Proposed Implementation

Gastric cancer is a major health problem worldwide, being the fifth largest cancer in terms of both diagnostic and mortality figures [1]. The prognosis of gastric cancer depends largely on the disease stage at diagnosis. The survival rate is less than 10% when diagnosed at an advanced stage but is as high as 85% if detected at an earlier stage [2]. GC diagnosis is critical due to the low detection rate of early‐stage tumors. Common biomarkers such as AFP, CEA, and CA50 have positive rates of less than 40% [3]. Therefore, searching for new biomarkers to diagnose GC is critical, especially given that most patients are asymptomatic until the disease advances to advanced stages. Thus, we construct a novel sensor based on a new biomarker-G3BP1. We are eager to make our project a reality and provide doctors and patients with a novel piece of technology that enables accurate and timely diagnosis of GC.

We propose to design and produce a test kit with the constructed sensors and transfected reagents. There are two detecting sensors (pMIR-EIF3B-HSU plasmid carries the luciferase gene and pCMV-EGFP-EIF3B-HSU plasmid carries the GFP gene) and two controls (pMIR-EIF3B plasmid and pCMV-EGFP-EIF3B plasmid). Our kits will provide a convenient and effective method for GC diagnosis with high sensitivity and specificity for doctors to use.

When people want to screen for GC, the most common method is upper endoscopy. In this process, the doctor will insert a flexible tube into the patient’s GI tract and observe its stomach lining tissues. To use our technology, the doctor will need to use a biopsy to collect epithelial cells from the patient’s stomach. This process will be minimally invasive as very few cells are needed. Moreover, for GC patients in metastatic stages, circulating tumor cells (CTC) could be isolated from peripheral blood. After obtaining samples, the doctor will culture the patient’s cells (primary stomach epithelial cell or CTCs) in two petri dishes for a few days until the concentration reaches 60%, which will be observed under a microscope. Afterward, the doctor can use the neofect reagent contained in our test kits to transfect the two plasmids into the patient’s cells in the two petri dishes. The cells need to be cultured for another 48 hours for transcription and translation of the genes contained in our sensors. This process is shown in Figure 1.

Regarding the cells transfected with the pMIR-EIF3B-HSU plasmid, transcription will produce an mRNA that codes for the luciferase protein and forms a highly structured 3’UTR (HSU) region from EIF3B gene [4]. G3BP1 is an HSU mRNA-degrading protein overexpressed in GC tumor cells [5]. In healthy cells, low G3BP1 concentration will lead to slow degradation of luciferase mRNA, producing a large number of luciferase proteins. In GC tumor cells, high G3BP1 expression will lead to fast degradation of luciferase mRNA, producing a small number of luciferase proteins. To visualize the luciferase concentration in the patient’s stomach cells, 20 μl cell lysis buffer can be used to lyse the cells and release the luciferase proteins. Then, the doctor will add 20 μl of luciferase substrate into the system. Afterward, a luminometer will be used to measure the relative firefly luciferase activity. The above steps need to be repeated for the pMIR-EIF3B plasmid, which has no HSU region, as a control to ensure a successful operation. The same process will be carried out for the Renilla substrate for both plasmids as a control group to calculate the relative luciferase activity. Lastly, the doctor will compare the relative luciferase activity between the group with pMIR-EIF3B-HSU plasmid and previously confirmed threshold values from healthy cells and tumor cells at different stages to see if there is a significant difference and hence diagnose GC. This process is shown in Figure 2.

Regarding cells transfected with the pCMV-EGFP-EIF3B-HSU plasmid, transcription will produce an mRNA that codes for the GFP protein and has a highly structured 3’UTR (HSU) region from EIF3B gene [4]. In healthy cells, low G3BP1 concentration will lead to slow degradation of GFP mRNA, producing a large number of GFP proteins. In GC cells, high G3BP1 expression will lead to fast degradation of GFP mRNAs, producing a small number of GFP proteins. To visualize the GFP concentration in the patient’s stomach cells, the doctor can use a fluorescence microscope, which measures the cells’ fluorescence values and reflects GFP concentration. Lastly, the doctor will compare the fluorescence value of the patient with a threshold value decided by previous tests. Fluorescence values above this threshold indicate healthy cells and vice versa. This process is shown in Figure 3.

In the future, our team will measure fluorescence values and luciferase activity in GC tumor cells and healthy stomach cells from patients to determine proper threshold values to minimize the probability for false positive and false negative reports. Furthermore, we may also consider developing a cell-free system and an adenovirus infection system to meet the needs of different doctors and patients. Compared to existing systems, the cell-free system do not need transfection, which is more accessible and robust [6]. Meanwhile, considering a high transfection efficiency of adenoviral vector for primary cells, recombination adenoviral vector also can be used for diagnosis in the future [7]. Both systems will require less instruments, enabling our method to be used at less-developed regions. Nonetheless, these two systems are not fully developed, requiring the endeavor of future scientists.