spread of bacteria in the body. Of course, the bacteria can also be introduced into the body by intravenous administration, which has also been tested. A special delivery method is an intranasal route, where the main target is the respiratory tract. External solutions such as ointments or special patches may be used for skin tumors, while subcutaneous solutions may be used for subcutaneous tumors. The latter can of course be used against cancer cells elsewhere in the body [6].

One of the best-studied methods is oral administration [7][8]. This method has also been used in in vivo experiments, where it has been shown that oral administration can translocate bacteria from the gastrointestinal tract and deliver them to the tumor. The results also showed that the systemic presence of the bacteria was generally not increased, i.e., the results so far indicate that oral administration is safe and does not pose an increased risk. In our questionnaire for the Human Practices section, we sought to assess the attitudes of end-users regarding which method of delivery they prefer. Our results showed that oral forms (capsules, tablets) are the most accepted. After surveying people's opinions on the topic, we definitely wanted to ask the experts' opinions as well. (More information on Human practices and Interview page). After our conversation with Dr. David T Riglar, we were more and more certain that oral administration was our goal in the future. However, we constantly came up with ideas for additional dosing methods, such as suppositories or fecal transplantation. In order to find out whether these dosage forms fit our project, we consulted Dr. Árpád Patai, a gastroenterologist, who provided us with a lot of useful information. He pointed out that in the case of rectal administration, the bacteria would be absorbed by the rectum's blood vessels, and then transported to other organs of the body via the circulatory system. Therefore, if the target is colon cancer, this is not necessarily the best option, but this method can also be used in the case of other types of cancer. Our other idea to target our bacteria was fecal transplantation, which is used to cure Clostridioides difficile infection (CDI). Although we were afraid that this method would cause resentment among the patients, according to Dr. Patai's opinion, most people would also accept this method of administration.

Overall, we would administer our product in oral form (capsule, tablet), however, depending on the type of cancer, other earlier listed methods are also possible, such as suppository or fecal transplantation.

Lighting

Perhaps one of the most critical elements of our project in terms of applicability is how we can induce our bacteria with blue light. Of course, we have tried to find a solution to that as well. Several lighting systems have been developed to use, such as LEDs or lasers. In such approaches, it is worth considering the form in which the light is to be used, for example, the tissue penetration of it. It has the advantage that it does not necessarily require medical supervision, as for certain types of cancer or tumors (e.g., skin cancer), the patient can even perform the illumination themselves. When we designed our project, we tried to create the system in a way that would not require an invasive procedure, but it became clear to us that this was not feasible for all types of cancer. This could be the case, for example, with colon cancer, which is one of the main targets of the project.

In order to make the possible colonoscopy procedure as efficient as possible, we tried to come up with a special device that could both detect cancer cells and illuminate them with blue light. Our idea was to combine in a single fiber-optic system a unit capable of processing a fluorescent signal and emitting blue light, thereby inducing the cells.

Reading the literature, we came across a method of fluorescence endoscopy that would be perfectly suited to our aims [9][10][11]. The basis of these systems is the fiber optic endoscope, also used in clinical practice [12]. By changing different optical filters from the light source, we can create white light and excitation light with different wavelength ranges, which can be used to excite fluorescent biomarkers. In addition, the excitation light can be used to induce the BLADE system using the appropriate optical filter set. The system also includes other optical elements and two cameras. A color camera is needed for white light imaging and a high-sensitivity electron-multiplier-CCD (EM-CCD) camera for fluorescence detection. With this system, it is also possible to perform fluorescence imaging in different spectrums and blue light induction. The method is also suitable for the quantitative detection of fluorescent signals in tissues, thus providing extra information about the tumor size [13]. Early diagnosis is of paramount importance in colorectal cancer, and such an efficient tool, which would allow the detection of cancer cells with high efficiency, even in a single intervention, could greatly improve survival rates.

One of the unplanned but more successful elements of our project has been the hardware, which has many advantages. The blue-light illumination system designed for the laboratory has several advantages over its competitors, making it suitable for research and industrial use. It can be functional for research and testing of light-indicated systems like ours. For details, visit our Hardware section.

Safety

From the design stage of our project, we put a strong emphasis on security and tried to create a system with minimal risk factors. Of course, every form of treatment has its risk factors, and our system is no exception. There are two main elements of safety considerations. One is to avoid bacteria entering the bloodstream as much as possible. The other major risk factor is blue-lighting illumination. Unnecessarily prolonged exposure to blue light can damage not only the helper bacteria but also the surrounding tissue. However, these hazards can be easily avoided by proper use of the product and by following the safety instructions. (Please see the Safety page.)

