Design
MscS Calcium Channel
By referencing the project of iGEM16_Slovenia group, we selected the calcium ion channels MscS (bacterial Mechanosensitive Channel ) (BBa_K1965000) and hTRPC1 (Transient Receptor Potential Channel 1) (BBa_K1965002) to induce calcium overload. These receptors respond to physical stress on the membrane and can be activated by ultrasound stimulation (Hurst et al., 2007; Kunichika et al., 2004). By introducing the 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 (Hu et al., 2024). Hence, reduce the nonspecific killing effect. According to this objective, we designed three plasmids to investigate how the MscS calcium channel influences cancer cell growth.
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 1.1 Plasmid information for the MscS calcium channel.
hTRPC1 Calcium Channel
Since MscS is a bacteria protein, we also used hTRPC1 (Transient Receptor Potential Channel 1) (BBa_K1965002) as an alternative to induce the calcium overload. hTRPC1 could also respond to physical stress on the membrane and lead to calcium ion influx (Hurst et al., 2007; Kunichika et al., 2004). By introducing this calcium ion channel into cancer cells, it could also generate calcium overload after ultrasound stimulation. We designed two plasmids to investigate how the calcium channel hTRPC1 influences cancer cell growth in this project.
1358: LentiV-hTRPC1/HA: EGFP/Neo (BBa_K5353064)
1108: LentiV-Lamp2b/hTRPC1/HA: Neo (BBa_K5353065)
Figure 1.2 Plasmid information for the hTRPC1calcium channel.
hNOS2 (Nitric Oxide Synthase 2)
In addition to directly exploiting the calcium ion channels, we also propose to use a signaling molecule to increase the intracellular calcium level. hNOS2 (Nitric Oxide Synthase 2) 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). We designed this plasmid to investigate how hNOS2 influences cancer cell growth.
1356: LentiV-hNOS2/Flag: Puro (BBa_K5353066)
Figure 1.3 Plasmid information for hNOS2.
Results
1. Midi-Prep Plasmid DNA preparation
To amplify the plasmids to generate sufficient amounts of genetic material for further analysis and manipulation, we used the midi-prep preparation kit. After plasmid isolation and purification. We qualified DNA concentrations and the results are shown in Table 1. The results suggested the plasmids were successfully produced. The concentrations were good enough for further experiments (generally >100 ng/ml). The A260/A280 and A260/A230 ratio also suggested the purity of the plasmids was excellent.
Table 1. DNA concentrations extracted and purified.
2. Checking of the plasmid insert by Polymerase Chain Reaction (PCR)
We tested the target DNA segments in the plasmids by PCR. After PCR amplification, we conducted gel electrophoresis. The amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively.
Compare the size of the bands to the fast DNA ladder running alongside the samples (Figure 2.1 and Figure 2.2), helping us to determine the amplified DNA fragment sizes practically. Theoretically, the amplicon size of the four targeted sequences are: Lamp2b (103 bp) , MscS (225 bp), hTRPC1 (162 bp) and hNOS2 (142 bp), respectively, which is aligned to our results.
The PCR product (1358: LentiV-hTRPC1/HA: EGFP/Neo) gel electrophoresis failed (Figure 2.1) in the first trial. In the second attempt, we can observe the pcr products (1358: LentiV-hTRPC1/HA: EGFP/Neo; 1364: LentiV-MscS/HA: EGFP/Neo) with correct band sizes (Figure 2.2). In summary, the result from PCR confirmed all plasmid inserts are correct.
Figure 2.1 Gel electrophoresis results of 1833, 1362, 1358, 1108, 1356.
Figure 2.2 Gel electrophoresis results of 1358 and 1364.
3. Validate the lentivirus infection by an apotome fluorescence microscope imaging
After plasmid preparation and PCR verification, we transfected the plasmids together with DVPR and VSV-G into the HEK293T cells for the packaging of lentivirus. The transfection process begins with the assembly of the lentiviral components, including the target DNA, the DVPR (packaging plasmid) and the VSV-G (envelope plasmid) within HEK293T cells. These components work synergistically to encapsulate the genetic material within the lentiviral vector, a crucial step in ensuring successful gene delivery to target cells.
Once the lentivirus is generated, we collect and utilize them to infect a fresh batch of HEK293T cells. This infection process is carefully orchestrated to introduce the modified genetic material into the recipient cells. By infecting the new HEK293T cells with the lentivirus, our objective is to create the genetically modified exosome with the target protein that we have designed.
To validate the infection efficiency, we inserted the EGFP or mCherry into different plasmids. Therefore, the successful infection could be visualized through fluorescence microscopy. As shown in Figure 3.1 to 3.4, the fluorescence signal confirms that the lentiviral vectors (1364, 1833, 1362, and 1358) had effectively delivered the desired genetic material to the target cells, leading to the expression of the gene construct.
