Team:BNUZH-CHINA/Design
Overview
Some cancers are difficult to cure and can only be managed. Cancers not only cause pain for patients, but also elicit profound sadness and shock among their families. The BNUZH-China 2023 team is addressing this issue by utilizing attenuated Salmonella typhimurium VNP20009 as a chassis and introducing custom-built plasmids capable of inducing ferroptosis in tumor cells.
To further enhance the tumor-specific targeting ability of bacteria, we engineered VNP20009 to express carcinoembryonic antigen (CEA)-specific single chain antibody fragments (scFv) on the bacteria surface.
Upon reaching the tumor tissue, the engineered bacteria will respond to the microenvironment by activating anaerobic promoter (pnirB). The engineered bacteria will then secrete glucose oxidase into the tumor cells through the type III secretion system (T3SS), resulting in production of H2O2 and hydroxyl radicals to facilitate lipid peroxidation.
In addition, we planned to mediate the silencing of SLC7A11 gene after the engineered bacteria invade the tumor cells. This process will lead to a decrease of glutathione and inactivation of GPX4, further accelerating the occurrence of ferroptosis in tumor cells.
To ensure biosafety, we designed a safety circuit based on toxin-antitoxin system to prevent the loss of two functional plasmids and enable the engineered bacteria to self-destruct upon treatment with doxycycline. At this point, our system successfully completed the healing process of cancer.
To further enhance the tumor-specific targeting ability of bacteria, we engineered VNP20009 to express carcinoembryonic antigen (CEA)-specific single chain antibody fragments (scFv) on the bacteria surface.
Upon reaching the tumor tissue, the engineered bacteria will respond to the microenvironment by activating anaerobic promoter (pnirB). The engineered bacteria will then secrete glucose oxidase into the tumor cells through the type III secretion system (T3SS), resulting in production of H2O2 and hydroxyl radicals to facilitate lipid peroxidation.
In addition, we planned to mediate the silencing of SLC7A11 gene after the engineered bacteria invade the tumor cells. This process will lead to a decrease of glutathione and inactivation of GPX4, further accelerating the occurrence of ferroptosis in tumor cells.
To ensure biosafety, we designed a safety circuit based on toxin-antitoxin system to prevent the loss of two functional plasmids and enable the engineered bacteria to self-destruct upon treatment with doxycycline. At this point, our system successfully completed the healing process of cancer.
Chassis Organism
Before selecting our chassis bacteria, we listed the functions that our chassis bacteria should satisfy to
make it as a more effective carrier. We expected that the engineered chassis possess the specificity and
safety to induce ferroptosis of tumor cells. The functions that we needed in our chassis are:
It should be a kind of low-toxicity Gram-negative bacteria: We planned to use the bacterial surface display technology1 to display single chain antibody fragment (scFv) on the surface of the Gram-negative bacteria. Besides, a trace amount of endotoxin released from Gram-negative bacteria can cause a rise in body temperature. The febrile response triggered by endotoxin will last until the pathogen is completely eliminated2. We would like to find a bacterial strain that can be delivered to tissues without causing health risks.
It should possess the tumor targeting ability: Anaerobic bacteria and parthenogenetic anaerobes can be significantly more prevalent in tumors, up to 10,000 times, due to the hypoxic conditions and diminished immune surveillance found in tumor tissues. We required a specific type of bacteria capable of surviving and proliferating exclusively within the hypoxic region of solid tumor tissues in order to induce effective anti-tumor responses.
It should be a kind of bacteria that can express proteins and perform secretory delivery well: We devised a plan to introduce glucose oxidase (GOx) into tumor cells, aiming to induce Fenton reaction and initiate starvation therapy in order to impede cancer cell proliferation. Hence, we sought to utilize bacteria with the capability of either secreting proteins externally or directly injecting them into host cells.
It should be able to efficiently induce gene silencing in host cells: SLC7A11 and GPX4 are core regulators of the ferroptosis pathway, and the down-regulation of them leads to ferroptosis in cells. We planned to construct a short hairpin RNA (shRNA) expression vector for SLC7A11 genes, then to transfer it into the engineered bacteria, and finally to infect the cells to achieve cross-kingdom RNA interference (RNAi). We expected our engineered bacteria to silence the target genes.
