| NU_Kazakhstan - iGEM 2023

Expectations and Hypothesis

The Proof-of-concept experiment involves conducting an experiment with a fully completed system on cancer tumor cells and obtaining data, the analysis of which will lead to a clear answer to the question, "Will the Cellcare system actually work?". Of course, we expect that the experiment will provide us with data that will confirm the efficacy of our product against cancer cells based on the fact that the genetically modified bacteria will release the toxin Colicin E1, which will pass through the pores of the hydrogel and will directly reduce the size of the solid tumor carcinoma.

Nevertheless, the hypothesis of the Proof of concept experiment would go like the following. If the number of live A549 cells exposed to a hydrogel filled with genetically modified Colicin E1 toxin-producing bacteria in response to an inducing concentration of lactate is significantly reduced compared to control samples, which are
1. A549 cells growing freely in DMEM media,
2. A549 cells growing in DMEM media cells growing in the presence of hydrogel NOT filled with bacteria
3. A549 cells growing in DMEM media in the presence of hydrogel filled with normal NOT genetically modified bacteria,
4. A549 cells growing in DMEM media in the presence of hydrogel filled with normal NOT genetically modified bacteria in the presence of the amount of lactate that is normally present in the tumor microenvironment, then it can be argued that indeed, our system has a positive therapeutic effect on the A549 lung cancer cell line. However, it should be noted that in case of success, this will prove the efficacy of Cellcare Therapeutics only on A549 cells because other cell lines and, even more so, actual tumors may differ significantly in morphology, microenvironment, and other various parameters that may affect the efficacy of the system on cancer cells. To obtain more detailed, accurate, and unambiguous results, our system should be tested on dozens of different cancer cell lines.

Goal

Our goal is to create a hydrogel-based living bacterial therapeutics for efficient, safe, and targeted treatment of solid carcinoma tumors.

General Protocol

The protocol for proof of concept can be found in the experiments page, but the idea is as follows. It is necessary to prepare different control samples in order to exclude the probability of one or another event that may prevent us from proving the efficiency of our whole system. It was mentioned earlier what kind of controls we need. More specifically:

1. A549 cells growing freely in the DMEM media sample are used to prove that our system has a significantly smaller number of viable cells in comparison to average growth conditions.
2. A549 cells growing in DMEM media cells growing in the presence of hydrogel NOT filled with bacteria. This sample is used to show that the decrease in the number of viable cells in our system is not only due to possible cytotoxicity of the thermosensitive hydrogel on A549 but that there are other factors, such as bacteria within the system.
3. A549 cells growing in DMEM media in the presence of hydrogel filled with normal, NOT genetically modified bacteria. This sample is used to show that the reduction in the number of viable cells in our system is significantly different from the same sample but with normal bacteria. The goal is to show that colicin is indeed produced and that it has a significant effect on cell viability compared to cancer cells, which are not affected by colicin
4. A549 cells growing in DMEM media in the presence of hydrogel filled with usual NOT genetically modified bacteria in the presence of the amount of lactate that is normally present in the tumor microenvironment. Here, it is necessary to prove that the decrease in the number of viable cells is not related to the amount of Lactic acid in the medium.
First of all, you need to remove the media that was there before, then try to detach them from the surface. Then, pour them into a tube, centrifuge, replace the supernatant with DMEM, and run them evenly into 24 trans-well plates. Then apply triplicates on top of our controls and, finally, a hydrogel loaded with genetically modified bacteria. Then, after incubation, the number of live cells should be evaluated on the Cell Counter.

Why our product will work

We expect our product to work, and these expectations are supported by numerous studies on the effect of Colicin E1 toxin on cancer cells, as well as our modeling, which showed the results you can find in Figure 1, Figure 2, and Figure 3.

Figure 1. Concentration of Colicin E1 in the tumor environment against time.

Figure 1 shows that the synthesis of Colicin E1 by the genetically modified bacteria in response to the tumor lactate-containing microenvironment increases dramatically, and translation of the mRNA of Colicin E1 will last for some time.

Figure 2. Concentration of Colicin E1 mRNA against time.

Figure 2 is directly proportional to the graph in Figure 1 and may indicate that in response to the cancer cell microenvironment, the plasmid we transformed would show a dramatic increase in transcription rate because colicin transcription is activated by the lactate sensor ALPaGA.

Figure 3. Concentration of Lactate in the environment against time.

Figure 3 shows us a significant decrease in Lactic acid concentration with the onset of transcription and translation of Colicin E1 because the synthesized toxin kills Lactic acid-producing cancer cells. Consequently, its concentration will decrease for this reason.

Safety issues

This experiment is absolutely safe for students to perform because A549 cells, genetically modified E cells. Coli BL-21 cells, or hydrogel or media are not dangerous to humans and fall under the BSL-1 category. You can read more about safety on our wiki page.

Usability as product

Suppose the experiments prove the effectiveness of the system against cancer cells. In that case, as mentioned earlier, it makes sense to see the effect of Cellcare Therapeutics on other cancer cell lines and perhaps from some perspective on living tumors. But even after this one experiment, if successful, we can already think about commercialization and entrepreneurship. You can read more about all our ideas and existing initiatives on the Entrepreneurship page.