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Cancer Research 

Its easier than It sounds I promise 

Since August of 2018, I have been given the opportunity to research, learn, and create my own Cancer Treatment through Western Reserve Academy under Dr. Robert Aguilar in conjunction with the Cleveland Clinic. After 3 years I am highly proficient in lab work. I need little to no guidance doing things such as making media, pouring plates, setting up PCR reactions, pipetting, completing electrophoresis, thawing, growing and identifying 4t1 breast cancer cells, Cyropreserving Cells, and can properly follow most SOPs.

Pantoea Agglomerans As an Anti-Tumorigenic Agent

BELOW IS A PAPER COMPLETED IN MAY OF 2020 ABOUT THE PROGRESS OF THE CANCER TREATMENT THUS FAR

Pantoea Agglomerans As an Anti-Tumorigenic Agent

Elizabeth S. Krapf1 and Erika D. Chagin2, Chloe Beggs3, Avery Brewer4, Robert Aguilar4

  1. Western Reserve Academy

  2. Western Reserve Academy

  3. Western Reserve Academy

  4. Western Reserve Academy

  5. Western Reserve Academy

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INTRODUCTION

 

4T1 cells are highly tumorigenic cells derived from the mammary glands of mice that can randomly metastasis into organs such as the brain, lymph nodes, liver, and lungs (Pulaski and Ostrand‐Rosenberg, 2000). Their metastasis is very similar to that of human breast cancer, which is prevalent in 1 in 8 women (Pulaski, B.A. and Ostrand‐Rosenberg, S., 2000). In fact, about 42,170 women die from breast cancer a year (American Cancer Society, 2020). Bacterial treatments, although proven successful by Dr. William B. Coley through the 1890s, have been overlooked, and the current treatments include radiation, chemotherapy, or mastectomy, although these methods have very detrimental side effects including anemia, infertility, and death.  

 

Pantoea agglomerans  (P. agglomerans) a gram-negative bacillus bacteria that is typically isolated from dust, and plant surfaces (Dutkiewicz, Mackiewicz, Kinga, et. all, 2016). P. agglomerans are known to release microvesicles, containing anti-tumorigenic and pro-inflammatory endotoxins which stimulate the release of cytokines such as interferon (IFN) and tumor necrosis factor alpha (TNF-α) (Śpiewak, Dutkiewicz, 2008). These cytokines are also produced by the immune system to target infections and tumors, therefore by using P. agglomerans as a therapeutic agent, an immune reaction releasing cytokines is simulated when the real immune system’s cytokines do not respond to tumor cells. However, the ability of these microvesicles to produce enough anti-tumorigenic endotoxins to shrink a tumor considerably is unconfirmed.

If P. agglomerans is proven to shrink tumor growth by releasing cytokines, its anti-cancer properties and potential as a vaccine will also be proven. By starting in-vitro, different concentrations of P. agglomerans will be cultured with 4T1 cells to observe the possible shrinkage. A dramatic loss of tumor mass is expected as the bacteria actively secrete endotoxins. The importance of IFN and TNF-α in triggering an immune response to destroy pathogens suggests a high likelihood that the cytokines will work similarly when combined artificially versus natural release. 


In order to gauge P. agglomerans  , P. agglomerans will be cultured in wells containing 4T1cells. The growth or shrinkage of 4T1 confluency will be measured under a microscope. Cell viability will then be mesured. We will end by tessting its efficany in-vivo with 4t1 inoculated Nu/Nu mice.

MATERIALS AND METHODS

 

Identifying Pantoea agglomerans 

When trying to find the correct method for culturing, the best media was decided to be tryptic soy agar and broth. The tryptic soy agar was made and P. agglomerans was cultured on this agar in a petri dish, culturing the bacteria on LB agar as a control. Also, the bacteria was grown on a plate with 25ul of ampicillin, a plate with 25ul of kanamycin, a plate with 25ul of oxytetracycline, and a plate without antibiotics to test its antibiotic-resistant qualities. The most growth with tryptic soy agar plates was without antibiotics, and P.

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agglomerans was not resistant to any of the tested antibiotics. The gram-stain required two samples of bacteria and photos of gram-positive versus gram-negative bacteria for comparison. Our bacteria strain was gram-negative, supporting the hypothesis that P. agglomerans bacteria had been acquired. 

 

Culturing  P. agglomerans on Agar Plate

For culturing purpeses a tryptic soy agar was used.  5g/L glucose, 10g/L tryptic soy broth, and 40g/L glycerol where combined in a 250 ml in a sterilized Erlenmeyer flask. Using aseptic technique, Frozen P. agglomerans were thawed in a water bath at 37 celcius with gentle agitation and periodic tube inversion. Once thawed, a prepared agar plate was used to sreak P. agglmomerans and cultured for 1-2 days at 37 degreies celius  in order to isolate colonies. Later the agar recipe was altered to a tryptic-soy agar, which consisted of 5g/L glucose, 10g/L tryptic soy broth, and 40g/L glycerol. 

