Cancer, as a major problem in the path of medical progress, is threatening the lives of many people. While some cancers can be effectively cured with timely detection and treatment, brain tumors still rank first in the world in terms of incidence and mortality, making them one of the most difficult categories of all tumors to treat.

Glioma, one kind of brain tumor, is the main subject of our project. Data show that glioma affects 5 to 8 out of every 100,000 people worldwide. In China, there are 45,000 new cases of glioma each year, second only to pancreatic cancer and lung cancer as serious life-threatening malignant diseases. The more established treatments for glioma today include surgical resection, radiation therapy, and chemotherapy. Among them, surgical resection is more difficult and risky, because the glial matter is spread all over the brain, and the brain structure is more complex, which makes the scope of surgery suffer from limitations and a lot of risks. Radiation therapy is based on the principle of targeting and killing cancer cells by radioactive rays, which can also cause considerable side effects. Therefore, in the Chinese National Health Care Commission's Guidelines for the Treatment of Glioma, both radiotherapy and chemotherapy are recommended as an adjunct to surgical treatment to minimize the possible side effects of each treatment.

Among chemotherapies, temozolomide is by far one of the most efficacious drugs, and strictly speaking, no alternative to it has emerged on the market. However, the drug has a clear limitation for glioma patients, namely the development of drug resistance after long-term administration.

There is no drug on the market that can fully solve the temozolomide resistance or replace temozolomide, and all the progress is at the theoretical and experimental stage, so our project can fill this gap.

Our project is aimed to supply glioma temozolomide-resistant cells and PDRG-1 knockdown Glioblastoma Cells for the research of reducing TMZ resistance, as well as providing proliferation services to pharmaceutical companies, drug research institutes, university laboratories, and drug manufacturers in China, as well as to pharmaceutical representatives, pharmaceutical researchers, and biochemistry student mentors. The three benefits are that first the knocked down PDRG-1 increases the effectiveness of reducing temozolomide resistance in gliomas, second saving the buyers the tedious process of screening glioblastoma, and third increasing the efficiency of the buyers in developing drugs by proliferating our cells. The target sales groups of our products are the research institutions studying temozolomide resistance, major pharmaceutical companies manufacturing temozolomide, and university laboratories using cells as education materials.
Our products (glioma temozolomide-resistant cells and PDRG-1 knockdown Glioblastoma Cells) could speed up the development on TMZ drug resistance studies and finally contribute to the treatment of Glioma and glioma patients.

TMZ-resistant cell line could be used for the TMZ- resistance research. T98G and U118 cells were selected for developing TMZ-resistant cell line.

Experimental method:
The drug concentration of starting TMZ was set with IC50 of each cell line, the medium containing TMZ was changed every two days, and the same concentration of TMZ was maintained for two weeks. The new IC50 was calculated, the TMZ concentration was increased, and the solution was changed in the same way for another two weeks. This was repeated until IC50 was increased to 500uM. After the successful preparation of TMZ-resistant cells, the culture was maintained at a low concentration of TM billion (different cell concentrations were different). Cell lines were expanded and preserved after establishment.

Laboratory safety has always been a key issue, especially under the impact of the epidemic. Inside the laboratory, we strictly obey the regulations and comply with iGEM's safety-related requirements. The strain we used in this experiment is the Escherichia coli strain (DH5α) which is non-toxic and non-pathogenic.
Especially, among our experimental materials, lentivirus GV248 was used to establish cell lines. GV248 is derived from HIV. It is the second-generation product of the lentivirus vector, which includes three independent vectors: packaging plasmid (pspax2), envelope plasmid (pmd2.G), and transfer plasmid (lenticas9-mCherry). For the sake of experimental safety, we directly choose to ask the company for lentivirus packaging.
To ensure our product will be properly used, we will require the necessary license or papers to prove our customers are qualified to use our product and will be used for research only.

Before implementing our solution in the real world, we are still facing some challenges in commercializing the product.

(1)Potential challenges in supply chain and product development
Firstly, the product transportation chain is not mature enough in this kind of industry. Secondly, so far we haven't tested the stability of genetic information of our PDRG-1 knockdown glioblastoma cells in constant replication.

(2)Pressure from other pharmaceutical R & D achievements
China vigorously develops technology industries, especially medical cell engineering research, and in the medical industry, the development speed is fast, and there are many iterative products, which will be easily replaced. Our cell line products are specifically targeted for the knockdown of temozolomide-resistant glioma blasts, but the iterative nature of the product will probably lead to a decrease in sales which brings a lot of pressure on product development and upgrading. In our previous interview, Zhang Liang, a professor at Shanghai Jiaotong University said: "Nowadays, major pharmaceutical companies in the world are frantically researching upgraded drugs to replace temozolomide, but no research has passed the third phase of clinical trials. If a drug to replace temozolomide is developed on the market, this product will lose its commercial value, but it still has scientific research value."

Our future plan put strength on the promotion of the products and based on the four stages of our experiments, we decided to develop our business strategy as follows.

(1) Offline popular science advertisement:
We have advertised our products and the principles of our lab around popular business circles in Shanghai by holding lectures, which promoted the knowledge level of glioma and TMZ resistance for people.

(2) Online promotional activities through social media
Our company has built our own social media matrix, including WeChat public account, Weibo, TikTok number, video number, little red book, and Twitter.

(3) PR activities
We worked with PR firms in the pharmaceutical industry, like Doctor Clove, Nutshell Health, other Internet celebrity experts in oncology, and medical health we-media bloggers.

Of course, as we mentioned in the Challenge part, we will not stop developing and upgrading our technology by conducting more tests on our PDRG-1 knockdown Glioblastoma cells to further study the further investigate the PDRG signaling pathway to gain a deeper understanding of the molecular mechanisms of TMZ tolerance development and thereby inspire new research directions seeking to eliminate or moderate TMZ tolerance. We believe that if we can fully understand the mechanism of PDRG1 in the TMZ-resistance process, we could provide new ways to treat GBM.

(1) Research Report on temozolomide industrial chain. n.p.2020.Web. 20,July, 2022. https://3g.163.com/dy/article/DNV81P340516D8N5.html.
(2) How much is the incidence rate of brain tumors? A quick understanding of brain tumor related knowledge .n.p.2020.Web. 20, July, 2022.
(3) Advantages and disadvantages of radiotherapy. n.p.n.d. Web.22, July, 2022. https://www.vsdiffer.com/proscons/pros-and-cons-of-radiation-therapy.html.