Cycle 1
Define
Cancer remains a global challenge and a persisting leading cause of death despite extensive efforts to combat it. The complexity of the disease, along with the drug resistance of cancer cells has made finding an effective therapeutic approach became an elusive goal. Classical approaches, such as chemotherapy and radiotherapy, are limited by their non-specificity and drug resistance. While recent advancements in immunotherapy have significantly benefited some patients, a substantial percentage of patients do not respond to these treatments. Therefore, a novel strategy that improves specificity and reduces the likelihood of drug resistance is urgently needed.
Design
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. This strategy involves introducing calcium ion channels (MscS or hTRPC1) and an NO signaling activator (hNOS2) using exosomes as the delivery vehicle.
Referring to the iGEM16_Slovenia group and we selected the calcium ion channels MscS (bacterial Mechanosensitive Channel ) (BBa_K1965000) and hTRPC1 (Transient Receptor Potential Channel 1) (BBa_K1965002) that respond to physical stress on the membrane and can be activated by ultrasound stimulation (Hurst et al., 2007; Kunichika et al., 2004) (Figure 1.1). By introducing these channels into cancer cells, we aim to generate calcium overloading. Ultrasound stimulation will precisely control cell death by opening the channels and leading to calcium ion influx (Hu et al., 2024).
Figure 1.1 MscS and hTRPC1 calcium channels function (iGEM16_Slovenia).
To deliver our target proteins to cancer cells, we propose using exosomes as the carrier vehicle (Salunkhe et al., 2020) (Figure 1.2). We will utilize the exosomal transmembrane protein lamp2b (BBa_K5353000), fused with our target proteins, to improve the loading of target proteins into the exosomes. In fact, Lamp2b has been widely used in the manufacture of engineered exosomes for targeted drug or other substances delivery (Qiao et al., 2023) (Figure 1.3).
Figure 1.2 Role of lamp2b in mediating autophagosome-lysosome fusion.
Figure 1.3 Example working model of the engineered exosomes with lamp2b.
Building
We constructed six plasmids corresponding to MscS, hTRPC1, and hNOS2 respectively (1364, 1833, 1362, 1358, 1108, and 1356 is our plasmid coding number we label).
MscS Calcium Channel
1364: LentiV-MscS/HA: EGFP/Neo (BBa_K5353061)
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa_K5353062)
1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa_K5353063)
Figure 1.4.1 Plasmids information of the MscS calcium channel.
Figure 1.4.2 Plasmid information of 1364.
Figure 1.4.3 Plasmid information of 1833.
Figure 1.4.4 Plasmid information of 1362.
hTRPC1 Calcium Channel
1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa_K5353064)
1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa_K5353065)
Figure 1.5.1 Plasmids information of the hTRPC1 calcium channel.
Figure 1.5.2 Plasmid information of 1358.
Figure 1.5.3 Plasmid information of 1108.
Test and Experiments
To achieve the objective of this project and the elaboration, please refer to the “Protocol” and “Results” section for the detailed experimental protocol and results. Here are some important experiments summary and conclusions.
1. Midi-Prep Plasmid DNA preparation and PCR
The designed DNA was constructed and we used the midi-prep preparation kit to conduct the plasmid isolation and purification of the plasmid DNA (Table 1). The inserts in each construct were further validated by the PCR reaction (Figure 1.6.1 and Figure 1.6.2).
Table 1. DNA concentrations extracted and purified.
The amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively.
Plasmids Checked:
1364: LentiV-MscS/HA: EGFP/Neo (BBa_K5353061)
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo (BBa_K5353062)
1362: LentiV-Lamp2b/MscS/mCherry: Neo (BBa_K5353063)
1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa_K5353064)
1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa_K5353065)
1356: LentiV-hNOS2/Flag: Puro (BBa_K5353066)
Figure 1.6.1 Gel electrophoresis results of 1833, 1362, 1358, 1108, 1356.
Figure 1.6.2 Gel electrophoresis results of 1358 and 1364.
