Everyone Is Different

Protein Analytics

Some cancer patients are given the same diagnosis. Despite this, the pathomechanism in their cells can be different. High-performance analytics from Bayer Technology Services help in identifying these differences. The final goal is to personalize patients’ treatment.

Each person is unique – we are quick to recognize that. But it is harder to recognize the differences in cell molecular activity in an individual. Yet, once this is achieved, it might be possible to customize therapy.

The statistics are merciless. Colon cancer claims over 600,000 lives worldwide – year in and year out. It is the fourth most common cause of death among all types of cancerous tumors. Yet many patients would live longer if they could be diagnosed early enough and treated with existing, readily available drugs. The problem is, however, that not every drug has the same effect on every patient. For this reason, physicians would love to be able to test a drug’s likely effectiveness for a particular patient in advance.

And this is a task for OncoTrack. OncoTrack is a project financed by the EU-wide Innovative Medicines Initiative, headed by Dr. David Henderson of Bayer HealthCare and Prof. Hans Lehrach at the Max Planck Institute of Molecular Genetics. Collaborating on this project are close to eighty scientists from both industrial and academic research. One of the ways they are developing a test is closely examining tumor cells from some thirty patients with colon cancer.

This involves employing a very special technique supplied by Bayer Technology Services. The researchers call it RPPA, which stands for reverse-phase protein array. Bayer Technology Services has developed its own RPPA platform based on know-how gained several years ago through its acquisition of a Swiss company, Zeptosens.

Each tumor can appear differently on the cellular level. Tissue samples (above) provide information. A detection procedure from Bayer Technology Services makes certain protein patterns visible and shows which signal paths are impaired (below).
Each tumor can appear differently on the cellular level. Tissue samples (above) provide information. A detection procedure from Bayer Technology Services makes certain protein patterns visible and shows which signal paths are impaired (below).

What makes RPPA so interesting is that it can identify a whole series of different proteins from a single sample and, at the same time, even register variations in the concentrations of a single protein compared to control samples. This technology has a myriad of applications in a number of fields, such that Bayer CropScience also makes use of it in joint projects. Dr. Mathias Gehrmann is well aware how concerted evidence of several proteins can be of great significance, particularly in cancer patients. He works at Bayer Technology Services in the field of Enabling Technologies in the Bioanalytics group, which is also responsible for the RPPA platform. “The composition of the proteins in the cells provides information about whether a signal path is active or not,” he explains. When scientists refer to a signal path they mean a type of molecular cascade that ultimately transmits specific commands. The messages are transferred by chemical means from one molecule to the next, which are, as a rule, proteins, until the messages eventually reach their destination, for example, the cell nucleus. When such signal paths are out of control, it can lead to uncontrolled cell division, even though this is not physiologically necessary, or to a breakdown in apoptosis, also known as programmed cell death. Both of these malfunctions are typical of many tumor cells. 

The special problem among tumor patients is that even when people are suffering from the same type of cancer, it does not necessarily mean that the same signal paths are disrupted. Or, as the biochemist, Dr. Henderson expresses it, “Two people can have colon cancer that is completely different on the cellular level.” This is also one of the reasons why some medications are effective with one patient and not with another. Many drugs have been developed to have a regulatory effect on specific signal paths – but have no effect on others.

This is where RPPA comes into play. The particular signal paths that are dysfunctional in a specific tumor are also expressed in the degree to which a certain protein has been modified. RPPA technology can determine the extent of this modification – and, as Dr. Gehrmann puts it, do it “for a lot of proteins in a single go.” As he sees it, this method is even “a whole lot faster and more sensitive” than other comparable methods.

Scattered blue light makes two lines visible (left). The arrays are then prepared along these lines such that a laser beam is directly channeled into the planar waveguide. This is also where the samplesare to be found, which have been added to the array using a special device (right).
Scattered blue light makes two lines visible (left). The arrays are then prepared along these lines such that a laser beam is directly channeled into the planar waveguide. This is also w

The researchers use antibodies to identify a protein in a sample, since an antibody can be found for almost every protein that it bonds well with. This means that a whole series of antibodies can be used in protein analytics, depending on how many proteins are to be identified in a sample. If these proteins are actually present, the corresponding antibody gets caught, as if on a fishing hook.

