I have isolated tumor-initiating (cancer stem) cells from a solid tumor and have hypothesized that our molecule of interest mediates the differentiation of these cancer stem cells into neo-vascular tissue. I plan to incubate them in a hypoxic environment and measure the production of vascular markers and tube formation as described in the literature as markers for vasculogenesis with and without siRNA knockdown or inhibitor treatment
However, I can not find a clear idea about what media or factors I should add. There are many protocols for maintenance of these cells once they grow, but which media should I be growing them in? To force differentiation I see many researchers use VEGF, but A) I'm planning to measure VEGF production as an output for formation of these cells and B)I don't want to force them to become vascular cells, I want to see if they do it on their own.
If anyone has any direction or advice I would really appreciate it!
EDIT: I'm not starting with bone marrow or mesenchymal ES cells, these are de-differentiated astrocytes.
I would like to talk about the experience with human breast cancer MCF7 cells, however, I have no experience with a solid tumour.
Firstly, your culture system to be used will be hypoxia condition. This may be enough to induce differentiation pre-tumour cells without any additional peptides or chemicals among the mixed cell population of your sample. That is mimicking the in vivo niche condition forming spontaneous cell-cell and cell-ECM interactions. Some cells in your culture condition would make their own way from the mesodermal origin of cells hidden the mixed cells.The mesodermal cells could differentiate into endothelial ball of cells filled with fluid. However, number of those cells will be very small among the cell population. By increasing density, and so the hypoxia state, you could see floating cells of ball. or enclosed shape of cells with a space on the dish bottom under low density. Obviously you are going to use biomarkers for the detection to be verified.
Second, I have seen this types of fluid-filled ball of cells with MCF7 cells which may contain stem-like phenotypes in the cell line. This happened in the medium we routinely used. You could use a similar medium people used for their culture of the origin of the solid tumour. that you are interested in. By the way, MCF7 is so-called epithelial cells, which are also heterogenous cells. They tend to show subtle EMT or MET under certain condition (niche gradually formed during proliferation and differentaition). Your mixed cells would show considerable selection process during culture, becoming much less heterogenous cells during culture condition and duration of culture.
It may be possible to demonstrate what's in your mind although it would be formidable task. So, my advice are to eliminate all the odds you may come across and perhaps to select cells from the tissues after chracterization for proper main experiments. Characterize the probable cell lineages within the dispersed cell suspension from the solid. then isolate by MACS or FACS for rather uniform cell population. It sounds like looking for a needle in a haystack.
You will see the light if you use less heterogenous cells. You can tell exactly what is possible or not with the characterized cells. The cells in neuroblastoma and glioma cell line are very uniform. Just compare your suspended cells prepared from the solid before you do anything. Good luck to you!
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