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Cryopreservation of P. agglomerans

 Using aseptic technique, cyropreseved  P. agglomerans were thawed in a water bath at 37 c with gentle agitation and periodic tube inversion. Once thawed, a sterilized inoculating loop was lightly dipped into the conical tube and covered with P. agglomerans before steaking on a agar plate. The plate was then placed upside down in a static incubator at 37 celcius for up to 3 days. An overnight culture was performed by pipetting 50 ml of broth into a 125 ml flask, inoculating the overnight culture using a sterile pipette tip. The flask was placed in an incubator shaker overnight. 150 ul of the  P. agglomerans was combined with 850 ul of glycerol using a pipette in a cryotube. The cryotube was vortexed and then stored at -80 celcius.

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GRAM STAIN 

A drop of the suspended colonie was placed directly on the slide. However, if the culture was taken from a petri dish a drop of water would be placed on the slide with a minute amount of P. agglomerans  from an isolated colony. Using a bunsen burner and forceps the bacteria was heat fixed to the slide. Applying heat to fix P. agglomerans to the slide was chosen over letting the smear air dry because the heat helps adhere bacteria to slides. Crystal violet was liberally applied over the fixed culture for 60 seconds before being gently rinsed off with a distilled water. Iodine (Potassium Iodide Solution)  was then liberally applied to the smear and allowed to sit for 60 seconds before being gently rinsed with distilled water. Decolorizer was liberally to the slide and was rinsed off after 5 seconds. When the solvent that flowed over the slide was no longer collared, the plate-rinsing stopped. Saffron O solution was then applied as a counterstain for 50 seconds before being rinsed off with distilled water. The slide was then jostled  to remove excess water and air dried. Once adequate, the dried slide was then placed under a microscope. If the smear is pink it is gram negative, if the smear is purple it is gram positive and if the smear is a mixture of pink and purple the bacteria is contaminated. P. agglomerans is a gram negative rod bacteria.

 

RESULTS

Culturing P. agglomerans 

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Plates made with tryptic-soy agar, where streaked yielded the best isolated colonies. When cultured in an incubator static at  37 celcius for up to two days. P. agglomerans proved resistant to Ampicillin, Penicillin and Kanamycin. The injection of 25 ul of the aforementioned antibiotics seemed to have no effect on the growth of the colonies.

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Identifying P. agglomerans VIA Gram Stain

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A gram stain was used to test to see if the bacteria in question was in fact P. agglomerans. After the Procedure the heat fixed bacteria was in fact gram negative or pink in color. P. agglomerans is a gram negative rod bacteria. The color is due to the differences in the physical and chemical properties of the bacteria's cell wall. The cell wall around gram- negative bacteria prevents some of the dye from adhering. The slide placed under the microscope was a pink in color meaning it, like P. agglomeranns, was gram negative.

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DISCUSSION

Thus far, the answers to our hypothesis are unknown because we have yet to begin our experiments with P. agglomerans besides identification. Considering identification, our hypothesis that our bacteria was P. agglomerans was not entirely proven to be correct. The negative gram-stain test and growth on tryptic soy plates provided evidence that our bacteria could likely be P. agglomerans, but does not solidify this. Because gram-negative bacteria encases such a broad range, we cannot use this to prove or disprove our hypothesis. Our next step was to further identify our bacteria to prove our hypothesis, but we were unable to continue our experiment. Once we successfully identify our bacteria we will be able to continue our experiment by beginning to culture the bacteria with 4T1 cancer cell in wells, and our question will ultimately be answered depending on its anti tumergenic affects.

REFRENCES

  1. Pulaski, B.A. and Ostrand‐Rosenberg, S. (2000), Mouse 4T1 Breast Tumor Model. Current Protocols in Immunology.

  2. https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471142735.im2002s392.    American Cancer Society (2020), How Common is Breast Cancer? American Cancer Society https://www.cancer.org/cancer/breast-cancer/about/how-common-is-breast-cancer.html

  3.     Dutkiewicz, J., Mackiewicz, B., Kinga Lemieszek, M., Golec, M., & Milanowski, J. (2016). Pantoea agglomerans: a mysterious bacterium of evil and good. Part III. Deleterious effects: infections of humans, animals and plants. Ann Agric Environ Med, 197-205. https://doi.org/10.5604/12321966.1203878

  4.  Śpiewak, R., Dutkiewicz, J. (2008). IN VITRO STUDY OF PRO-INFLAMMATORY AND ANTI-TUMOUR PROPERTIES OF MICROVESICLES FROM BACTERIAL CELL WALL OF PANTOEA AGGLOMERANS. Ann Agric Environ Med., 15(1), 153-161. http://www.aaem.pl/IN-VITRO-STUDY-OF-PRO-INFLAMMATORY-AND-ANTI-TUMOUR-PROPERTIES-OF-MICROVESICLES-FROM,90204,0,2.html#ungrouped

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