Anthony Park

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Dr. Bartlett
Biology 23000: Biology of the Living Cell
6 October 2016
The idea of using bacteria to cure cancer has been talked about and attempted since the
1890s1. In the paper, Synchronized cycles of bacterial lysis for in vivo delivery,2 researchers
attempt to use bacteria as a platform for fighting cancer in vitro, which means in a test tube3 as
well as to regulate them in vivo, meaning in the organism. This paper was to address the problem
of future cancer research as well as an alternative to or a compliment to traditional methods of
chemotherapy. Currently, the prevailing method of cancer treatment, chemotherapy, is a method
of cancer treatment which has the potential to mildly to severely harm healthy cells around the
tumor4. This necessitates research in alternative methods of cancer treatment. Furthermore, there
are benefits to having a biological agent instead of a chemical agent such as more complex
reactions against tumor cells as well as a kill switch which can limit bacterial growth.
Therefore, the major problem the researchers are trying to tackle is to find an alternative
treatment to cancer using bacteria and genetic engineering as a basis.

The methodology of the experiment is described in depth in the article. In order to make
sure that other scientists can check their work, they describe how their strain was grown. They
grew their bacterial strain in an LB medium, which stands for Luria broth5. This is a medium
thats commonly used to grow bacteria in labs5. The hlyE gene, which was the gene that they
engineered within the bacteria due to known anti-tumor properties2, was made using polymerase
chain reaction. This procedure uses a known strand of DNA that needs to be copied, primers, and
DNA polymerase. These are all just molecules or proteins that allow the DNA to be copied over
and over again until enough is created to be usable to scientists6. These genes as well as those

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that were just synthesized such as mouse CCL21 and CDD-iRGD, which highlight different parts
of the body to the bodys natural immune system and force cancer cells to kill themselves2, were
then put into the bacteria through the use of plasmids. The genes were cloned into plasmids.
Plasmids are circular strands of DNA that bacteria naturally transfer to each other7. That way,
bacteria can take in these artificial plasmids into themselves and incorporate it into its own DNA.
The bacteria are now ready for being put into a test subject.

In order to actually test the bacteria against tumor cells, the bacteria were injected in
female mice that were given tumors from a known mouse cancer cell line. The bacteria that were
modified, the lysis strain, gave off light 100x less than an unmodified strain of the same bacteria.
First, the bacteria were tested in an in vitro (test tube) environment to understand how the
bacteria would deliver the necessary chemicals to have the anti-tumor effect as well as to
understand effective that delivery method is. Bacterial lysis, the act of destroying and breaking
open the bacteria8, proved to be a good method in delivering the necessary chemicals,
specifically the anti-tumor toxin generated by the hylE gene. In vivo, since the bacteria are being
destroyed, the luminescence can be measured to see how its reacting with the tumors and if its
properly working inside the animal. By comparing this to tumor activity, they can tell if the
bacteria are exhibiting the anti-tumor properties that researchers are looking for.

During the testing, researchers gave the treatment orally to the mice by itself or with
another established chemotherapy treatment. This was due to previous studies that showed that
the bacterial treatment could enhance existing treatments2. Furthermore, a triple-strain dose was
administered on the mice. The triple-strain dose was the dose which included the main lysis

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line which had the hylE gene but also the ones that had CCL21 and CDD-iRGD. This was due to
the fact that researchers noticed that the triple-strain dose was more effective than each of the
single ones administered alone.

AHL, a molecule whose production is controlled by a gene, luxI, can be looped in a

positive feedback. This means that the more of the molecule is produced, the more that the gene
makes it produce9. It also creates a negative feedback loop with the bacterial lysis/destruction,
meaning that the more of it is produced, the slower the effect becomes9. These combinations
allow the bacterial growth in the mice to be regulated as need be.

