Dear Colleagues,
We plan to isolate and characterise extracellular vesicles released by dying cells during radiotherapy of head and neck squamous cell carcinoma. Given the duration of radiotherapy (about 6-7 weeks), we are thinking about the optimal time when the maximum wave of cell death occurs in the tumour (Ideally, we'd like to capture the first wave of cell death :)). Do you have any advice on when to take samples after radiotherapy? In this case, would plasma, serum or whole blood be better for the isolation of extracellular vesicles?
Thank you for your answer.
Kind regards,
Jan
Dear Dr. Jan Balvan
The tumor cell fate decisions such as programmed cell death and non-programmed cell death induced by radiotherapy are related to various factors such as tumor cell type, time and dose of radiation and tumor microenvironment. Radiotherapy does not kill cancer cells immediately, and the tumor cell death may persist for days or even months after radiotherapy.
Some cells may die relatively rapidly through apoptosis. However, most cells do not manifest evidence of damage until mitosis occurs, and several divisions may ensue before actual cell death which is termed as mitotic cell death. This means that the cell dies when it attempts to divide. So, if a cell does not divide, then it does not die, but just sits there. At the same time, if a cell doesn't divide, it also cannot grow and spread. For tumors that divide slowly, the mass may shrink over a long, extended period after radiation stops. For instance, the median time for a prostate cancer to shrink is about 18 months (some quicker, some slower). For colon cancers, some may grow more quickly, and others may grow more slowly and this will affect how they are killed by radiation.
The cellular doubling time which is typically three to five days for head and neck cancer also influences the rapidity with which a tumor will shrink. For this reason, most tumors do not show immediate shrinkage after starting radiotherapy. While radio responsive tumors start to shrink in a few days, most head and neck cancers may take weeks or longer to shrink. Some low-grade, slowly proliferating tumors histologically appear to be viable for prolonged periods after irradiation. So, it would be difficult to predict the first wave of cell death.
To select the optimal time when the maximum wave of cell death occurs in the tumor, I would suggest you include radiological scanning as this would help you to record the tumor shrinkage, which in turn could assist in the selection of the optimal time. However, shrinkage may not always be seen right away even when cells may still be dying in response to radiation, sometimes causing an inflammatory response that can even make a mass look larger.
The second part of your question.
In such case, plasma sample would be better for the isolation of extracellular vesicles. You may collect blood samples in citrate tubes and centrifuge at 1000 x g for 10 mins followed by 2500 x g for 10 mins. Following centrifugation, it is important to immediately transfer the liquid component (plasma) into a clean microcentrifuge tube using a pipette. The samples should be maintained at 2-8°C while handling. If the plasma is not analyzed immediately, store the plasma samples at –80°C in aliquots. Avoid multiple freeze-thaw cycles.
You may want to refer to the article attached below. It may be helpful.
https://www.nature.com/articles/s41416-021-01567-8
Regards,
Malcolm Nobre
Dear Dr. Nobre.
Thank you very much for your comprehensive reply and the lovely article you have enclosed. The information you have provided is very important to us.
Best,
Jan
Different cell types have different rates of division and repair capabilities. Cancer cells, which often divide rapidly, may be more susceptible to the effects of radiation. The total dose and the fractionation of the dose can influence the timing of cell death. Higher doses can lead to more immediate cell death, while lower doses might take longer to have an effect. Tumor cells that are not receiving adequate oxygen (hypoxic cells) can be more resistant to radiation and may take longer to die. Cells are more sensitive to radiation at certain phases of their cell cycle. Cells in the mitotic phase are typically more susceptible to radiation-induced death.
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