Team:Nanjing China Bio/background

1. Cancer introduction and treatment
Current situation
Cancer, also known as malignant neoplasm, is a disease caused by abnormal mechanism controlling cell growth and proliferation. Apart from incontrollable growth, tumor cells invade surrounding normal tissues. Cancer is a joint name for a broad class of malignant neoplasm.
Cancer cells not only reproduce unlimitedly, consuming large amount of nutrition in the patient's body, but also secrete many toxins, causing a series of symptoms. Worst of all, these cells could transfer to the whole body through lymphatic metastasis, blood metastasis, and implantation metastasis, resulting in emaciation, weakness, anemia, inappetence, fever, and severe damage to viscera. Patients will eventually die of organ failure.
Cancer affects human health seriously, posing a great threat on human life. According to WHO, 12 million people were diagnosed with cancer globally every year. And the number of patients dying from the disease is predicted to reach 17 million in 2030. Therefore, cancer is considered the first cause of death. Listed in the following are lists and discussions of several cancer therapies at present.

Specialized cancer therapies and the future direction of researches.
(1) Progress in chemotherapy
The main methods for curing cancer at present are chemotherapy and radiotherapy. Since Goodman et al. tried nitrogen mustard to cure lymphatic tumors in 1942, chemotherapy became common. Chemotherapy is to transport drugs to tumor surroundings using different administrations. Anti-cancer drugs nowadays are from diverse sources, and have different mechanisms. However, they share a common feature of low specifity. They kill certain amounts of normal cells while killing tumor cells, causing toxic side effects like alopecia, chromosome damage, and harm to urinary system. These side effects of anti-cancer drugs greatly reduce the effects of clinical treatment. What are worse, researches revealed that cells can escape from chemotherapy. Cancer therapies at the present stage are lack of drugs possessing significant effects and less toxicity. Even the patient is diagnosed, he could hardly get effective treatments, not only placing heavy economic burdens upon numerous families, but also torturing patients both physically and mentally.
(2) Radiotherapy
Radiotherapy uses high-energy electromagnetic radiation to act on life, changing the structures of biomolecules to achieve the aim of destroying cancer cells which are sensitive to radioactive rays. The radioactive rays applied in clinical treatment are X-ray and γ-ray. Future researches focus on exploring and applying new radioactive sources, accurate location of irradiation site, treatment plans and enhancing sensitivity towards radioactive rays.
(3) Surgery: including radical operations, palliative operations, and exploratory operations sharing one purpose of eliminating tumor tissues completely.
There are no breakthroughs in cancer surgery. The main obstacles are lack of accurate detection method for the range of tumor invasion and the explanation for different reactions of immune system. Though surgical techniques have been greatly developed, the trend for surgery is to narrow the extent of operations to ensure the completeness of organ functions.
(4) Immunotherapy
There is one certain effective immunotherapy at present, with local injection of BCG (Bacillus Calmette-Guerin) to cure melanoma. Transfer factors and immune ribonucleic acid (iRNA) have certain curative effects as well. Some new immune drugs have yet to be identified and product standardized.

2.　Bacteria therapy
Nowadays there are three major problems in the tumor therapy, which are inadequate tissue penetration, low tumor targeting and limited toxicity to all cancer cells. These drawbacks prevent effectual treatments and are associated with increasing morbidity and mortality. However, bacteria can carry out the ideal functions to overcome these limitations. Therefore the bacteria therapy has several advantages superior to common chemotherapy.1
1)It's self-propulsion and can penetrate into tumor regions that are inaccessible to passive therapies.
2)The bacteria are able to sense the local environment and make decision about when and where to release the drugs.
3)When the bacteria carry the reporter gene, they can be easily detected and can provide information about the state of tumors and the effects of the treatment.
4)Anaerobic bacteria can highly colonize the hypoxic areas of tumors, invading the tumor cells. The invading bacteria can reproduce unlimitedly escaping from immune cells. 2
5)These bacteria can not only inhibit the growth of tumor tissues through parasitism in tumor cells, but also secrete natural toxin to kill surrounding cancer cells.
6)Bacteria have large genome and can carry multiple exogenous genes. Researchers can also modify bacteria with gene engineering and synthetic biology techniques to improve their lethality upon tumor cells. 3,4
7)When bacteria spread in human bodies, they could be controlled effectively by antibiotics. Unlike virus, most bacteria parasitic in tumors cannot survive in cells. Their function is to deliver relevant target anti-cancer genes. Meanwhile, bacteria occupy certain parts both outside and inside of cells, competing for nutrition with tumor cells.
8)If we combine bacteria therapy with traditional chemotherapy, targeting on both internal and external parts of tumors, we could get more ideal curative effects. 2
Once the bacteria penetrate into the tumor, bacteria can replicate unimpededly by the macrophage and neutrophil clearance mechanisms that normally serve to eliminate them. At the same time, they can sensitize the immune system to induce tumor clearance.
Dating back to 19th century, researchers have found out that some tumors of cancer patients shrank after infections of bacteria. In 1860s, a German surgeon tried to use Streptococcus to cure a female patient with neck tumor who was near death, pioneering in curing cancer with bacteria therapy. After that, bacteria therapy started to be applied in tumor treatment. However, people could not take control of those bacteria drugs at that time, and terrible side effects occurred sometimes. In some cases, the tumors didn't reduce and the patient died of bacteremia. This side effect became the biggest inhibition for the development of bacteria treatment. Therefore, US Federal Regulators cracked down this dangerous therapy a century later and demanded more clinical trials to improve bacteria treatment, hoping to decrease this side effect.

