Hi Rodi,

I'm not sure that this is quite what you are after, but since doxorubicin works by intercalating into DNA and fluoresces- if you can homogenise your cell sample - you can actually just put your cells through a flow cytometer and measure fluroescence in the FL-2 channel (at least on a FACSCalibur). I don't know the exact fluorescent parameters im afraid. I discovered this by accident after treating cells with doxorubicin and then putting them through the cytometer a few days later. I do however work on suspension cells, so am not sure of the feasibility of doing this with your solid tumour cells/mice. I very much doubt the dox fluorescence would be strong enough to do any live imaging of your mice.

Hope that helps!

Vicky

Hello Rodi,

Generally it is not difficult to measure labelled molecules such as labelled doxorubicin (dox) in tumors. You could dissect out the tumor, cut off a piece of material, weigh the amount, homogenize and measure with appropriate tools, such a fluorescent plate reader in your case. Then you would get arbitrary signal per weight unit (for example U/mg). With a U/mg dox standard curve you could follow accumulation time but only in different mice, for example in dose-uptake studies and get the total amount of dox per mg tumor per time point.

However, it becomes a complex problem if you want to make a distinction between how much dox was actually taken up by the tumor, i.e. how much dox has passed the blood vessel wall and accumulates in the actual tumor compartment (as apposed to the amount of dox passively passing through the tumor vasculature), this is doable but has many tricky issues and I'm not sure whether fluorescence would be the way to go. I'm not sure whether this is what you're after but if it is then let me know and I can maybe help you further.

Good luck!

Hi Rodi,

I'm not sure that this is quite what you are after, but since doxorubicin works by intercalating into DNA and fluoresces- if you can homogenise your cell sample - you can actually just put your cells through a flow cytometer and measure fluroescence in the FL-2 channel (at least on a FACSCalibur). I don't know the exact fluorescent parameters im afraid. I discovered this by accident after treating cells with doxorubicin and then putting them through the cytometer a few days later. I do however work on suspension cells, so am not sure of the feasibility of doing this with your solid tumour cells/mice. I very much doubt the dox fluorescence would be strong enough to do any live imaging of your mice.

Hope that helps!

Vicky

Dear Tijs thank you , as the purpose of my project is to improve the accumulation in tumor i have to refere to the dose efficiency so you right i need to run the assay in a different mice which make the results not stable sometimes .also do you think that i have to take the whole tumor and weight it ,homogenize and measure or a part of that will be enough ?

Dear Victoria thank you , you right I'm already using flowcytometry for detecting doxorubicn in vitro actually but i have not tried yet with cells extracted from tumors,also this method could be one of the semi quantification maybe .concerning the live imaging, why do you think that doxorubicn flourescence is not strong enough ? indeed i was thinking of using night ow 981 in vivo imaging using the flourescence filter

Hello Rodi,

Generally it is good to take only part of the tumor for quantification, but this is only possible with enough tumor material. You can then use the other part for other purposes such as sectioning/immunohistochemistry staining, biochemistry etc. If the tumors are smaller you may want to use them only for uptake quantification; how much tumor material you minimally need would depend on the lower limit of your detection method. This is a thing you could and should test empirically in vitro with your labeled dox in cells prior to starting the animal studies! Do you have any idea of how much dox would be expected to accumulate in the tumor cells? This could also help to estimate the minimal amount of tumor tissue required. If not, a pilot study with few animals may be warranted to determine threshold of detection and accuracy of the methodology.

Also make sure to weigh all parts of and only the tumor parts, so be careful and precise with your dissection as taking 'normal' tissue along will twist your results. It may be a good idea here to try out Vicki's suggestion of homogenization (with trypsin/EDTA treatment) and flow cytometry.

I would not recommend trying live imaging for quantitative purposes as it is extremely difficult due to excitation/emission issues in deeper tissues. Even with a two-photon microscope you only get 1mm into the tissue and most experimental tumors I know of are much larger in diameter than that. Also to use it for quantification purposes is tricky.

You're right in that the use of several mice per treatment group is required due to inter-and intratumoral variation. You need to use statistics to determine the significance of your results.

Best wishes,

Tijs