Based on all these requirements, we chose the attenuated S. typhimurium strain VNP20009 as our chassis microorganism. This strain was chosen for the following reasons:
It is a kind of low-toxicity bacteria. With the deletions of the purI and msbB genes, VNP20009 is purine dystrophy and without terminal myristylation of lipid A, which reduce the pathogenicity and inflammatory response caused by the strain. VNP20009 has completed phase I clinical trials in patients with metastatic melanoma and renal cell carcinoma. That validates the safety of VNP20009 in clinical practice3.
It is a kind of Gram-negative bacteria that preferentially accumulates in tumor tissue. Because of the nutrient-rich nature of the tumor cells, as well as the facultative anaerobic character and preferential localization in the tumor microenvironment of VNP200094. We planned to use this nutrient-deficient strain and engineer VNP20009 to display specific single-chain antibody using bacterial surface display technology, allowing the engineered bacteria to exhibit enhanced targeting ability.
It is a kind of bacteria that can express GOx and allows GOx to be transported directly from the bacterial cytoplasm to the target cell cytoplasm. VNP20009 releases proteins into tumor cells via its type III secretion system (T3SS). T3SS needle complex allows effector proteins from external bacteria to be injected directly into the cytoplasm of the host cell5.
It is a kind of bacteria that make RNA interference stabilize and persistently repress gene expression. VNP20009 can be phagocytosed into Salmonella-containing vesicles (SCVs) by host cells and replicate in vesicles. The T3SS needle complex of VNP20009 can penetrate SCV membranes, allowing proteins to be secreted into the host cytoplasm. We found that the HlyA gene encodes Listeriolysin-O (LLO), a cytolysin that enables the escape of bacterial plasmids and contents.
Based on our motivation, methodology and approach to induce ferroptosis in tumor cells using engineered bacteria, we can divide our therapeutic component into four parts to enhance comprehension:
- Engineered bacteria delivery & targeting
- Generation of H2O2 to promote Fenton reaction
- shRNA induced SLC7A11 gene silencing
- Toxin-antitoxin system
It should be a kind of low-toxicity Gram-negative bacteria: We planned to use the bacterial surface display technology1 to display single chain antibody fragment (scFv) on the surface of the Gram-negative bacteria. Besides, a trace amount of endotoxin released from Gram-negative bacteria can cause a rise in body temperature. The febrile response triggered by endotoxin will last until the pathogen is completely eliminated2. We would like to find a bacterial strain that can be delivered to tissues without causing health risks.
It should possess the tumor targeting ability: Anaerobic bacteria and parthenogenetic anaerobes can be significantly more prevalent in tumors, up to 10,000 times, due to the hypoxic conditions and diminished immune surveillance found in tumor tissues. We required a specific type of bacteria capable of surviving and proliferating exclusively within the hypoxic region of solid tumor tissues in order to induce effective anti-tumor responses.
It should be a kind of bacteria that can express proteins and perform secretory delivery well: We devised a plan to introduce glucose oxidase (GOx) into tumor cells, aiming to induce Fenton reaction and initiate starvation therapy in order to impede cancer cell proliferation. Hence, we sought to utilize bacteria with the capability of either secreting proteins externally or directly injecting them into host cells.
It should be able to efficiently induce gene silencing in host cells: SLC7A11 and GPX4 are core regulators of the ferroptosis pathway, and the down-regulation of them leads to ferroptosis in cells. We planned to construct a short hairpin RNA (shRNA) expression vector for SLC7A11 genes, then to transfer it into the engineered bacteria, and finally to infect the cells to achieve cross-kingdom RNA interference (RNAi). We expected our engineered bacteria to silence the target genes.
Based on all these requirements, we chose the attenuated S. typhimurium strain VNP20009 as our chassis microorganism. This strain was chosen for the following reasons:
It is a kind of low-toxicity bacteria. With the deletions of the purI and msbB genes, VNP20009 is purine dystrophy and without terminal myristylation of lipid A, which reduce the pathogenicity and inflammatory response caused by the strain. VNP20009 has completed phase I clinical trials in patients with metastatic melanoma and renal cell carcinoma. That validates the safety of VNP20009 in clinical practice3.
