Design

System Overview

The utilization of detection of circulating tumor cells for diagnosis of metastasis has become a hot topic during the last decade. put excel figure, Several methods were already developped and are starting to be used regularly in practic. One big disadvantage of such systems, like for example Cell Search, is that they are based on the detection of one individual surface marker of cancer cells, wihch can be lost during in metastasis references. Other existing systems base their CTC detection on cancer type specific markers, reducing their applicability to one type at a time maybe find examples. The system we developped Harun help deals with these constraints by detection of two general cancer markers that are specific to a broad range of cancer typesreferences.

Figure 1. Simplified scheme of the system as it will be in the talk.

The two cancer specific signals which our MicroBeacon CTC detection sysetm is based on are the elevated lactate production rate due to the Warburg effect and sensitivity to sTRAIL, an apoptosis trigger specific to cancer cells. In a first step, the samples of potential cancer cells are incubated with sTRAIL for several hours, before introducing them into actual test environnement. For detection of the rarely occuring CTC we rely on single cell analysis to avoid false negative diagnosis caused by failure of detection of one single CTC amongst many healthy cells. To achieve this, we want the setup of our test to be a water-in-oil emulsion in a microfluidic chip. Once singel cells are separated in the chip, increased lactate production by cancer cells triggers production of quorum sensing molecules in bacteria in the vincinity of the respective cancer cell. Due to the previous treatement with sTRAIL, the cancer cell is also apoptotic and therefore displays phosphatidylserine, the surface marker our MicroBeacons can detect and bind. Quorum sensing between the bacteria bound to the same cell allows them to detect colocalization on the surface of the mammalian cell, which in turn leads to the expression of green fluorescent protein (GFP), the output which indicates positive testing for cancer cells. This setup makes it necessary for a mammalian cell to display two independent markers that characterize it as cancerogenic, making our testing system highly specific, yet broadly applicable.

Figure 1.General scheme of the functioning of MicroBeacon system.

Influence of human practices

The most important point considering the development of a novel method improving existing methods, is to find its position in practice and to make sure the added value exceeds the added expenses. This is why we decided to interview medical doctors and find out more about their needs and wished towards a novel method of CTC detection. It soon became clear that the main emphasis lays in a system which could lead to an improved treatment and better prognosis for patients. The interviewed doctors valued devices with high specificity and selectivity, although they also pointed out how an improvement in the detection system should be linked with an improvement in the treatment. It would not be sufficient to simply detect cancer cells if this would not bring us closer to curing the patients. Anoher comment reassuring us in the choice of establishing a genreally applicable test was that up to date there are many screening tests, but not all types of cancers are equally easy to detect.

Considering their answers, we decided to make a general system for detection of CTC integrating two different signals which will provide selectivity. High sensitivity is achieved by tuning our system towards low levels of leakyness for our AND gate, choosing the best possible promoter from our designed collection of promoters according to the modeling data and experimental characterization.

Genetic circuit

The genetic circuit we implemented combines two signals in an AND gate, producing a fluorescent output only if both signals are detected. Below you can find a briefe description of the different parts of our system. For more details on the BioBricks we used, please see our Parts Overview.

Figure 2.Genetic circuit for a co-localization with cancer cells and a lactate detection system.

Lactate sensor: Fold Change Sensor Topology

In order to be able to differentiate between lactate levels produced from cancer cells as opposed to healthy cells, we cannot rely on steady state conditions, as they might never be reached. Therefore, instead of measureing absolute values, we decided to detect differences in the lactate production rate. To this end we set up a lactate sensor displaying a fold change sensor topology. Find out more about how we designed this topology using natural E. coli repressory mechanisms.

Detection of apoptosis by quorum sensing

Detection of apoptosis by E. coli is not a trivial task since there are no well established two-component signalling cascades available for E. coli. This is why we rely on apoptosis detection by quorum sensing upon colocalization of MicroBeacon bacteria on apoptotic cells. We implemented the LuxR-system in our bacteria. In previous iGEM projects, it was shown that leakyness of this system poses a rather big problem. We tried to reduce leakyness by introduction of AHL degradation via constitutive expression of the AHL-Lactonase AiiA.

AND Gate

To combine the two signals in an AND gate, we had to decide what setup would lead to better results: sequential or parallel arrangement. Thanks to our model we could decide on having a sequential setup, filtering first for elevated lactate production and only allowing quorum sensing if the first signal is present. In this fashion we were able to further reduce leakiness, which is caused mainly by LuxR. In our system, LuxR is only present if the first signal is detected, therefore preventing non-specific firing of GFP in any other situation.