Is it available to use IVIS in your study?

1. Histopathology,

2. Immunohistochemistry.

there are specific marker for determining primary origin.

2-3 months is enough time for metastasis. you have to look first to the lungs tissue. It is easy to recognise the tumour by prepare a well stained slides.

There are particular markers for metastasis; depending on the type of cancer induced for maximum accuracy.

Additionally, you may perform a molecular profile to determine if the cancer has spread to the lungs; lung cancers have changes in the ALK gene.

Thanks for all your responses. Seems like using the lung digest method and subsequent culturing/histopathology will be ideal in my case as we don't have IVIS or PET-CT facility.

I once had a similar problem you now have. What you can do:

1. Generate tumor cells that stably express luciferase and then inject them. Wait.

2. Prepare lysates of individual tissues, e.g. draining lymph nodes (don't forget to keep some for IHC)

3. Measure luciferase activity in each sample

4. Normalize (by total protein conc. etc)

5. You can now also stain the remaining tissues using IHC with an anti-luciferase antibody if you don't have another marker

I used Promega's Glo luciferase substrate which worked well.

Similar to this:

http://www.jleukbio.org/content/84/4/965.full.pdf

I forgot to mention in my previous answer that there's another way you could measure metastasis to other tissues (in case you don't want to generate stable cell lines): by quantitative real time PCR. If you use for example human cancer cells, you could design primers that selectively amplify human GAPDH but not murine GAPDH. You would also have to make a standard curve so that you can quantify it. You could then normalize for total mRNA or cDNA concentration.

Like the luciferase method, this is highly sensitive. Northern blot or western blot would be by far not sensitive enough as you will probably only have a few hundred tumor cells that initially metastasized.

Similar to this:

http://www.nature.com/nature/journal/v410/n6824/abs/410050a0.html

Also, I don't think that your mentioned lung digestion approach will work because if the tumor cells metastasize via the lymphatic system, you will not have lung metastases but metastases in the draining lymph nodes. And if you have lung metastases, you will also not know how many tumor cells a) survive the digestion process, b) you lose during the digestion c) don't grow in culture and d) if cells isolated from different samples will grow at different rates once put back in culture as metastasis to other organs can favor specific tumor cell clones in one mouse/experiment but not in the other. Also, attachment to the lung capillaries plays an important role in lung metastasis and depends on the tumor cell size, tumor cell adhesion molecules etc, which again can be dependent on the cell cycle etc).

Also, quantifying the metastases by IHC is not reliable and is very tedious because you would have to prepare hundreds of sections of each organ to make sure you get all of the micro metastases as a section is a few micrometers thick and the size of an organ like the lung can be more than a centimeter.

Hi Adrian,

RT-PCR or luciferase studies would have been perfect if it was a cell line induced tumor. But, in this case, I am using a chemical carcinogen to induce tumor in mice. So I think those approaches are not feasible.

2 months time is quite sufficient for metastasis, but it depends on the type of cancer. Usually malignant ones get metastasized within very few weeks while benign don't get metastasized. Once the tumor is formed in the body, it should be checked for its etiology and type. Also the rate of angiogenesis in the individual could be a determinant factor to deduce the chances of metastasis. What if some kind of biomarker could be standardized?