Proof-of-concept

Abstract

Our project aims to provide a safe, effective and practical approach to bringing and maintaining high levels of L-arginine in solid tumors, especially in colorectal tumors, to facilitate colon cancer immunotherapy. We engineered bacteria to overcome arginine inhibition of the arginine synthesis pathway by adding the argJ gene and deleting the argR gene. To ensure biosafety, we inserted the lysin gene into our engineered bacteria EcN to lyse the bacteria after they've done their work. Laboratory work has been done to verify the efficacy and safety of the engineered bacteria and the therapeutic effect of arginine on colorectal cancer cells. Our experiments showed that our engineered bacteria could produce large amount of arginine. Cellular experiments showed that high concentration of arginine inhibited the viability and anaerobic respiration of colon cancer CT26 cells, indicating the anti-cancer effect of arginine supplementation. High level of arginine also decreased PD-L1 expression of colon cancer cells, which can result in the improvement of the efficacy of PD-L1 related immunotherapy. We also verified that the EcN bacteria engineered with the pGLO-Lysin plasmid were lysed in response to arabinose so that the safety use of the vehicle was ensured.

 

1 Aim of the project

Our project aims to provide a safe, effective and practical approach to bringing and maintaining high levels of L-arginine in solid tumors, especially in colorectal tumors, to facilitate colon cancer immunotherapy. To use high concentration arginine locally to synergize the antitumor effect of immunotherapy on solid tumors such as colorectal cancer, our project uses probiotic E.coli Nissle 1917 (EcN1917, EcN) as a therapeutic carrier to produce a large amount of L-arginine in the tumor, which can enter the digestive tract through the mouth and colonize tumor sites in the digestive tract. We engineered bacteria to overcome arginine inhibition of the arginine synthesis pathway by adding the argJ gene and deleting the argR gene. To ensure biosafety, we inserted the lysin gene into our engineered bacteria EcN to lyse the bacteria after they've done their work. The experiments aimed to verify the efficacy and safety of the engineered bacteria and the therapeutic effect of arginine on colorectal cancer cells. Probiotic EcN has a tumor-targeting effect and can specifically accumulate in tumor tissue as a targeting transporter.

Our product is a powder agent, which can be rapidly colonized in the intestinal tract by oral administration and can stay in the tumor tissue in the intestinal tract for a long time, which has the potential to treat digestive system tumors and has a positive effect. Therefore, this direction can be developed in future clinical applications.

 

2 Functions to be proved

We need to verify the functionality of our product from the following two aspects.

2.1  Arginine production

To test the ability of the engineered bacteria to produce arginine, the rate of arginine synthesis and the amount of arginine produced by our EcN1917^∆argR (argJ) (EcN1917 with argR knockout and argJ gene insertion) were measured by an amino acid analyzer after incubation, and native EcN1917 was used as a control.

 

2.2  Lysis control

To test the function of the lysin gene in our engineered EcN1917, we added arabinose to the EcN1917 colonies and analyzed bacterial growth curves to detect the lysis.

 

2.3  Arginine against cancer cells

To verify the anti-cancer effect of arginine, we treated colon cancer CT26 cells with different concentrations of arginine, and tested the viability, metabolism and PD-L1 expression of the cells.

 

2.4  The engineered EcN anti-cancer

To verify the anti-cancer effect of our engineered EcN, we co-cultured EcN1917^∆argR with the colon cancer CT26 cells, and tested the viability of the CT26 cells.

 

 

3 Summary of the proof

Our experiments showed that our engineered bacteria could produce large amount of arginine. EcN bacteria engineered with the pGLO-Lysin plasmid were lysed in response to arabinose. And cellular experiments showed that high concentration of arginine inhibited the viability and anaerobic respiration of colon cancer CT26 cells, indicating its anti-cancer effect. High level of arginine also decreased PD-L1 expression of colon cancer cells, which can result in the improvement of the efficacy of PD-L1 related immunotherapy.  

 

4  Experiments:

4.1  Arginine production

The production of L-arginine of the engineered bacteria was detected by Hitachi amino acid analyzer. Wild type EcN1917 was used as control. The 24- and 48-hour fermentation broth of the bacteria were collected and broken by ultrasonic crusher, and detected by automatic amino acid analyzer. The results showed that EcN1917^∆argR (argJ) had a yield of 3.6 mM L-arginine.

 

 

Figure 1  L-Arginine production of ECN1917 WT (upper) and ECN1917^∆argR (argJ) (bottom) after incubation for 48h.

 

4.2  Lysis control

Bacteria solution of EcN1917 transformed with our pGLO-Lysin plasmid were treated with 30mM arabinose for 10 hours. The bacteria were lysed significantly as shown below (Figure 2).  

 

 

Figure 2 Effect of arabinose on EcN1917 transformed with pGLO-Lysin plasmid (Left: bacteria solution without arabinose, right: bacteria solution treated with 30mM arabinose for 10h)

 

4.3  Arginine effect on cancer cells

To verify the anti-cancer effect of arginine. We had several cellular experiments on colon cancer CT 26 cells. MTT assay showed that arginine affected the viability of colon cancer cells through concentrations dependent manner (Figure 3-A). As a substrate for endogenous NO synthase, arginine can regulate the tumor microenvironment through the NO pathway. It is generally believed that increasing NO level can increase the blood flow to tumors and alleviate hypoxia in tumors. In order to further explore the effect of L-arginine on the anaerobic metabolism of tumor cells, different concentrations of L-arginine were incubated with CT26 cells to detect the level of lactic acid in the cell supernatant. The experimental results showed that arginine at low concentrations (10 and 20 mM) down-regulated the level of lactate metabolism in tumor cells without affecting cell viability (Figure 3-B). When the concentration of arginine reached 50 mM, it had a certain inhibitory effect on cell viability, and the cells’ regulation of lactate level was limited at this time. Since tumor metabolism is closely related to the tumor immunosuppressive microenvironment, tumor cells can promote T lymphocyte apoptosis by upregulating PD-L1 expression. To verify that arginine can regulate the tumor immunosuppressive microenvironment, we detected the mRNA expression level of PD-L1 in CT26 cells after arginine exposure by RT-qPCR. The results showed that arginine at a concentration of 50 mM could significantly down-regulate the mRNA expression level of PD-L1 in CT26 cells (Figure 3-C). These experiments indicated that arginine at high concentrations (100 and 200 mM) could exert a direct tumor-killing effect. At low concentrations, arginine could regulate tumor microenvironment by down-regulating the expression level of lactic acid and the mRNA expression level of PD-L1 in tumor cells, which was beneficial for tumor immunotherapy.

 

Figure 3 (A) Effect of arginine on the viability of CT26 cells (colon cancer cells): the higher concentration of arginine, the lower survival rate in percent of CT26 cells ; (B) Effect of arginine on the lactic acid level of CT26 cells ; (C) Effect of arginine on the PD-L1 mRNA expression of CT26 cells

 

4.4 The engineered EcN anti-cancer

We also co-cultured CT26 cells with the EcN1917^△argR strain at MOIs of 25, 50,100, and 200 (number of bacteria cells: number of CT26 cells) for 3 hours, and evaluated the viability of the CT26 cells by MTT assay, with wild type EcN1917 (EcN1917) used as control.  As shown in Figure 11, EcN1917^△argR-argJ could significantly decrease the viability of CT26, as compared to  EcN1917, which indicating that arginine producing EcN1917 was capable to exert anti-cancer effect against colon cancer cells.

 

Figure 4: Effect of EcN1917^△argR on the viability of CT26 cells (MOI: multiplicity of infection, the ratio of the number of bacteria to the number of target cells)