2. Transfection, Infection and Fluorescence Microscope Imaging
To validate the infection efficiency, we inserted the EGFP or mCherry into different plasmids. Therefore, the successful infection could be visualized by fluorescence microscopy.
1364: LentiV-MscS/HA: EGFP/Neo
Figure 1.7.1 Expression of green fluorescent protein of 1364 HEK293T cell.
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo
Figure 1.7.2 Expression of green fluorescent protein of the 1833 HEK293T cell.
1362: LentiV-Lamp2b/MscS/mCherry: Neo
Figure 1.7.3 Expression of red fluorescent protein of 1362 HEK293T cell.
1358: LentiV-hTRPC1/HA: EGFP/Neo
Figure 1.7.4 Expression of green fluorescent protein of 1358 HEK293T cell.
3. Confirm the production of exosomes by TEM
The size of the exosomes is extremely small, approximately 30–140 nm, membrane-enclosed molecules.
The halo-like structure is the exosomes (Figure 1.8.1 and Figure 1.8.2), and the light dots shown on the background are salts. The normal and well-functioning exosomes are standard round, while the broken and unhealthy exosomes with an elliptical collapse structure were also observed (Figure 1.8.3).
Figure 1.8.1 Exosomes with normal structures.
Figure 1.8.2 Exosomes tangled together.
Figure 1.8.3 Exosomes with elliptical collapse structure.
4. DLS Measurement to determine the size of the exosomes
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.
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo
Exosome Sample 1833-1 (Figure 1.9.1):
The peak of the size (d. nm) is 186.6 nm and more than 70% of exosomes range from 141.8 nm to 220.2 nm, which is similar to the size of TEM imaging.
Figure 1.9.1 Exosome size distribution of sample 1833-1.
1362: LentiV-Lamp2b/MscS/mCherry: Neo
Exosome Sample 1362-3 (Figure 1.9.2):
The peak of the size (d. nm) is 372.2 nm.
Figure 1.9.2 Exosome size distribution of sample 1362-3.
1108: LentiV-Lamp2b/hTRPC1/HA: Neo
Exosome Sample 1108-1 (Figure 1.9.3):
The peak of the size (d. nm) is 20.93 nm.
Figure 1.9.3 Exosome size distribution of sample 1108-1.
5. BCA Assay
The Table 2 shows the absorbance values at 562 nm for various concentrations of BSA standards. By plotting the absorbance values against the known protein concentrations, a standard curve is generated. This curve is then used to determine the protein concentration in unknown samples on the basis of their absorbance readings.
Table 2. The absorbance value (562 nm) of BSA and exosome samples.
Figure 1.10 The BCA standard absorbance curve.
According to the equation of the BCA standard absorbance curve, we calculated the total protein concentration in exosomes (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) samples through their absorbance values. The amount of proteins in three repeat extraction of 1833 is arranged from 0.067 to 0.280 mg/ml. The result suggests the first extraction (1833-1) has the best concentration.
6. Western Blot
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 1.11. Western Blot of CD81 from 1833 exosome samples.
7. Cancer Cell Apoptosis Assay
The Apoptosis Assay by flow cytometry using the Annexin V staining method is a technique to detect and quantify apoptotic cells within a cell population. It depends on Annexin V staining, propidium iodide (PI) staining, and flow cytometry analysis.
Cell apoptosis-A2780
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 1.12.1 Results of necrosis and apoptosis of A2780 under normal and exosome treated conditions.
Cell apoptosis-NCI-H1299
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 1.12.2 Results of necrosis and apoptosis of NCI-H1299 under normal and exosome treated conditions.
Learn
1. Midi-Prep Plasmid DNA preparation
At the beginning, the plasmid DNA extraction yields a low-concentration and impure DNA sample. Therefore, instead of directly amplifying the DH5α, we incubated the DH5α on LB agar and selected the single colony to amplify. This helps us obtain purer and higher-concentration DNA samples.
Figure 1.13.1 DH5α Amplification.
Figure 1.13.2 Selection of a single colony.
