| NU_Kazakhstan - iGEM 2023

Problem

One of the foremost obstacles encountered in skin cancer treatment is the incapability to diagnose and promptly initiate treatment without allowing the cancerous tumors to progress to a more severe stage1. The reason for this problem to be important is that skin cancer is known for its expeditious growth rate, as it shifts from highly treatable to hardly treated stage¹. As fluorescent imaging microscopes show promising perspectives in skin cancer detection, this technology is restricted to fluorescent markers, principles of detection, and equipment setup².

Fluorescence imaging can also potentially track the progress of cancer treatment³, which will eventually help to detect cancerous tumors at early stages and take timely treatment without severe health consequences. The method of tracking tumor size depletion is limited in versatility and convenience to the patients. Given that most cancer tracking methods that use fluorescent tags are tied to preoperative injections that last for 24 to 48 hours on average, which may cause patient discomfort with frequent injections. In addition, the cost of such equipment might pose a great obstacle to researchers with limited funding and equipment available (for instance, the cost of fluorescent imaging system devices varies from $8000⁴ to $10000⁵). All of these factors, individually and collectively, affect researchers' capabilities, limiting them in resources, convenience, and apparatus features.

Solution

With our hardware, we aim to create a compact, and affordable device, which will be able to detect bacteria we designed for cancer treatment in the tumor sites of the patients. The advantages of this approach include localized injection of bacteria and rapid fluorescence of cancerous tumors. The designed bacteria in pairs with the proposed device are mainly focused on the detection of skin carcinoma, as this cancer type poses major threats if not cured timely and is most suitable for our hardware design technically. Any fluorescent labeling approach is suitable for our project hardware. Based on our design, we suggest and encourage future iGEM teams to modify our plasmid, deleting the colicin E1 gene and adding a fluorescent agent instead. Another option is to modify our hydrogel, changing its composition or synthesizing nanogels, which could be more specific and applicable for fluorescent labeling purposes.

Previously, iGEM NEFU_China and iGEM Cornell teams in 2020 proposed similar devices, and we decided to build on their hardware and further extend the application of this promising technology by suggesting new labeling approaches and exploring possible future implications in more detail.

Hardware principle of work

The device applies the principles of fluorescent microscopy to detect the presence of fluorescent tags synthesized by bacteria, as they settle in the sites of tumor

Figure 1. The visual presentation of the work principle of a hardware device.

The fluorescent tags that could be used for our purposes are limited, and we speculate that the following agents could have been our variants. One of the promising agents for cancer detection using fluorescent staining is BLZ-100, which targets annexin A2 and matrix metalloproteinase 2⁶, which are specific markers of cancer cells that distinguish them from normal cells. This drug is administered intravenously and reaches the target cells of tumors, which include carcinoma³. The drug has also been tested on HNSCC cell line and mouse models⁷, as well as a clinical trial⁶ showing its effectiveness in detecting carcinoma by fluorescence imaging. Another fluorescent agent that responds to changes in the acidity of the medium is ONM-100, which is used in preoperative fluorescence detection of carcinoma cancer⁸.

As the fluorescent tag is chosen, next comes its detection on the designed apparatus. The main factor that plays a role in this part is the wavelength needed to excite the fluorescent agent. Following that, as our fluorescent tags emit the light in response to our excitation, we collect the data and further analyze it, visualizing the surrounding tumor.

Design

The tumor fluorescence detection apparatus consists of three important components: the LED light source, the coaxial light path, and the fluorescence collection site.

a. LED light source serves to excite the fluorescent proteins, producing a wavelength shorter than that emitted by the excited proteins⁹
b. A coaxial light path consists of three filters that serve as paths for outgoing and incoming light rays. The main feature is the passage of outgoing LED light with a 45-degree refraction through a dichroic filter and direct passage through a low pass filter. The first is an extremely important part of the design, due to the ability of this filter to pass both long and short-wavelength light rays simultaneously¹. Low-pass and high-pass filters allow the device to filter out light of long and short wavelengths, respectively¹⁰.
c. The fluorescence collection site is the acquisition of reflected light from the skin area being scanned, the source of which is a cluster of fluorescent proteins at the site of the tumor.