During the design of the project, we tried to assess the expectations of potential end-users, mainly based on the responses to our survey. In this, we found that respondents put a high value on safety. This was also confirmed by the fact that, in the evaluation of the joint questionnaire with the Wageningen_UR team, we also found that safety was definitely a top priority. We, therefore, tried to find a way of developing our product that would help us to create an even safer result. So-called Minicells have recently emerged in scientific research [14]. These are small cells without chromosomes that cannot reproduce but can produce a very high level of protein. Minicells are created from bacteria containing the min mutant gene, and they have several properties that benefit us. For example, they cannot divide, so there is no risk of them causing systemic disease. They also have the bonus that they can easily take up plasmids and produce them as they carry only a fraction of their genome. This helps us to produce the proteins we need and to run our systems more efficiently. Their uses can be many and varied, but they have been the main focus of recent attention in drug delivery and vaccine research. There are also known minicell-based formulations that have been successfully used in Phase I clinical trials. This knowledge may facilitate the potential licensing mechanism for our product. In view of the above, we have come to the decision that we would definitely use minicell technology in the future to develop our therapeutic and diagnostic system, taking full advantage of its benefits. From safety to increased protein production [15].

Challenges and future

We cannot say at this stage what resources and opportunities will be available to us in the future, but if there is an opportunity to take the project forward and develop it further, we would like to take it.

The first step would be to optimize the intensity and duration of the light induction. For this, we have the help of our hardware illumination system, which allows the simultaneous analysis of many parallel samples. This also helps us to work quickly and efficiently.

In the future, we would also like to improve the project we invented, to which our interviews have significantly contributed. One of the main comments Dr. Árpád Patai had one main observation, that the targets we chose (CEA, EGFR) are not expressed in all types of cancer. Therefore, we started looking for additional options in the literature, mainly for colorectal cancer, as this is one of the main targets of our project. Hence, we started looking for different options in the literature, mainly for colorectal cancer, as this is one of the main targets of our project. By searching the literature, we found several other possible targets, such as VEGFR, HER2, IGF-1R, or DR5 [16][17]. We would definitely expand our Display System with these targets in the development of our product. By combining all of these, we can increase the efficacy of our system and offer people an even more universal therapy.

At present, both in Hungary and in the European Union, there are very strict regulations on the use of GMOs in pharmaceuticals, which we will also have to comply with. In Hungary, the National Institute of Pharmacy and Nutrition (OGYÉI) is responsible for the authorization of medicines, and its current regulation is in line with the EMA guidelines [18]. The regulation allows for the testing and marketing of GMO-based medicinal products, provided that all competent authorities comply with the requirements, which mainly aim to assess the safety of GMOs, their environmental impact, and possible side effects. All such products must undergo a scientific risk assessment and cannot be placed on the market without a proper scientific basis [19].

[1] https://www.ksh.hu/stadat_files/ege/hu/ege0025.html Accessed October 8, 2022.

[2] https://gco.iarc.fr/causes/obesity/tools-pie Accessed October 8, 2022.

[3] Gupta, K. H.; Nowicki, C.; Giurini, E. F.; Marzo, A. L.; Zloza, A. Bacterial-Based Cancer Therapy (BBCT): Recent Advances, Current Challenges, and Future Prospects for Cancer Immunotherapy. Vaccines 2021, 9, 1497. https://doi.org/10.3390/vaccines9121497.

[4] Forbes, N. S. Engineering the Perfect (Bacterial) Cancer Therapy. Nature Reviews Cancer 2010, 10, 785–794. https://doi.org/10.1038/nrc2934.

[5] Sedighi, M.; Zahedi Bialvaei, A.; Hamblin, M. R.; Ohadi, E.; Asadi, A.; Halajzadeh, M.; Lohrasbi, V.; Mohammadzadeh, N.; Amiriani, T.; Krutova, M.; Amini, A.; Kouhsari, E. Therapeutic Bacteria to Combat Cancer; Current Advances, Challenges, and Opportunities. Cancer Medicine 2019, 8, 3167–3181. https://doi.org/10.1002/cam4.2148.

[6] Shende, P.; Basarkar, V. Recent Trends and Advances in Microbe-Based Drug Delivery Systems. Daru: Journal of Faculty of Pharmacy, Tehran University of Medical Sciences 2019, 27, 799–809. https://doi.org/10.1007/s40199-019-00291-2.

[7] Jia, L.-J.; Wei, D.-P.; Sun, Q.-M.; Huang, Y.; Wu, Q.; Hua, Z.-C. Oral Delivery of Tumor-Targeting Salmonella for Cancer Therapy in Murine Tumor Models. Cancer Science 2007, 98, 1107–1112. https://doi.org/10.1111/j.1349-7006.2007.00503.x.