It is a kind of Gram-negative bacteria that preferentially accumulates in tumor tissue. Because of the nutrient-rich nature of the tumor cells, as well as the facultative anaerobic character and preferential localization in the tumor microenvironment of VNP200094. We planned to use this nutrient-deficient strain and engineer VNP20009 to display specific single-chain antibody using bacterial surface display technology, allowing the engineered bacteria to exhibit enhanced targeting ability.
It is a kind of bacteria that can express GOx and allows GOx to be transported directly from the bacterial cytoplasm to the target cell cytoplasm. VNP20009 releases proteins into tumor cells via its type III secretion system (T3SS). T3SS needle complex allows effector proteins from external bacteria to be injected directly into the cytoplasm of the host cell5.
It is a kind of bacteria that make RNA interference stabilize and persistently repress gene expression. VNP20009 can be phagocytosed into Salmonella-containing vesicles (SCVs) by host cells and replicate in vesicles. The T3SS needle complex of VNP20009 can penetrate SCV membranes, allowing proteins to be secreted into the host cytoplasm. We found that the HlyA gene encodes Listeriolysin-O (LLO), a cytolysin that enables the escape of bacterial plasmids and contents.
Based on our motivation, methodology and approach to induce ferroptosis in tumor cells using engineered bacteria, we can divide our therapeutic component into four parts to enhance comprehension:
- Engineered bacteria delivery & targeting
- Generation of H2O2 to promote Fenton reaction
- shRNA induced SLC7A11 gene silencing
- Toxin-antitoxin system
Targeting
Although genetically modified S. typhimurium VNP20009 is a useful vehicle for cancer therapy and vaccine
development, it still exhibits limited tumor targeting in vivo6. This
implies that it is necessary to
enhance the ability of engineered bacteria to target tumor cells.
Carcinoembryonic antigen (CEA) is abundantly expressed in a wide range of human carcinomas, including gastrointestinal tract, pancreatic, non-small cell lung and breast cancers, thus constituting a common therapeutic target7. The OmpA protein is one of the main outer membrane proteins of Gram-negative bacteria , which can serve as a carrier for the expression of foreign antigens on the surface of Gram-negative bacteria including Salmonella spp. A method that takes advantage of efficient targeting of OmpA to the outer membrane and allows C-terminal fusion of passenger proteins to be displayed is the Lpp-OmpA expression system8.
Based on the aforementioned studies, we proposed the inducible expression of high-affinity CEA-specific single chain antibody fragments (scFv) into use on the surface of the bacteria.
Carcinoembryonic antigen (CEA) is abundantly expressed in a wide range of human carcinomas, including gastrointestinal tract, pancreatic, non-small cell lung and breast cancers, thus constituting a common therapeutic target7. The OmpA protein is one of the main outer membrane proteins of Gram-negative bacteria , which can serve as a carrier for the expression of foreign antigens on the surface of Gram-negative bacteria including Salmonella spp. A method that takes advantage of efficient targeting of OmpA to the outer membrane and allows C-terminal fusion of passenger proteins to be displayed is the Lpp-OmpA expression system8.
Based on the aforementioned studies, we proposed the inducible expression of high-affinity CEA-specific single chain antibody fragments (scFv) into use on the surface of the bacteria.
Figure 1. The Lpp-OmpA-scFv expression pathway
Generation of H2O2 to promote
Fenton reaction
Ferroptosis is an iron-dependent programmed form of cell death that is strongly associated with abnormal
iron metabolism and lipid peroxide accumulation. Lipid-based reactive oxygen species/phospholipid
hydroperoxide (PLOOH) are thought to be the executor molecules of ferroptosis9. We designed to use
the
engineered attenuated Salmonella VNP20009 to express the proteins relating to this metabolism pathway.
After the engineered bacteria VNP20009 targeting tumor cells, the proteins will be injected into tumor
cells through the type III secretion system (T3SS) to induce ferroptosis in tumor cells, and then achieve
the aim of killing tumor cells.