2. Checking of the plasmid insert by Polymerase Chain Reaction (PCR)
The gel electrophoresis of the PCR product (1358: LentiV-hTRPC1/HA: EGFP/Neo) in Figure 1.6.1 failed, maybe due to overloading the DNA sample in the PCR reaction or error in electrophoresis.
3. Validate the lentivirus infection by an apotome fluorescence microscope imaging
During the transfection process, we encountered multiple failures in our initial attempts. At first, HEK293T cells were on the brink of death due to low density, while in other instances, the lentivirus packaging failed because of the high density of HEK293T cells, which affected the cell-cell interaction. And the optimal cell density for successful lentivirus packaging is typically 70%-80%, ensuring the production of well-functioning lentiviral vectors.
During the infection process, we faced challenges in establishing a stable cell line during the infection process, as G418 did not kill uninfected HEK293T cells. Subsequently, we discovered that G418 requires sustained and prolonged duration to exert its effect, playing a role after approximately the fifth to sixth day with the appropriate concentration of 1000 (μg/ml).
During the imaging process, we discovered that cell density significantly impacts imaging quality, with optimal results achieved when cells are arranged in a single layer (Figure 1.7.2). When cells are closely packed, the fluorescence doesn't show the cell structures (Figure 1.7.3). Additionally, it is crucial to change the medium before capturing images to wash away the dead cells.
4. DLS Measurement to determine the size of the exosomes
The results show the significance difference of size distribution between the different samples. The reason may be that we keep the exosomes extracted in the 4℃ refrigerator for a long time, about three weeks, while it is suggested to use the exosomes within one week or keep them in the -80℃ refrigerator for long-term storage. The long time storage may result in abnormal exosome structures or exosomes flocked together to fuse and form larger structures. In addition, because there are different target proteins that are inserted into the exosome, which can also affect the structure and formation of the exosome.
5. Exosomes imaging by TEM, measured by BCA Assay
The major challenge at the current stage is the concentration of exosomes. Therefore, we do need to increase the yield of the exosome and to produce a more concentrated exosome. Further optimization must be carried out, such as adjusting the culture environment by using specific media additives, hypoxic conditions, or altering the pH, which can stimulate cells to produce more exosomes. For concentrating exosomes, we could try to perform ultrafiltration or ultracentrifugation.
6. Western Blot
Perhaps this is because the exosome concentration is too low, and therefore the protein concentration is too low to be detected by western blot.
Furthermore, the oversized MscS, a weak electric field, and inadequate electrophoresis duration all lead to bands that were too large to migrate fully, resulting in incomplete separation on the gel.
7. Cancer cell apoptosis
Cell apoptosis-A2780
With the exosome treated, there are more A2780 cells in necrosis, but there is no difference in apoptosis. Although the exosome concentration in one well of a six-well plate is just approximately 0.056 mg/ml, the results still show that the cancer cell death is affected and more A2780 cells appeared resulting in necrosis. We think with higher exosome concentration and better ultrasound stimulation, there will be more cancer cells to die.
Cell apoptosis-NCI-H1299
With the exosome treated, there are slightly more NCI-H1299 cells in apoptosis (even the results are not significant due to only a single experiment), but there is no change in necrosis. We think the modest response may be due to the low exosome concentration and lack of enough membrane stress stimulation by the ultrasound. Repeat of the experiment is required to confirm the effect of the engineered exosomes.
Cycle 2
Define
According to the feedback from different interviews, most opinions suggest that in addition to using the ion channels, we need to look for another alternative to induce calcium overload. Therefore, we proposed to modulate the NO signaling hNOS2 to enhance the calcium overload efficiency.
Design
hNOS2 (Nitric Oxide Synthase 2) (BBa_K5353003) is an enzyme that induces NO signaling by catalyzing the reaction: L-arginine + O2 + NADPH + H+ → NO + L-citrulline + NADP+ + H2O. Up-regulation of the NO signal can induce the opening of the RyR calcium channel in the endoplasmic reticulum, dramatically increasing intracellular calcium levels (Ziolo et al., 2001) (Figure 2.1).
