Graduation Year

2020

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Graduate School

Major Professor

Subhra Mohapatra, Ph.D.

Committee Member

Shyam Mohapatra, Ph.D.

Committee Member

Eleni Makoutsa, Ph.D.

Keywords

Bioluminescence Imaging, CRC, Orthotopic tumor model, Transfection

Abstract

CRC is the second most common type of cancer in women and the third most common type of cancer in men. It accounts for 9.7% of all cancers in terms of people diagnosed. Measuring the real-time effect of chemotherapeutic drugs has been a major obstacle in developing a tumor model mouse. Tracking the regression of cancer cells after chemotherapy could be an effective way to resolve this issue. Therefore, cancer cells expressing the constitutive luciferase reporter gene can be a potential solution. The biggest advantage of the luciferase (Luc) reporter is that it is detectable at very low concentrations in cell cultures, as well as in animal imaging. Here, we have analyzed different transfection methods to find out the least toxic and most effective technique. We have generated HCT116, a human colon carcinoma cell line, and MC38, a mouse colon carcinoma cell line expressing the luciferase gene constitutively. The optimization of four different transfection reagents was done. HCT116 and MC38 cells were finally transfected with luciferase using Lipofectamine 3000 which had the highest efficiency. Transfected cells were treated with G418 for two consecutive weeks. Single-cell colonies were selected using determined antibiotic concentration and expanded. Stably transfected cell lines were tested for luciferase expression using a luciferase assay. Stability studies were performed for luciferase expression over 3 passages. HCT116-Luc and MC38-Luc cells were grown on a 3D scaffold to form tumoroids that mimic in the Vivo tumor microenvironment; tumoroids were tested for luciferase activity. HCT116-Luc and MC38-Luc cells were injected in mice subcutaneously and orthotopically to form tumors and were tested after 7 days for luciferase activity post addition of D-luciferin intraperitoneally. We have been able to generate stable Luciferase reporter HCT116 and MC38 colon cancer cell lines and bioluminescence Imaging was performed in mice to study colon tumor progression. In conclusion, the luciferase reporter cell lines can be used for studying the efficacy of chemotherapeutic drugs in colorectal tumor regression.

Download

Share

COinS