As an important nutrient, glucose plays a vital role in tumor growth. The proliferation of tumor cells is primarily dependent on aerobic glycolysis, which leads tumor cells to be more sensitive to changes in glucose concentration than normal cells. Therefore, we planned to inject glucose oxidase (GOx) into tumor cells through the T3SS system when the engineered bacteria VNP20009 successfully infected tumor cells5. Glucose oxidase (GOx) can catalyze the reaction of glucose and oxygen in cells to produce hydrogen peroxide (H2O2) and gluconic acid. The consumption of glucose can cut off the nutrient source of cancer cells, thereby inhibiting their proliferation and achieving the effect of starvation treatment.
While achieving the starvation therapeutic effect, the hydrogen peroxide produced by this reaction can be used as one of the reactants to mediate the Fenton reaction. The essence of the Fenton reaction is that redox-active Fe2+ catalyze hydrogen peroxide (H2O2) to produce hydroxyl radicals (·OH) and other reactive oxygen species (ROS). The accumulation of ROS will directly promote the production of PLOOH. If the reduction of PLOOH into corresponding phospholipid alcohols (PLOH) by GPX4 and GSH didn’t work efficiently, these substances will continue to oxidize the polyunsaturated fatty acyl moieties in phospholipids (PUFA-PL) to produce more PLOOH. This chain reaction may eventually disrupt the integrity of the cell membrane, leading to the breakdown of the organelle and cell membranes10.
However, at the same time, the antioxidant mechanism in tumor cells may also inhibit the occurrence of ferroptosis and decline its effect. System Xc-/GSH/GPX4 axis mediate ferroptosis defense in tumor cells, thereby maintaining the infinite proliferation of cells11. So, we also designed shRNA modules to silence SLC7A11 to better achieve the effect of killing tumor cells.
As an important nutrient, glucose plays a vital role in tumor growth. The proliferation of tumor cells is primarily dependent on aerobic glycolysis, which leads tumor cells to be more sensitive to changes in glucose concentration than normal cells. Therefore, we planned to inject glucose oxidase (GOx) into tumor cells through the T3SS system when the engineered bacteria VNP20009 successfully infected tumor cells5. Glucose oxidase (GOx) can catalyze the reaction of glucose and oxygen in cells to produce hydrogen peroxide (H2O2) and gluconic acid. The consumption of glucose can cut off the nutrient source of cancer cells, thereby inhibiting their proliferation and achieving the effect of starvation treatment.
While achieving the starvation therapeutic effect, the hydrogen peroxide produced by this reaction can be used as one of the reactants to mediate the Fenton reaction. The essence of the Fenton reaction is that redox-active Fe2+ catalyze hydrogen peroxide (H2O2) to produce hydroxyl radicals (·OH) and other reactive oxygen species (ROS). The accumulation of ROS will directly promote the production of PLOOH. If the reduction of PLOOH into corresponding phospholipid alcohols (PLOH) by GPX4 and GSH didn’t work efficiently, these substances will continue to oxidize the polyunsaturated fatty acyl moieties in phospholipids (PUFA-PL) to produce more PLOOH. This chain reaction may eventually disrupt the integrity of the cell membrane, leading to the breakdown of the organelle and cell membranes10.
However, at the same time, the antioxidant mechanism in tumor cells may also inhibit the occurrence of ferroptosis and decline its effect. System Xc-/GSH/GPX4 axis mediate ferroptosis defense in tumor cells, thereby maintaining the infinite proliferation of cells11. So, we also designed shRNA modules to silence SLC7A11 to better achieve the effect of killing tumor cells.
Figure 2. The SopE-FLAG-GOx expression pathway
Figure 3. GOx is injected into the tumor cells by T3SS for promoting Fenton reaction.
shRNA Induced SLC7A11 gene silencing
As one class of cystine transporter proteins, SLC7A11 (Solute Carrier Family 7 Member 11) is also one
of
the most critical upstream regulators of ferroptosis. Recent studies have revealed that SLC7A11 drives cell resistance to ferroptosis and plays an important role in many diseases such as tumors.