Figure 2.1 NO release to stimulate RyR channel opening.
For hNOS2 (1356: LentiV-hNOS2/Flag: Puro), because of the limitation of the size of the lentivirus plasmid, the insert will be too large, if we combine lamp2b together with hNOS2. Therefore, we aim to carry out a second transfection of hNOS2 after generating the stable HEK293T cell line with the 1833, 1362, or 1108 plasmid. In this way, our objective is to observe whether hNOS2 can enhance the effect of MscS or hTRPC1 on calcium overload.
Building
hNOS2 (Nitric Oxide Synthase 2)
1356: LentiV-hNOS2/Flag: Puro (BBa_K5353066)
Figure 2.2.1 Plasmid information of the hNOS2.
Figure 2.2.2 Plasmid information of 1356.
We have extracted the plasmid DNA and checked the plasmid insert by PCR. We will continue the experiments following the two to three three weeks.
Cycle 3
A. Enhancing the specificity of exosome in targeting cancer cells
Define
Precisely targeting cancer cells is crucial in effective therapy. Enhancing exosome targeting poses a significant challenge. Strategies to improve exosome targeting efficiency could involve exploring advanced delivery mechanisms, enhancing specificity through surface modifications, optimizing targeting ligands, and leveraging innovative technologies to ensure accurate and effective delivery to cancer cells.
Design
To achieve the precision, we plan to introduce the iRGD peptide tag to our exosomes. Known for its tumor-penetrating properties, iRGD facilitates improved targeting specificity and efficiency in the delivery of payloads to cancer cells. By incorporating the iRGD tag, we aim to optimize the targeting ability of exosomes, potentially enhancing therapeutic outcomes through the delivery of more calcium channels from MscS and hTRPC1 to cancer cells.
Building
Figure 3.1 Plasmid information of exosomes with iRGD.
B. Increase the yield and concentration of the exosome collection
Define
As we mentioned before, because of the low concentration of exosomes, we cannot detect the CD63 and HA-tag successfully by western blot. More importantly, higher exosome concentrations can lead to more potent therapeutic effects. Therefore, how to improve the exosome production and exosome concentration is an important content.
Design
Enhance exosome production by optimizing cell culture conditions such as media composition, pH, and temperature. Utilize efficient isolation methods like ultracentrifugation, size exclusion chromatography, or commercial kits to concentrate exosomes from biological samples. Moreover, ultrafiltration techniques are employed to concentrate exosomes by selectively passing them through membranes based on size.
Tips for Ultracentrifugation Process:
Choose an appropriate rotor and centrifuge tubes that can withstand the high speeds and forces generated during ultracentrifugation.
Carefully load the sample into the centrifuge tubes without introducing air bubbles, ensuring balanced loading across all tubes.
Start the ultracentrifuge and gradually increase the speed to the desired level, typically reaching speeds exceeding 100,000 x g or higher.
C. The use of ultrasound stimulation to control the calcium overload killing
Define
We assume the MscS and hTRPC1 could be precisely controlled by the ultrasound stimulation. However, the optimal frequency of the ultrasound to open the two calcium channels is 310kHz, while ultrasound frequencies that are typically above 20 kHz can potentially damage cancer cells. Typically, these frequencies fall within the range of high-intensity focused ultrasound (HIFU). Furthermore, ultrasound frequencies can potentially cause damage to normal cells in the range of high-intensity focused ultrasound (HIFU) above 20 kHz. Therefore, how to improve the sensitivity of the MscS and hTRPC1 to the ultrasound is very important.
Design
Use pulsed ultrasound instead of continuous waves to modulate calcium channel sensitivity. Pulsed ultrasound can exert controlled effects on calcium channels, enhancing their response to stimulation.
Employ focused ultrasound techniques to target specific areas more precisely. Focused ultrasound can concentrate energy on the target region, potentially increasing the sensitivity of calcium channels in that area.