1364: LentiV-MscS/HA: EGFP/Neo
Figure 3.1 Expression of green fluorescent protein of 1364 HEK293T cell.
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo
Figure 3.2 Expression of green fluorescent protein of the 1833 HEK293T cell.
1362: LentiV-Lamp2b/MscS/mCherry: Neo
Figure 3.3 Expression of red fluorescent protein of 1362 HEK293T cell.
1358: LentiV-hTRPC1/HA: EGFP/Neo
Figure 3.4 Expression of green fluorescent protein of 1358 HEK293T cell.
For the plasmid 1356 and 1108, because the target protein we insert is too large, we did not add the fluorescence protein on them. We would detect them with western blotting through the HA tag (1108: LentiV-Lamp2b/hTRPC1/HA: Neo) and flag tag (1356: LentiV-hNOS2/Flag: Puro).
4. Confirm the production of exosomes by TEM
Since the TEM imaging process is complicated and expensive, we just sent our exosome samples coding 1833 (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) to the company in Guangzhou, China to do the TEM imaging.
We used the supernatant of the successfully infected HEK293T cell to isolate the exosomes. The size of the exosomes is extremely small, approximately 30–140 nm, membrane-enclosed molecules. Also, since we used the exoEasy Maxi Kit (QIAGEN, cat. no. 76064) to extract them and did not concentrate the exosomes using ultrafiltration, the exosome concentration is a little low.
The figures below show the shape and size of our exosomes, the halo-like structure is the exosomes (Figure 4.1 and Figure 4.2), 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 4.3).
Figure 4.1 Exosomes with normal structures.
Figure 4.2 Exosomes tangled together.
Figure 4.3 Exosomes with elliptical collapse structure.
5. DLS Measurement to determine the size of the exosomes
Dynamic light scattering (DLS) is a powerful technique used in the field of nanotechnology and biophysics to measure the size of particles in a solution, and Dynamic light scattering (DLS) is an optical analysis method for measuring the size and distribution of sub-micron particles. Therefore we used DLS to provide valuable insights into the size distribution and polydispersity of exosomes.
In DLS, a laser beam is directed into a sample containing exosomes. As these particles move randomly as a result of Brownian motion, they scatter light. When the fluctuations in scattered light intensity are analyzed over time, DLS can determine the hydrodynamic size of exosomes in the sample. The technique is highly sensitive to small particles, making it ideal for studying exosomes, which typically range in size from 30 to 150 nanometers.
The results of the different samples of exosome size distribution are as follows (Figure 5.1 to Figure 5.3).
1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo
Exosome Sample 1833-1 (Figure 5.1.1):
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 5.1.1 Exosome size distribution of sample 1833-1.
Exosome Sample 1833-2 (Figure 5.1.2):
The peak of the size (d. nm) is 390.8 nm.
Figure 5.1.2 Exosome size distribution of sample 1833-2.
Exosome Sample 1833-3 (Figure 5.1.3):
The peak of the size (d. nm) is 14.23 nm.
Figure 5.1.3 Exosome size distribution of sample 1833-3.
Exosome Sample 1833-4 (Figure 5.1.4):
The peak of the size (d. nm) is 43.22 nm.
Figure 5.1.4 Exosome size distribution of sample 1833-4.
1362: LentiV-Lamp2b/MscS/mCherry: Neo
Exosome Sample 1362-1 (Figure 5.2.1):
The peak of the size (d. nm) is 493.7 nm.
Figure 5.2.1 Exosome size distribution of sample 1362-1.
Exosome Sample 1362-2 (Figure 5.2.2):
The peak of the size (d. nm) is 31.14 nm.
Figure 5.2.2 Exosome size distribution of sample 1362-2.
Exosome Sample 1362-3 (Figure 5.2.3):
The peak of the size (d. nm) is 372.2 nm.
Figure 5.2.3 Exosome size distribution of sample 1362-3.
11108-1: LentiV-Lamp2b/hTRPC1/HA: Neo
Exosome Sample 1108-1 (Figure 5.3.1 ):
The peak of the size (d. nm) is 20.93 nm.
Figure 5.3.1 Exosome size distribution of sample 1108-1.
Exosome Sample 1108-2 (Figure 5.3.2):
The peak of the size (d. nm) is 10.24 nm.
Figure 5.3.2 Exosome size distribution of sample 1108-2.
Exosome Sample 1108-3 (Figure 5.3.3):
The peak of the size (d. nm) is 16.14 nm.
Figure 5.3.3 Exosome size distribution of sample 1108-3.
