Contribution
Team UM-Macau 2024: Contribution on BBa_K1965000
In contrast to the previous project using MscS for environmental issue, this year we integrated MscS with Lamp2b, into a plasmid for eukaryotic use, generated exosomes from HEK293T, and transferred them to eukaryotic cancer cells, where ultrasound was used to induce calcium overload in the cells, which in turn mediated cancer cell death. Unlike this 2016 project, this year UM-Macau has taken the use of MscS beyond prokaryotes and used it for the first time to induce eukaryotic calcium influx - we have used a new plasmid that is suitable for eukaryotes and successfully validated its potential for inducing calcium overload in cancer cells, undoubtedly expanding the scope of application of MscS exponentially from BBa_K1965000.
Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. By introducing the MscS channels into cancer cells, we aim to generate calcium overload upon ultrasound stimulation. The ultrasound stimulation could precisely control cell death in tumors by opening the channels and leading to calcium ion influx. We utilized the exosomal transmembrane protein lamp2b, fused with our target proteins, to enhance the loading of MscS into the exosomes.
Figure 1.1 Plasmids information of the MscS calcium channel.
Figure 1.2 Plasmid information of 1364.
Figure 1.3 Plasmid information of 1833.
Figure 1.4 Plasmid information of 1362.
We used the supernatant of the successfully infected HEK293T cell to isolate the exosomes. And we confirm the production of exosomes by TEM. The halo-like structure is the exosomes, and the light dots shown on the background are salts.
Figure 1.5 Exosomes with normal structures.
We used DLS to provide valuable insights into the size distribution and polydispersity of exosomes, which typically range in size from 200 to 300 nanometers.
Figure 1.6 Exosome size distribution of sample 1833-1.
In our Western blot experiments, we could detect the CD81 exosome marker in the samples 1833-1 (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo).
Figure 1.7 Western Blot of CD81 from 1833 exosome samples.
The A2780 ovarian cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in necrosis, but there is no difference in apoptosis.
Figure 1.8 Results of necrosis and apoptosis of A2780 under normal and exosome treated conditions.
The NCI-H1299 lung cancer cells treated with the exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) have an increase in apoptosis (although not significant due to a single experiment), but there is no change in necrosis.
Figure 1.9 Results of necrosis and apoptosis of NCI-H1299 under normal and exosome treated conditions.
Team UM-Macau 2024: Contribution on BBa_K1965002
For BBa_K1965002, which was used in 2016, we applied it to humans in a new way. Using the same expression vector as MscS in this year's project, we also tested hTRPC1 in exosomes and two cancer cell lines in a very different application scenario than in the 2016 iGEM project. After a series of experimental validations, UM-Macau has demonstrated with preliminary results the potential of hTRPC1 for cancer-related applications, building on the parameters of hTRPC1 in 2016 and providing an important reference for future iGEMers in the field of biomedicine and oncology. Of course, in our experiments up to this point, since our experiments related to hTRPC1 have not yet reached the same amount of data as MscS, we do not yet have detailed data specifically around the use of hTRPC1 channels to kill cancer cells here, and we plan to complete the data in the future before sharing it with future iGEMers through other means.
Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. By introducing the hTRPC1 channels into cancer cells, we aim to generate calcium overload upon ultrasound stimulation. The ultrasound stimulation could precisely control cell death in tumors by opening the channels and leading to calcium ion influx. We utilized the exosomal transmembrane protein lamp2b, fused with our target proteins, to enhance the loading of MscS into the exosomes.
Figure 2.1 Plasmids information of the hTRPC1 calcium channel.
Figure 2.2 Plasmid information of 1358.
Figure 2.3 Plasmid information of 1108.
We used the supernatant of the successfully infected HEK293T cell to isolate the exosomes. And we confirm the production of exosomes by TEM. The halo-like structure is the exosomes, and the light dots shown on the background are salts.
Figure 2.4 Exosomes with normal structures.
We used DLS to provide valuable insights into the size distribution and polydispersity of exosomes.
Figure 2.5 Exosome size distribution of sample 1108-1.
As we have mentioned in the first paragraph in this part about our contribution on hTPCR1, the amount of data on this part is relatively less than the data of MscS. And we will use the exosomes containing hTRPC1 to investigate its function to cancer cell death in the future.
