| Popis: |
Earlier work showed transfer of cytoplasm between co-cultured cancer cells and fibroblasts. Cancer cell changed upon uptake of fibroblast contents, with altered morphology and increased cell volume, internal cell complexity and cell migration velocity. The biomechanical basis for transfer was revealed as hydrodynamic, and was termed 'cell-projection pumping'. An important role for cell-projection pumping in driving cancer cell diversity and disease progression was proposed. Assays used in earlier studies averaged the response of many thousands of cancer cells. This may have obscured important effects only detectable at the single cell level. The current thesis used methods for single cell analysis of the effects of cell-projection pumping from fibroblasts to cancer cells. A novel Cartesian Plot analysis method was developed that related transfer of fluorescent contents between co-cultured cancer cells and fibroblasts, to fluorescence in control cells cultured alone. This identified five separate cell sub-populations in co-cultures, and gave a numeric score for transfer to each cell. Results confirmed the morphological effects of transfer earlier seen. The method was extended to study the effect of cell-projection pumping on global DNA methylation, and it was established that global DNA methylation of cancer cells was unaffected. A single-cell tracking method was developed for analysis of time-lapse recordings of co-cultures which confirmed the morphological effects and increased cancer cell migration earlier seen. Data also revealed for the first time, increased cancer cell division subsequent to uptake of fibroblast contents. Importantly, division of cancer cells abrogated the effect of cell-projection pumping, re-setting daughter cells to their native state. This was consistent with absence of changes in DNA methylation, and also supported the possible exploitation of inhibitors for cell-projection pumping as a novel anti-cancer treatment. |
