Attenuated bacterial strains have been investigated on the premise of selective tumor colonization and drug delivery potential for decades. Salmonella Typhimurium VNP20009 was derived from the parental strain 14028 through genetic modification and tumor targeting ability, being well studied for anticancer effects in mice. In 2001 Phase 1 Clinical Trials, patients diagnosed with melanoma were introduced with VNP20009, resulting in safe delivery of the strain and targeting to the tumor, however no anticancer effects were observed. Recently, it was discovered that VNP20009 contains a SNP in cheY, which encodes the chemotaxis response regulator of flagellar motor function, rendering the strain deficient in chemotaxis. Replacement of cheY with the 14028 wild-type copy resulted in a 70% restoration of phenotype in traditional chemotaxis capillary assays compared to the parental strain. We attempted to optimize the chemotactic potential of VNP20009 but were unable without reversing the attenuated state of VNP20009.
Due to the role of chemotaxis in bacterial tumor colonization and eradication remaining unclear, we aimed to compare VNP20009 and VNP20009 cheY+ primary tumor colonization and impact on metastasis in an aggressive 4T1 mouse mammary carcinoma model. Bacterial tumor colonization and metastatic potential of the cancerous cells to the lungs appear bacterial chemotaxis independent. Moreover, mice bearing tumors exposed to Salmonella exhibited increased morbidity that was associated with significant liver disease. Our results suggest that in our timeline VNP20009 may not be safe or efficacious when used in the context of immunocompetent animals with aggressive, metastatic breast cancer. In a novel approach, we aimed to understand the bacterial-cancer cell relationship within the tumor microenvironment, with an emphasis on gene expression changes occurring within the eukaryotic transcriptome. We employed the B16-F10 mouse melanoma model because VNP20009 is known to colonize and eradicate these tumors in mice. First, we optimized a timeline for Salmonella treatment of mouse melanoma, finding a dramatic delay in tumor growth between 2 and 7 days due to the presence of Salmonella. Additionally, we observed upregulation of the IFN-gamma signaling pathway within tumor tissue upon exposure to Salmonella after 7 days. In future studies, we aim to analyze the bacterial transcriptome in the tumor microenvironment to gain unique understanding and contribute to knowledge supporting bacterial-mediated cancer therapies. / Ph. D. / Bacteria have become our allies in the fight against cancer. Strains of Salmonella, normally thought of as a cause of gastrointestinal discomfort, are able to target cancer in the body and effectively shrink tumors in several animal models. Specifically, a strain of Salmonella Typhimurium called VNP20009, has shown great promise as an anticancer agent. Research on VNP20009 culminated in a Phase 1 Clinical Trial in which safe delivery of the strain and targeting to the tumor were achieved, however no anticancer effects were observed. We hypothesized further targeting of Salmonella could be achieved using chemotaxis, the coordination of flagellar driven movement with sensing environmental chemical gradients, akin to the nose of the bacterium. We discovered strain VNP20009 to be defective in chemotaxis, due to a genetic mutation that occurred during the strain’s construction. We were able to restore chemotaxis of the strain, at least partially, and discovered we could not further optimize chemotaxis without compromising the safety profile of VNP20009. We tested the effect of chemotaxis on tumor colonization in a mouse breast cancer model and found that the bacteria had an additive effect in causing liver disease and morbidity of the mice. We finally examined genome-wide gene expression changes occurring in the tumor microenvironment, as a response to anticancer agent VNP20009 colonization in a mouse melanoma model of cancer. Overall, this work contributes significantly to the understanding of VNP20009 chemotaxis and its tumor targeting abilities.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/84898 |
| Date | 22 August 2018 |
| Creators | Broadway, Katherine Marie |
| Contributors | Biological Sciences, Scharf, Birgit E., Caswell, Clayton C., Popham, David L., Behkam, Bahareh, Jensen, Roderick V. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/vnd.openxmlformats-officedocument.wordprocessingml.document |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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