As a key regulatory protein of ferroptosis, the main function of SLC7A11 is to transport extracellular cystine into the cells. Cystine is transported into the cell and reduced to cysteines, which is involved in protein biosynthesis, such as the production of prototype glutathione (GSH), and it also regulates the synthesis rate of glutathione peroxidase 4 (GPX4)12. The latter substance is able to reduce toxic peroxides with GSH as a cofactor, thus inhibiting cell ferroptosis13. When the expression of SLC7A11 gene is down-regulated, the content of cystine in tumor cells will be decreased and the cysteine metabolic pathway will also be inhibited, resulting in the decreased or even blocked synthesis rate of the GSH and GPX4. This will lead to the accumulation of lipid peroxide, destroy membrane lipid bilayer structure and increase cell membrane’s permeability, and eventually induce cell ferroptosis.
Tumor cells' metabolism is more dependent on SLC7A11 than normal cells. Based on this, we tried to silence SLC7A11 gene expression by delivering RNAi effector short hairpin RNA (shRNA) through the engineered attenuated Salmonella VNP20009 to tumor cells. In this module, we designed a functional plasmid containing a shRNA sequence used to down-regulate the SLC7A11 gene in tumor cells. When the engineered bacteria invade the tumor cells, they can release the shRNA into the cell and specifically degrade the corresponding mRNA, thereby blocking the translation of the target gene SLC7A11. Meanwhile, we added the HlyA gene encoding Listeriolysin-O protein to the functional plasmid. This pore-forming protein safeguards shRNA from degradation by allowing its rapid release from entry vesicles14, resulting in the improved efficiency of gene silencing.
As a key regulatory protein of ferroptosis, the main function of SLC7A11 is to transport extracellular cystine into the cells. Cystine is transported into the cell and reduced to cysteines, which is involved in protein biosynthesis, such as the production of prototype glutathione (GSH), and it also regulates the synthesis rate of glutathione peroxidase 4 (GPX4)12. The latter substance is able to reduce toxic peroxides with GSH as a cofactor, thus inhibiting cell ferroptosis13. When the expression of SLC7A11 gene is down-regulated, the content of cystine in tumor cells will be decreased and the cysteine metabolic pathway will also be inhibited, resulting in the decreased or even blocked synthesis rate of the GSH and GPX4. This will lead to the accumulation of lipid peroxide, destroy membrane lipid bilayer structure and increase cell membrane’s permeability, and eventually induce cell ferroptosis.
Tumor cells' metabolism is more dependent on SLC7A11 than normal cells. Based on this, we tried to silence SLC7A11 gene expression by delivering RNAi effector short hairpin RNA (shRNA) through the engineered attenuated Salmonella VNP20009 to tumor cells. In this module, we designed a functional plasmid containing a shRNA sequence used to down-regulate the SLC7A11 gene in tumor cells. When the engineered bacteria invade the tumor cells, they can release the shRNA into the cell and specifically degrade the corresponding mRNA, thereby blocking the translation of the target gene SLC7A11. Meanwhile, we added the HlyA gene encoding Listeriolysin-O protein to the functional plasmid. This pore-forming protein safeguards shRNA from degradation by allowing its rapid release from entry vesicles14, resulting in the improved efficiency of gene silencing.
Figure 4. The Listeriolysin-O expression pathway
Figure 5. Listeriolysin-O is crucial for inducing shRNA into cytoplasm.
Figure 6. Schematic diagram of engineered bacteria expressing shRNA
Figure 7. shRNA-mediated gene silencing and induction of ferroptosis
Anaerobic Expression
While anti-CEA scFv can enhance the targeting ability of VNP20009 towards tumor cells, we aimed to mitigate
the toxic effects of the engineered bacteria on normal tissue. To further enhance the safety of our project, we
needed to screen specific promoters that activate exclusively within the tumor microenvironment
(TME).
In solid tumors, the tumor tissue grows rapidly, leading to significant inflammation, and the vascular system within the tissue is incomplete. Consequently, the tumor microenvironment often exhibits overall oxygen deficiency. The nirB promoter (pnirB) is the promoter of the first gene of E. coli NADH-dependent nitrite reductase operon. This promoter is induced in anaerobic conditions and in response to nitrite and nitrate. Its synthesis mechanism has been thoroughly studied and has been chosen by numerous iGEM teams (e.g., Nanjing_China_Bio 2012, TecMonterrey 2013, etc.) to mitigate the toxic effects of bacteria on normal tissue.
Based on the aforementioned considerations, we selected the pnirB to initiate subsequent killing modules.