3　Salmonella
1)The bacteria we normally use today vary from obligate anaerobes (Clostridium and Bifidobacterium for instance) to facultative anaerobes (Salmonella and Escherichia for instance). In the late 20th century, some scientists from Yale chose an attenuated Salmonella Typhimurium as a new drug against solid tumors like melanoma. Compared to common microorganism, this Salmonella Typhimurium has the following unique advantages.
1)The modified bacteria can infect human and mice with high invasiveness and low pathogenecity. Concerning no ideal animal models at present and the infections caused by Salmonella in human and mice are similar, mouse models are usually used in researches to conduct the preclinical evaluation of the anti-cancer effect of Salmonella. 5
2)Being sensitive to common antibiotics helps to clear the bacteria from organisms with antibiotics to interrupt the treatment.
3)As anaerobic bacteria, Salmonella can survive in both hypoxia and necrotic areas of tumors to accumulate selectively in tumor microenvironment.
4)With a distinct genetic background, the bacteria can be easily genetic engineered and attenuated.
5)The experiments can be conveniently operated with oral administration.
6)Salmonella administrated orally can reach the solid tumors. With distant injection, the bacteria can target different tumors, and even track transferred tumor cells.
8)The bacteria can express effect genes, therefore express various therapeutic proteins, taking part in the combination therapy of enzyme prodrugs.
8)With low expenses, the bacteria can be applied in long-term treatments.
To date, there are a number of avirulent salmonella which have been typically impaired biosynthesis of aromatic compounds, purines or amino acids in the tumor therapy. They are used either to directly kill cancer cells or to deliver the expressed therapeutic proteins to tumor in the mouse models. For example, strain A1 and its derivative A1-R which has defects in leucine-arginine target a metastases model. Defects in guanosine5'-diphosphate-3'-diphosphate synthesizing an attenuated salmonella which is an effective vector against CT-26 tumors and metastasis. A defect in the aroA gene gives rise to SL7207 which has been used by several groups. VNP20009 which has defects in msbB and purI exhibited antitumor effects and has been used in the clinical trials. (Modification at the purI genes increases dependence on adenine and disruption in the msbB gene alters lipid A and greatly diminishes the potential to induce TNF-α and cause septic shock.)
As for the mechanism to guide the bacteria accumulation in tumor, we think various nutritional environments in a tumor may compensate the metabolic defects in these bacteria, thereby allowing effective growth in a tumor.

4　Promoter
In animal models, attenuated Salmonella prefer to colonize solid tumors. The concentration of bacteria in solid tumors is 1000 times of that of normal tissues, causing cancer tissues to shrink, even cure cancer. In fact, people used bacteria therapy to cure cancer from a long time ago (over 150 years). However, due to uncontrollable toxic effects to normal tissues, this method has been disregarded for decades.
Thanks to the development of molecular biology technologies, we can modify bacteria as we wish, thus tumor therapy undergoes a spectacular renaissance. In order to eliminate the toxic effects of bacteria upon normal tissues, we need to screen some promoters that express specifically only in tumor areas. As stated above, the central parts of tumor are highly hypoxic areas. Therefore if we screen out the anaerobic promoters that only function under hypoxic circumstances, we could make the genes for cytotoxic drugs specifically express in tumor tissues to decrease the damage on normal tissues. Through consulting on predecessors' work, we searched out several anaerobic promoters, ligating each of them to plasmid vectors with reporter using molecular techniques. Promoters highly expressed in hypoxic circumstances could be screened after transferring Salmonella Typhimurium with modified combined plasmids.
In addition, the Salmonella Typhimurium we used are VNP20009 from the lab of Professor Zichun Hua, a bacteria knocked out of chromogenes purI and msbB. The bacteria are also removed of antibiotic-resistance markers, becoming stable antibiotic sensitive strains.6
1 Jia, L. J. & Hua, Z. C. Development of bacterial vectors for tumor-targeted gene therapy. Methods Mol Biol 542, 131-154 (2009).
2 Dolgin, E. From spinach scare to cancer care. Nature medicine 17, 273-275, doi:Doi 10.1038/Nm0311-273 (2011).
3 Ganai, S., Arenas, R. B. & Forbes, N. S. Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice. Brit J Cancer 101, 1683-1691, doi:DOI 10.1038/sj.bjc.6605403 (2009).
4 Jia, L. J. et al. Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model. International Journal of Cancer 121, 666-674, doi:Doi 10.1002/Ijc.22688 (2007).
5 Forbes, N. S. Engineering the perfect (bacterial) cancer therapy. Nat Rev Cancer 10, 784-793, doi:Doi 10.1038/Nrc2934 (2010).
6 Clairmont, C. et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181, 1996-2002 (2000).