Figure 2. The schematic overview of the hardware model.

Limitations

The limitation our team faced was that there was no possibility to check our hardware in practice, as no experimentations on model organisms such as mice, or humans are allowed for us in the scope of this project.

Future Implementations

Because our hardware relies on bacteria that will produce fluorescent agents for a short time, it might be possible to deliver bacteria in a hydrogel without loss of efficiency and prolonging fluorescence of carcinoma sites. In addition, through means of engineering initial plasmid that could synthesize several fluorescent agents, it could be possible to detect a diverse amount of tumor markers, diagnosing or predicting other cancer types.

Figure 3. Proposed mechanism of work of multiple fluorescent agents delivered to the carcinoma tumor sites.

Since our project involves the delivery of bacteria to the cancer sites through the means of chitosan-based hydrogel, we theorize that hydrogel on its own could be used to deliver the fluorescent agents synthesized in vitro, providing flexibility for researchers. This could be achieved by modifying the hydrogel composition, suiting it for a tight fit to the cancer cells.

Figure 4. Proposed mechanism of work of hydrogel-delivered fluorescent agents to the carcinoma tumor cites.

1. Pan, J., Liu, Q., Sun, H., Zheng, W., Wang, P., Wen, L., Duan, J., Xuan, Z., Yu, X., Wang, S., Wang, X., Zhang, T., & Lu, W. (2020). A miniaturized fluorescence imaging device for rapid early skin cancer detection. Journal of Innovative Optical Health Sciences, 14(02).https://doi.org/10.1142/s1793545820500261

2. Liu, L., Yang, Q., Zhang, M., Wu, Z., & Xue, P. (2019). Fluorescence lifetime imaging microscopy and its applications in skin cancer diagnosis. Journal of Innovative Optical Health Sciences, 12(05). https://doi.org/10.1142/s1793545819300040

3. Woo, Y., Chaurasiya, S., O’Leary, M. P., Han, E., & Fong, Y. (2021). Fluorescent imaging for cancer therapy and cancer gene therapy. Molecular Therapy - Oncolytics, 23, 231–238. https://doi.org/10.1016/j.omto.2021.06.007

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6. Yamada, M., Miller, D. M., Lowe, M. G., Rowe, C., Wood, D., Soyer, H. P., Byrnes-Blake, K., Parrish-Novak, J., Ishak, L., Olson, J. M., Brandt, G., Griffin, P., Spelman, L., & Prow, T. W. (2021). A first-in-human study of BLZ-100 (tozuleristide) demonstrates tolerability and safety in skin cancer patients. Contemporary Clinical Trials Communications, 23, 100830. https://doi.org/10.1016/j.conctc.2021.100830

7. Baik, F. M., Hansen, S., Knoblaugh, S. E., Sahetya, D., Mitchell, R. M., Xu, C., Olson, J. M., Parrish-Novak, J., & Méndez, E. (2016). Fluorescence identification of head and neck squamous cell carcinoma and High-Risk oral dysplasia with BLZ-100, a Chlorotoxin-Indocyanine green conjugate. JAMA Otolaryngology-- Head & Neck Surgery, 142(4), 330. https://doi.org/10.1001/jamaoto.2015.3617

8. Martinez, A. (2022, February 7). Publication of ONM-100 Phase 1 - OncoNano Medicine. OncoNano Medicine. https://onconano.com/onconano-announces-publication-of-onm-100-phase-1-data-in-nature-communications/

9. Rijal, N., & Rijal, N. (2022). Fluorescence microscope: principle, types, applications. Microbe Online. https://microbeonline.com/fluorescence-microscope-principle-types-applications/

10. Fluorescence microscopy - explanation and labelled images. (n.d.). New York Microscope Company. https://microscopeinternational.com/fluorescence-microscopy/