[8] Cronin, M.; Morrissey, D.; Rajendran, S.; El Mashad, S. M.; van Sinderen, D.; O’Sullivan, G. C.; Tangney, M. Orally Administered Bifidobacteria as Vehicles for Delivery of Agents to Systemic Tumors. Molecular Therapy 2010, 18, 1397–1407. https://doi.org/10.1038/mt.2010.59.

[9] Ramezani, S.; Parkhideh, A.; Bhattacharya, P. K.; Farach-Carson, M. C.; Harrington, D. A. Beyond Colonoscopy: Exploring New Cell Surface Biomarkers for Detection of Early, Heterogenous Colorectal Lesions. Frontiers in Oncology 2021, 11. https://doi.org/10.3389/fonc.2021.657701.

[10] de Jongh, S. J.; Voskuil, F. J.; Schmidt, I.; Karrenbeld, A.; Kats-Ugurlu, G.; Meersma, G. J.; Westerhof, J.; Witjes, M. J. H.; van Dam, G. M.; Robinson, D. J.; Nagengast, W. B. C-Met Targeted Fluorescence Molecular Endoscopy in Barrett’s Esophagus Patients and Identification of Outcome Parameters for Phase-I Studies. Theranostics 2020, 10, 5357–5367. https://doi.org/10.7150/thno.42224.

[11] Yoo, S. W.; Nguyen, D.; Park, S.; Lee, H.; Lee, C.-M.; Lee, C.; Min, J.-J. Development of Dual-Scale Fluorescence Endoscopy for in Vivo Bacteria Imaging in an Orthotopic Mouse Colon Tumor Model. Applied Sciences 2020, 10, 844. https://doi.org/10.3390/app10030844.

[12] Mitsunaga, M.; Kosaka, N.; Kines, R.; Roberts, J.; Lowy, D.; Schiller, J.; Ishihara, Y.; Hasegawa, A.; Choyke, P.; Kobayashi, H. In Vivo Longitudinal Imaging of Experimental Human Papillomavirus Infection in Mice with a Multicolor Fluorescence Mini-Endoscopy System. Cancer prevention research 2011, 4, 767–773. https://doi.org/10.1158/1940-6207.CAPR-10-0334.

[13] Tjalma, J. J. J.; Koller, M.; Linssen, M. D.; Hartmans, E.; de Jongh, S.; Jorritsma-Smit, A.; Karrenbeld, A.; de Vries, E. G.; Kleibeuker, J. H.; Pennings, J. P.; Havenga, K.; Hemmer, P. H.; Hospers, G. A.; van Etten, B.; Ntziachristos, V.; van Dam, G. M.; Robinson, D. J.; Nagengast, W. B. Quantitative Fluorescence Endoscopy: An Innovative Endoscopy Approach to Evaluate Neoadjuvant Treatment Response in Locally Advanced Rectal Cancer. Gut 2019, 69, 406–410. https://doi.org/10.1136/gutjnl-2019-319755.

[14] Zhang, Y.; Ji, W.; He, L.; Chen, Y.; Ding, X.; Sun, Y.; Hu, S.; Yang, H.; Huang, W.; Zhang, Y.; Liu, F.; Xia, L. E. Coli Nissle 1917-Derived Minicells for Targeted Delivery of Chemotherapeutic Drug to Hypoxic Regions for Cancer Therapy. Theranostics 2018, 8, 1690–1705. https://doi.org/10.7150/thno.21575.

[15] Yu, H.; Khokhlatchev, A. V.; Chew, C.; Illendula, A.; Conaway, M.; Dryden, K.; Maeda, D. L. N. F.; Rajasekaran, V.; Kester, M.; Zeichner, S. L. Minicells from Highly Genome Reduced Escherichia Coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells. ACS Synthetic Biology 2021, 10, 2465–2477. https://doi.org/10.1021/acssynbio.1c00375.

[16] Françoso, A.; Simioni, P. U. Immunotherapy for the Treatment of Colorectal Tumors: Focus on Approved and in-Clinical-Trial Monoclonal Antibodies. DDDT 2017, 11, 177–184. https://doi.org/10.2147/DDDT.S119036.

[17] Hwang, K.; Yoon, J. H.; Lee, J. H.; Lee, S. Recent Advances in Monoclonal Antibody Therapy for Colorectal Cancers. Biomedicines 2021, 9, 39. https://doi.org/10.3390/biomedicines9010039.

[18] https://ogyei.gov.hu/gene_technology_/ Accessed October 8, 2022.

[19]https://health.ec.europa.eu/medicinal-products/advanced-therapies/genetically-modified-organism-gmo-aspects-investigational-medicinal-products_en Accessed October 8, 2022.