In solid tumors, the tumor tissue grows rapidly, leading to significant inflammation, and the vascular system within the tissue is incomplete. Consequently, the tumor microenvironment often exhibits overall oxygen deficiency. The nirB promoter (pnirB) is the promoter of the first gene of E. coli NADH-dependent nitrite reductase operon. This promoter is induced in anaerobic conditions and in response to nitrite and nitrate. Its synthesis mechanism has been thoroughly studied and has been chosen by numerous iGEM teams (e.g., Nanjing_China_Bio 2012, TecMonterrey 2013, etc.) to mitigate the toxic effects of bacteria on normal tissue.
Based on the aforementioned considerations, we selected the pnirB to initiate subsequent killing modules.
Figure 8. The anaerobic expression of SopE-FLAG-GOx
Figure 9. The anaerobic expression of Listeriolysin-O
Toxin-antitoxin system
Considering that our design module contains two functional plasmids, and that both of them are essential
to the engineered bacteria to play their roles, we must maintain both plasmids to ensure that the module
can be implemented successfully. However, without selection pressure, these plasmids may be
instable in vivo, resulting in the loss of functional plasmids. What’s more, when the condition of
illness improved after tumor regression, normal cells are at risk of being harmed by the engineered
bacteria. Therefore, it is necessary for artificially manipulate the initiation of engineered bacterial
suicide.
To this end, we designed a "logical suicide circuit" to maintain two plasmids in the project and to induce engineered bacteria to commit suicide through intake of doxycycline after the end of the mission.
Firstly, we introduced the "logical suicide circuit" in two plasmids (Figure 10). The pSilence
plasmid
used the constitutive promoter prpsM to express the tetR protein which can repress the promoter
pLtetO
well. In the pFenton plasmid, the pLtetO regulates the expression of LacI repressor, the promoter
pLacO
regulates the expression of antitoxin Sok and the trp promoter regulates the expression of toxin
Hok and protein Mok.
Sok is a small RNA which can specifically bind to the mRNA of Mok so as to block the translation of the Mok. Since the expression of Hok protein depends on the regulation of Mok protein, Sok RNA indirectly repress the expression of Hok protein and protect the engineered bacteria in the meanwhile.
When both pFenton and pSilence are exist, repressor tetR will be constitutively expressed to repress pLtetO so as to repress the production of LacI. In the absence of LacI, Sok will be steadily transcribed and prevent the proper folding of Hok to keep the engineered bacteria survival.
To this end, we designed a "logical suicide circuit" to maintain two plasmids in the project and to induce engineered bacteria to commit suicide through intake of doxycycline after the end of the mission.
Figure 10. Schematic representation of the "logical suicide circuit"
Sok is a small RNA which can specifically bind to the mRNA of Mok so as to block the translation of the Mok. Since the expression of Hok protein depends on the regulation of Mok protein, Sok RNA indirectly repress the expression of Hok protein and protect the engineered bacteria in the meanwhile.
When both pFenton and pSilence are exist, repressor tetR will be constitutively expressed to repress pLtetO so as to repress the production of LacI. In the absence of LacI, Sok will be steadily transcribed and prevent the proper folding of Hok to keep the engineered bacteria survival.
Induce Suicide
Once the engineered bacteria have completed the task, patients can take doxycycline (Dox) orally to induce the suicide of engineered bacteria. Dox is a tetracycline analog with anti-tumor properties, which can
combine with tetR and prevent tetR from repressing the production of LacI. Therefore, LacI will combine
with Lac operator to repress the transcription of Sok. At this point, Hok will properly fold into the toxin protein and kill the engineered bacteria.
Figure 11. Dox induce the suicide of engineered bacteria
Maintain Plasmids
In the first case, if the plasmid pSilence is lost, TetR will gradually degrade and will not be
produced
anymore. And then pLtetO will promote LacI gene which will repress the transcription of Sok. As a
result,
Hok will properly fold into the toxin protein and kill the engineered bacteria.
In the second case, if the pFenton is lost, like not passed down to the daughter cells, the TA system will
not work and kill the cells. Hok gene encodes a highly toxic transmembrane protein that is capable of
permeating the cell membrane, leading to drastically imbalanced plasma membrane electrochemical gradients
and cell death. Hok mRNA has a long half-life of 20 minutes while Sok has a half-life of only 30
seconds.
