Development of Bioluminescent Mammary Cancer Cells with Knocked Down Expression of OSM for Detection of Bone Metastasis in Vivo
Dr. Cheryl L. Jorcyk
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Oncostatin M (OSM) is a multifunctional cytokine belonging to the interleukin (IL)-6 subfamily. OSM was originally recognized by its ability to decrease breast and other tumor cell proliferation in vitro. Based on data from our lab, we hypothesize that OSM is a mediator of breast cancer metastasis to bone. For our model system, we are utilizing two cell lines, 4T1.2 and 66c14, which were derived from the mammary carcinoma of a Balb/c mouse. 4T1.2 cells express medium levels of OSM and are highly metastatic to bone, lymph nodes, and lungs when injected into the mammary fat pad of Balb/c mice. 66c14 cells express low levels of OSM and demonstrate low levels of metastasis that is restricted to the lymph nodes and lungs. A small hairpin RNA (shRNA) to OSM was designed and stably transfected into the cells in order to decrease or ‘knock down’ OSM expression. Enzyme-linked immunosorbent assay (ELISA) was used to quantify levels of secreted OSM protein in both cell lines and confirm knocked down OSM expression. Normal 4T1.2 and 66c14 cells, as well as knockdown 4T1.2 and 66c14 cells, will be injected into the mammary fat pad of female Balb/c mice. To visualize metastasis to bone in vivo, the cell lines are currently being transfected with pGL4, a vector containing a bioluminescence maker called luciferase. The luciferase gene was isolated from the firefly and encodes an enzyme, which along with its substrate luciferin, can be used to image the cells expressing luciferase in vivo. Our aim is to determine whether tumor cell-produced OSM is necessary for breast cancer metastasis to bone by measuring metastasis in cells that have knocked down expression of OSM. If our results confirm our hypothesis, inhibiting OSM and its signaling may be a new target for treating breast cancer metastasis to bone.