If the antibody molecule is previously coupled with a fluorescent dye, the protein can even be made visible. And what is even more important for these researchers is that it makes the relative concentration of the protein much more easily measurable when compared to (healthy) cells, because the stronger the light signal, the greater the amount of a specific protein in the sample.

By itself, that is nothing new. Actually, this is normal practice in protein analytics. Yet, RPPA technology is substantially different from other methods. In most of the procedures, the antibodies are first placed on a carrier plate, a so-called array, and then the sample is added. With the RPPA approach, it is exactly the opposite, first the sample and then the antibody – hence the term “reverse phase.”

The advantage is that “In this way, a large number of different samples can be examined at the same time on a single array,” explains bioimaging expert Daniel Rechsteiner, who played a decisive role at Bayer Technology Services in the further development of the RPPA method. The biochemist further emphasizes that this approach makes the analysis substantially more flexible because, once prepared, an array can handle any antibody. 

“Compared to other methods, our approach is a whole lot faster – and also more sensitive.”

Dr. Mathias Gehrmann

Bioanalytics, Bayer Technology Services

All this happens completely automatically and with a high sample throughput. The hardware needed is also supplied by Bayer Technology Services, with some of this highly specialized equipment having been developed inhouse. Among these is the optical procedure that makes proteins visible and measurable after bonding with antibodies. The RPPA approach by Bayer Technology Services also scores with a further advantage that is thanks to, as Rechsteiner calls it, a “twist of physics.” This is his way of describing what is known in scientific circles as an integrated planar waveguide. This is what causes the laser beam, which excites the dye on the captured antibodies, to propogate through only one layer of the array, the one that conveniently happens to be the layer with the sample molecules and antibodies (see box). “In this way, random signals can be circumvented and the sensitivity of the whole procedure is substantially increased,” emphasizes Rechsteiner. 

Shine a light

Light can not only be “contained” in round glass fibers (as used in modern broadband data transmission), but also in flat layers. And it is these planar waveguides (PWG) that Bayer Technology Services is using for protein analysis. Sample arrays are coated with a layer of tantalum pentoxide just 0.2 micrometers thick, through which a laser beam is directed diagonally (right). This leads to an electromagnetic field close by – causing all the dye molecules on the array to shine. And this is namely wherever antibodies have bonded to the proteins in the sample. The intensity of the fluorescence in each case depends on the amount of dye – and can be measured using a CCD camera. Other methods where the laser beam scans the array from above have two disadvantages compared to planar waveguides: to begin with, they are considerably less sensitive due to light scattering, and part of the laser beam is reflected by the array, concealing the signals sent by the luminescent dye.

Sensitivity in this case primarily means that decidedly small amounts of samples are enough to identify the proteins. In concrete terms, a mere 0.4 billionth of a liter of a sample is spotted on the array. That is such a minuscule amount that about 100,000 of these samples would fit into a single drop of water. Other methods require considerably more sample material.

But no matter how small the amount of sample may be, only those proteins can be identified for which the researchers have a matching antibody available. And they do not grow on trees but are in fact the result of further expert research. And this is yet another major advantage of the RPPA platform: the experts at Bayer Technology Services have an outstandingly large pool of antibodies for various applications. “For cancer alone, we have over 300 tests for specific tumor-related proteins,” Rechsteiner points out.

“This expertise will help us tobreak down the individual differencesin the formation of cancer.”

Dr. Christian Regenbrecht

Project head, OncoTrack, Charité University Medicine Berlin

“This expertise will help us to break down the individual differences in the formation of cancer,” says Dr. Christian Regenbrecht, molecular biologist at the pathological institute of the Berlin Charité hospital and head of an OncoTrack subproject. At the moment, patients are being sought whose tumor tissue can be examined. “We will use highly validated antibodies to identify, for example, specific protein modifications produced by dysregulated enzymes,” explains Dr. Gehrmann. This, in turn, will allow conclusions to be drawn about the individual, impaired signal paths. “Ideally, this will reveal which specific mechanism leads to cancer in which patient,” he says.

And that would be a vital clue in answering the question as to which medication can really help in which case. Thousands of lives may depend on this knowledge.

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  1. RegenbrechtSehr vielversprechend und Zukunftsträchtig.
    Allen Beteiligten weiterhin viel Erfolg!

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