If the pSilence is lost, both previously transcribed Hok mRNA and Sok will remain in the cell.
However,
Sok will degrade faster than Hok mRNA due to its shorter half-life. Without more transcribed
Sok, Hok mRNA
will be then translated and thus kill the engineered bacteria.
Figure 12. Schematic representation of pSilence lost
Figure 13. Schematic representation of pFenton lost
References
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2 Morris, D. D., Crowe, N., Moore, J. N. & Moldawer, L. L. Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro. Am J Vet Res 53, 1298-1301 (1992).
3 Low, K. B. et al. Construction of VNP20009: a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods in molecular medicine 90, 47-60 (2004).
4 Sznol, M., Lin, S. L., Bermudes, D., Zheng, L. M. & King, I. Use of preferentially replicating bacteria for the treatment of cancer. The Journal of clinical investigation 105, 1027-1030 (2000).
5 Costa, T.R. et al. Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13, 343-359 (2015).
6 Bereta, M. et al. Improving tumor targeting and therapeutic potential of Salmonella VNP20009 by displaying cell surface CEA-specific antibodies. Vaccine 25, 4183-4192 (2007). https://doi.org:10.1016/j.vaccine.2007.03.008
7 Marshall, J. Carcinoembryonic antigen-based vaccines. Semin Oncol 30, 30-36 (2003). https://doi.org:10.1016/s0093-7754(03)00233-1
8 Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444-10448 (1993). https://doi.org:10.1073/pnas.90.22.10444
9 Chen, X., Kang, R., Kroemer, G. & Tang, D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol 18, 280-296 (2021).
10 Jiang, X., Stockwell, B.R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nature Reviews Molecular Cell Biology 22, 266-282 (2021).
11 Fu, L.H., Qi, C., Lin, J. & Huang, P. Catalytic chemistry of glucose oxidase in cancer diagnosis and treatment. Chem Soc Rev 47, 6454-6472 (2018).
12 Koppula, P., Zhuang, L. & Gan, B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12, 599-620 (2021).
13 Zhang, Y. et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun 12, 1589 (2021).
14 Guo, H. et al. Targeting tumor gene by shRNA-expressing Salmonella-mediated RNAi. Gene Ther 18, 95-105 (2011).
2 Morris, D. D., Crowe, N., Moore, J. N. & Moldawer, L. L. Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro. Am J Vet Res 53, 1298-1301 (1992).
3 Low, K. B. et al. Construction of VNP20009: a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods in molecular medicine 90, 47-60 (2004).
4 Sznol, M., Lin, S. L., Bermudes, D., Zheng, L. M. & King, I. Use of preferentially replicating bacteria for the treatment of cancer. The Journal of clinical investigation 105, 1027-1030 (2000).
5 Costa, T.R. et al. Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat Rev Microbiol 13, 343-359 (2015).
6 Bereta, M. et al. Improving tumor targeting and therapeutic potential of Salmonella VNP20009 by displaying cell surface CEA-specific antibodies. Vaccine 25, 4183-4192 (2007). https://doi.org:10.1016/j.vaccine.2007.03.008
7 Marshall, J. Carcinoembryonic antigen-based vaccines. Semin Oncol 30, 30-36 (2003). https://doi.org:10.1016/s0093-7754(03)00233-1
8 Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444-10448 (1993). https://doi.org:10.1073/pnas.90.22.10444
9 Chen, X., Kang, R., Kroemer, G. & Tang, D. Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol 18, 280-296 (2021).
10 Jiang, X., Stockwell, B.R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nature Reviews Molecular Cell Biology 22, 266-282 (2021).
11 Fu, L.H., Qi, C., Lin, J. & Huang, P. Catalytic chemistry of glucose oxidase in cancer diagnosis and treatment. Chem Soc Rev 47, 6454-6472 (2018).
12 Koppula, P., Zhuang, L. & Gan, B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12, 599-620 (2021).
13 Zhang, Y. et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun 12, 1589 (2021).
14 Guo, H. et al. Targeting tumor gene by shRNA-expressing Salmonella-mediated RNAi. Gene Ther 18, 95-105 (2011).