From the results, we can confirm that the modified HEK293T cell can produce the exosomes successfully and we can also extract the exosomes from the supernatant using the exoEasy Maxi Kit.
However, 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℃ fridge for long-term storage. Keeping them for a long time may result in abnormal structure of the exosome or in the exosomes flocked together to fuse and form larger structures. In addition, because there are different target proteins that are inserted into the exosome, which may also affect the structure and formation of the exosome.
Overall, the DLS measurement and TEM imaging confirm the existence of exosomes, and we then conducted the BCA assay to measure the exosome concentration in our different samples.
6. BCA Assay
The Bicinchoninic Acid (BCA) assay is a widely used method to quantify the total protein concentration in a sample. This colorimetric assay is based on the ability of protein molecules to reduce Cu2+ to Cu+ in an alkaline medium, forming a purple complex with BCA. The intensity of this complex is directly proportional to the protein concentration and can be measured spectrophotometrically.
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 6. 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.
Moreover, according to research related to exosome measurement, we can assume the total protein concentration in the sample reflects the amount of proteins on the exosomes. However, the results do not reflect the target protein expression such as MscS, lamp2b, and hNOS2 in the exosomes.
7. Western Blot
Western blotting, also known as protein immunoblotting, is a widely used technique in molecular biology for detecting specific proteins in a complex sample. This method allows us to identify and quantify proteins based on their size and antigenicity.
In our Western blot experiments, we detected CD63 and CD81, which are exosome marker proteins, and also the HA-tag , which is linked to the MscS (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) and hTRPC1 proteins. We were able to detect the CD81 marker protein successfully (1833-1), this suggests the existence of the exosome (Figure 7). However, possibly due to the low exosome or target protein concentration, we did not detect the CD63 and HA tag successfully. Because our laboratory does not have the antibody to target the Flag-tag yet, the detection of Flag will be performed later.
Figure 7. Western Blot of CD81 from 1833 exosome samples.
8. 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.
Annexin V Staining:
Apoptotic cells exhibit phosphatidylserine (PS) residues on their outer membrane, a hallmark of early apoptosis. Annexin V, a calcium-dependent phospholipid-binding protein with high affinity for PS.
Propidium iodide (PI) staining:
PI is often used in conjunction with Annexin V to differentiate between apoptotic cells and necrotic cells. PI stains necrotic cells with compromised membrane integrity. Because propidium iodide does not penetrate living cells, it is commonly used to detect dead cells in a population. PI binds to DNA by intercalating between bases with little or no sequence preference.
Flow Cytometry Analysis:
After staining with Annexin V and PI, the cells were analyzed using flow cytometry. Flow cytometers detect fluorescence emissions from stained cells and provide quantitative data on the cell population.
Cell apoptosis-A2780
Figures (Figure 8.1.1, Figure 8.1.2 and Figure 8.1.3) below show the result of ovarian cancer cells A2780 treated with or without the exosome treatment (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo). With the exosome treated, there are more A2780 cells during necrosis, but there is no difference in apoptosis.
Since the number of exosomes produced by HEK293T cells is low, the exosome concentration in one well of a six-well plate is approximately 0.056 mg/ml. However, the results still show that the cancer cell death is affected and more A2780 cells appeared resulting in necrosis.
Figure 8.1.1 Flow cytometry of the control group of A2780.
Figure 8.1.2 Flow cytometry of the exosome treated group of A2780.
Figure 8.1.3 Results of necrosis and apoptosis of A2780 under normal and exosome treated conditions.
Cell apoptosis-NCI-H1299
The figures (Figure 8.2.1, Figure 8.2.2 and Figure 8.2.3) below show the result of the growth of lung cancer cells, NCI-H1299, and the cancer cells treated with/without our exosomes (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo). 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 believe the modest response may be due to the low exosome concentration and lack of enough membrane stress stimulation by the ultrasound.
Figure 8.2.1 Flow cytometry of the control group of NCI-H1299.
Figure 8.2.2 Flow cytometry of the exosome treated group of NCI-H1299.
Figure 8.2.3 Results of necrosis and apoptosis of NCI-H1299 under normal and exosome treated conditions.
Due to the time limit, we just investigated how the exosome containing MscS (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) affects cancer cell death. Also due to the very specialized instruments required for the ultrasound stimulation, we cannot induce the opening of the MscS and hTRPC1 calcium channels precisely as we proposed. But to our surprise, the A2780 group was treated with 0.056 mg/ml exosome (1833: LentiV-Lamp2b/MscS/HA: EGFP/Neo) seems to show that cancer cell death has been affected. We will try the other two kinds of the exosomes (1108: LentiV-Lamp2b/hTRPC1/HA: Neo), and (1362: LentiV-Lamp2b/MscS/mCherry: Neo) to treat the cancer cells later.
