Fusion Cells Cancer Vaccines Kildow

NOTES FOR DISCUSSION

Background** Basi**s: Try to find a way to kill off (lyse) breast tumor cells
Researcher’s Ideas: Dendritic Cells are cells that present antigens from particular malignant cells (in this case, cancer cells). DC’s have surface molecules called MHC molecules that carry fragments of an antigen from its internal compartment to the surface so they can be recognized by naïve T-Cells that generate into cytotoxic T lymphocytes (CTL) and kill of malignant cells.
Approach: They would like to fuse DC’s w/breast cancer cells in order to coexpress both the MHC molecules and the antigens presented in the cancer cells to proliferate T-cells and induce CTL against tumor cells-Studies have shown this to work in rodents.

Materials and Methods1. Breast Cancer cell Culture-Got MCF-7 breast cancer cells from biopsies and were grown, clean and cultured w/antibiotics
2. Preparation of DC’s, Monocytes, and T-Cells-Used Peripheral blood monocuclear cells (PBMC) from patients w/metastatic breast cancer. They separated these cells into monocytes, T-cells, and DC’s (GM-CSF stimulated DC’s w/MHC molecules)
3. Cell Fusion-MCF-7 and DC’s mixed together and “glued” by polyethylene glycol
4. Flow Cytometry-Basically like ELISA-they washed both the DC’s and MCF-7 cells and fusion cells with PBS and introduced it to antibody, washed it again, introduced it to another antibody and with this they ran a bidimensional analysis by fluorescence activation to see density and identification of particular antigens and surface molecules (see the dot graphs)
5. Immunohistochemistry-Introduced an anti (MUC1/cytokeratin) body to BC’s which stained if present, same were introduced to fusion cells and a anti(MHC)body which if both present would stain red and blue.
6. Autologous T-cell stimulation-DC’s, breast tumor cells, and Fusion cells were introduced to T-cells, and thymidine uptake was measured
7. CTL Assays-PBMC cells were cultured w/fusion/breast tumor cells to stimulate more T cells as effector CTL’s. They used these CTL’s and introduced them to many other cells (BT’s, monocytes, MCF-7, ovarian cancer cells etc to measure if the CTL’s would attack other cells besides the breast tumor cells. Also, an anti-MHC class I molecule was introduced to the fusion cells to see if CTL proliferation and destruction of tumor cells were MHC dependant
Results 4. Through the flow cytometry, they were able to get results showing that the Breast carcinoma cell (MCF-7) expressed its antigen MUC1 (and MHC I not shown) , but not MHC II and other surface molecules, while the DC’s expressed MHC II (and I) and other surface proteins, but not MUC1. The fused cells showed both expression of MHC II and MUC I which means that both cells were actually fused
5. With immunohistochemistry, they confirmed results above by showing (thru staining) that Breast carcinoma cells had MUC1 and cytokeratin (another antigen) and did not have MHC II, also showed that the fusion cells of autologous DC’s and tumor cells had both MUC1, cytokeratin and MHC II and confirmed w/ bidimensional flow cytometry.
6. T-cell proliferation was only seen when introduced to fusion cells. All other controls such as DC’s and tumor cells alone stimulated T-cell proliferation.
7. T-Cells that were cultured w/ fusion cells showed specific lysing of autologous tumor cells. T-cells cultured w/just tumor cells had not lyse effect on autologous tumor cells. T-cells stimulated by the fusion cells only lysed the breast tumor cells and not any other cells. Taking away MHC reduced lysing of tumor cells-shows the need of this molecule in DC’s to lyse tumor cells.

Conclusion-The fused cell is used as a vaccine to fight cancerous tumor cells in the body. However, this cannot be used right now as a vaccine because cells from one person cannot be put in another person. So, for this to work, fusions cells would have to be made from the cells of the person being treated. Also, this treatment can only be applied to certain cancer types in which the antigen has been identified. iBasically, these fusion cells stimulate T-cells which usually do not respond to tumor cells in the body. Used in antitumor immunotherapy.

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