Date of Graduation

5-2016

Document Type

Thesis

Degree Name

Bachelor of Science in Biomedical Engineering

Degree Level

Undergraduate

Department

Biomedical Engineering

Advisor

Zaharoff, David

Reader

Wolchok, Jeff

Second Reader

Jensen, Hannah

Third Reader

Kim, Michelle

Abstract

Hematogenous metastasis causes 90% of breast cancer-related deaths.Current therapies include chemotherapy and irradiation following surgery. These therapies are very harmful to the human body and do not elicit an anti-tumor immune response. To create a novel therapeutic, an autologous vaccine increasing the immunogenicity of non immunogenic breast cancer cell lines has been proposed.

To create this vaccine, 4T1 mouse mammary breast cancer cells have been selected as the desired cell line to treat. They are non immunogenic and highly invasive. In order to increase their immunogenicity, first projected, was the addition of cytokines to 4T1 cells to increase the expression of immunostimulatory molecules on the cells. TNF-α and IFN-γ, two pro-inflammatory cytokines, were added individually at a concentration of 50ng/mL to 4T1 cells. This concentration was chosen based on an MTS viability assay and ensures that the cytokines themselves are not killing the cells before an immune response is stimulated. Flow cytometry was used to evaluate the surface marker expression of major histocompatibility complex (MHC) I, MHC II, B7.1, B7.2, ICAM, and Fas. There was a minimal effect on each MHC and co-stimulator molecule resulting from TNF-α. IFN-γ developed in an increase in ICAM and MHC II. Neither of the cytokines created a large enough increase to have a large effect on the immunogenicity of 4T1 cells. Therefore, protein expression of 4T1 cells was reviewed.

4T1 cells express G-CSF in large amounts which causes myeloid derived suppressor cells (MDSCs) to accumulate within the spleen of a mouse. These MDSCs produce mature T cells although with high levels of G-CSF expressed they remain in the spleen undifferentiated. G-CSF was knocked out using CRISPR/Cas9 methods in order to increase the immunogenicity of 4T1 cells. The 4T1.GCSF- cell line underwent single colony expansion and the concentration of G-CSF within the cell line was examined using ELISA (n=3). After single colony expansion, G-CSF was undetectable in the 4T1.GCSF- cell line.

Next, the 4T1.GCSF- cell line will be used in an in-vivo prophylactic vaccination. 100,000 4T1.GCSF- cells will be irradiated and subcutaneously inserted into the mice as a primary vaccine on day 0 and a booster on day 10. On day 20 the mice will be challenged with live 4T1 cells and the tumor volume will be measured three times per week.

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