The results of this experiment were that in regards to the single strain bacteria, the hlyE
bacteria could kill tumors approximately 111 minutes after initial activity found in from detecting
the light from the bacteria. The bacteria that had the ability to trigger the bodys immune system
was the most effective amongst the single strain bacteria. By combining all three strains,
researchers found that tumor activity, and hence the effectivity of the treatment, was better than
chemotherapy over a forty-day span. However, this therapy didnt result in completely
destroying the tumors2.

Researchers also found that the bacteria tended to circulate in the mice for 18 days and
have a 30% reduction of tumors during that time. It also tended to penetrate in places where
normal drugs couldnt go to. They also found that with this treatment, the mice tended to live
50% longer than normal mice that had the tumor and didnt receive this treatment2.

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In conclusion, the researchers were able to determine that this form of treatment can be
used to deliver drugs to certain disease sites. The researchers note that certain bacteria have an
ability to colonize disease sites2. Furthermore, they note that because the bacterial population can
be controlled with the positive and negative feedback loops explained above, this design to
deliver drugs can be used to keep immune responses by the patient to a lesser extent than other
forms of delivery, which can cause inflammatory responses or other undesirable immune
responses2. They also note that new bacterial drug delivery can be used by controlling how fast
and how much the bacteria grow and die.

For future direction, there is a desire to maintain the bacterial cycle within the patient.
Since bacteria can mutate, there are pressures to get rid of the plasmid that the procedure relies
on to deliver the drug. So one of the future research aims that the researchers for this paper note
is decreasing the bacterias vulnerabilities to mutation10. Another obvious future direction is to
modify this procedure to be used in humans as they have only been tested in mice so far2, 10.
Furthermore, since the bacteria are used due to their reaction when encountering tumor cells,
further research in alternative means to deliver more potent drugs can be explored as opposed to
being limited to the toxins produced by the hlyE gene. If bacteria were introduced other toxin
producing genes through the same method, a study could be conducted determining the potency
of various known anti-tumor toxins.

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1. Offord, C. (2016, April). Fighting Cancer with Infection, 1891. Retrieved September 25, 2016,
from http://www.the-scientist.com/?articles.view/articleNo/45593/title/Fighting-Cancer-withInfection--1891/
2. Din, M. O., Danino, T., Prindle, A., Skalak, M., Selimkhanov, J., Allen, K., . . . Hasty, J.
(2016). Synchronized cycles of bacterial lysis for in vivo delivery. Nature, 536(7614), 81-85.
doi:10.1038/nature18930
3. Differences between in vitro, in vivo, and in silico studies. (2013, January 03). Retrieved
September 25, 2016, from http://mpkb.org/home/patients/assessing_literature/in_vitro_studies
4. Chemotherapy. (n.d.). Retrieved September 25, 2016, from https://www.cancer.gov/aboutcancer/treatment/types/chemotherapy
5. Making LB Agar Plates. (n.d.). Retrieved September 25, 2016, from
https://www.addgene.org/plasmid-protocols/bacterial-plates/
6. Polymerase Chain Reaction (PCR). (n.d.). Retrieved September 25, 2016, from
http://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
7. Plasmid / plasmids. (n.d.). Retrieved September 25, 2016, from
http://www.nature.com/scitable/definition/plasmid-plasmids-28
8. lysis. (n.d.) Miller-Keane Encyclopedia and Dictionary of Medicine, Nursing, and Allied
Health, Seventh Edition. (2003). Retrieved September 25 2016 from http://medicaldictionary.thefreedictionary.com/lysis
9. Homeostasis: Positive/ negative feedback mechanisms (2013, May 18). Retrieved September
26, 2016, from http://anatomyandphysiologyi.com/homeostasis-positivenegative-feedbackmechanisms/
10. University of California - San Diego. (2016, July 20). Synthetic biology used to limit bacterial
growth and coordinate drug release. ScienceDaily. Retrieved September 25, 2016 from
www.sciencedaily.com/releases/2016/07/160720135647.htm