Barcode of tumor proteins

How to make a protein portrait of a cancer cell

Kirill Stasevich, CompulentaKnowing what mutations are in the cell is important, but not enough: after all, these mutations must pass into proteins.

And protein synthesis itself obeys a lot of rules and regulatory signals, and, say, some mutation can simply be realized because it will be blocked at the level of polypeptide synthesis. In addition, the cell responds to some important and urgent signals primarily at the level of translation, that is, at the level of protein synthesis on mRNA. Therefore, if you want to imagine the molecular "physiognomy" of a cell as fully as possible, you need to know not only its DNA, but also RNA and a set of proteins.

This is especially important in the case of cancer cells, which can change their protein spectrum at different stages of the development of the disease and during treatment. Here, the more we know, the better, but there is one problem with malignant cells: on the one hand, you need as much information about them as possible, on the other – you need to be able to understand this sea of information. If we talk about the mutational portrait of different tumors, then only recently scientists have managed to find a method to determine which mutation belongs to cancer (and which one), and which one does not.

In relation to cancer proteins, scientists can greatly benefit from the method described in Science Translational Medicine by Ralph Weissleder and his colleagues from the Massachusetts General Hospital (Ullal et al., Cancer Cell Profiling by Barcoding Allows Multiplexed Protein Analysis in Fine-Needle Aspirates).

Its essence is as follows: cells are passed through a microcapillary device containing antibodies against the type of cells that need to be caught, then the caught cells are bathed in a cocktail of several dozen antibodies to which pieces of DNA 70 nucleotides long are attached. This DNA is taken from the tomato genome – and chosen so as not to overlap with any human sequence in any way. In addition, there is a site in these tomato DNA that is cleaved by ultraviolet light and enzymes.

Cells bathed in a mixture of antibodies (there were 90 of them in the experiment) are washed from the immunoglobulins that did not bind to them, after which they are treated with splitting reagents and ultraviolet light. The resulting DNA stubs that were left with antibodies (hanging on the actual cellular proteins) can now contact other DNA sites that will be added to them now. These new pieces of DNA are linked to several fluorescent proteins.

As a result, a complex multi–level "sandwich" is obtained: first, the antibodies that caught and fixed the cancer cells, then these cells themselves, on top of them - a bunch of different antibodies that should clarify, "draw" the protein "facial features" of cancer cells, and already fluorescent proteins are superimposed on these antibodies through a DNA bridge. The intensity of their glow can be determined quite accurately, and by this parameter it is easy to understand what kind of proteins and in what quantity a cancer cell has.

Traditional methods that can be used to determine the protein composition of a cell require a lot of resources and time. For them, you either need to collect a lot of fabric material, or somehow amplify (amplify) the signal - both can distort the result. In the new method, neither one nor the other is needed. All reaction components can be created by biotech companies, the process itself takes several hours, which again cannot be compared with conventional methods of immunohistochemistry, which can take several days.

But the authors call the most important advantage of the new technology that it can be used to identify many proteins at once (recall: 90 antibodies were used in the experiment, and this is not the limit). When a tumor begins to be treated, many changes occur in its cells, including those aimed at overcoming therapy, but conventional methods are able to catch only about ten protein markers, and therefore many details of the "personal life" of tumor cells will be hidden from doctors. It is clear that if there are not 10, but 100 such markers, then the picture will turn out to be more detailed: with the help of specific antibodies, you can quite accurately see the protein profile of a cell – something like its own barcode.

Cancer cells and their protein "barcode" obtained with the help of several dozen antibodies
(photo by Ralph Weissleder / Massachusetts General Hospital).

Finally, in this way, it is possible to determine not only proteins, but also DNA and RNA, and in the same cells, without separating one from the other. In general, there are solid advantages compared to the usual procedure, when a tumor biopsy is taken and long manipulations with the extracted material begin.

In addition, we are not obliged to limit ourselves to tumor cells alone: this method, which the authors called ABCD-platform (antibody barcoding with photocleavable DNA), is useful for the study of any cellular material, so its prospects are not limited to oncomedical use alone. We can say that a complex methodological problem has been successfully solved here, which has long been trying to solve in a variety of biotechnological fields and which has recently become especially relevant – how to get the maximum of reliable (and visual) information from a minimum of material.

However, for the time being, it is necessary to talk about the prospects of the new method with caution: not too many samples of cancer cells were used in experiments, and, perhaps, in a larger study with a large number of patients, this technology will have some weaknesses that will add to the work of its authors.

Prepared based on the materials of the General Clinical Hospital of Massachusetts:
'Barcode' profiling enables analysis of hundreds of tumor marker proteins at once.

Portal "Eternal youth" http://vechnayamolodost.ru17.01.2014