Team:ETH Zurich/Design

"What I cannot create I do not understand."
- Richard Feynmann

Design

System Overview

Figure 1. CTC are a rising topic in the literature. The number of publications including the term CTC is rising continuously.

The detection of circulating tumor cells for diagnosis of metastasis has become a hot topic during the last decade. Several methods were already developed and are starting to be used regularly in practice. One big disadvantage of such systems, for example Cell Search, is that they are based on the detection of one individual surface marker of cancer cells, which can be lost during epithelial-mesenchymal transition in metastasis [Autebert 2015]. Other existing systems base their CTC detection on cancer type specific markers, reducing their applicability to one type at a time [Msaouel 2014, Ilie 2014]. The system we developed deals with these constraints by detection of two general cancer markers that are specific to a broad range of cancer types [Hanahan 2011, Johnstone 2008].

Figure 2. Two general cancer markers (apoptosis induced by sTRAIL and enhanced lactate producion) are integrated into an AND gate with GFP as an output.

The two cancer specific signals on which our MicroBeacon CTC detection sysetm is based 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 the actual test environment. For detection of the rarely occurring 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 envision the setup of our test to be a water-in-oil emulsion in a microfluidic chip, where potential cancer cells and MicroBeacon E. coli are combined in water droplets. Once single cells are separated in the chip, increased lactate production by cancer cells triggers production of the quorum sensing molecule AHL (N-Acyl homoserine lactone) in the accompanying bacteria within the same droplet. Due to the treatment with sTRAIL, the cancer cell is apoptotic and therefore displays phosphatidylserine, the general 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 GFP output in turn indicates positive testing for cancer cells. This setup makes it necessary for a mammalian cell to display two independent markers that characterize it as carcinogenic, making our testing system highly specific, yet broadly applicable.

Figure 3.General scheme of the MicroBeacon system: Upon incubation with sTRAIL, apoptosis is induced in cancer cells whereas healthy cells stay unharmed. In a second module, the TRAIL-treated samples are combined with MicroBeacon bacteria in a microfluidic chip. Upon binding to apoptotic cancer cells via Annexin V that is expressed on the MicroBeacon surface, our bacteria will integrate signals derived from our two cancer markers, leading to a fluorescent signal. This fluorescent signal indicates the presence of a cancer cell in the sample. The detection of an elevated lactate production rate of a cancer cell and the detection of colocalization with other MicroBeacons on an apoptotic cancer cell are combined in an AND gate which provides a stable binary signal. (1) Normal and cancer cells are both incubated with sTRAIL. (2) Apoptosis is induced only in cancer cells by sTRAIL and causes phosphatidylserine to flip to the outer membrane. (3) Mammalian cells and E. coli are distributed in a nano-well plate resulting in single-cell analysis. (4) E. coli with membrane-expressed annexin V bind to exposed phosphatidylserine only on apoptotic cancer cells. (5) Higher lactate production in the apoptotic cancer cells initiates quorum sensing between the E. coli cells.(6) A green fluorescent protein is produced by those E. coli cells, which detect both lactate and quorum sensing signals.

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 wishes when it comes to a novel method of CTC detection. It soon became clear that a major preference was 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. Another comment reassuring us in the choice of establishing a generally 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 design 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 leakiness for our AND gate, choosing the best possible promoter from our designed collection of promoters according to the modeling data and experimental characterization.

We also informed the general public about synthetic biology and more specifically about our project. We were pleased to see that most people would welcome this innovation into their lives.

Figure 4. Survey with EPFL: As an application of synthetic biology, would you support the use of modified bacteria for medical diagnosis? (in % of 70 persons asked)

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 brief description of the different parts of our system. For more details on the BioBricks we used, please see our Parts Overview.

Figure 5.Genetic circuit for implementation of MicroBeacon-based CTC detection. The elevated lactate production rate of cancer cells is detected via a synthetic promoter responsive to LldR and lactate. For signal amplification, we include a fold change sensor topology into this module. The output of the lactate module is LuxR, the transcription factor necessary for quorum sensing. In the presence of LuxR, leakiness of plux leads to production of AHL by LuxI. AHL molecules diffuse freely through membranes and once they reach a neighboring cell they will fully activate LuxR. this in turn further amplifies AHL production and, in parallel, GFP-production is induced. An AND gate between the lactate module and the quorum sensing module is formed by allowing the second module to be active only in the presence of a positive output of the first one.

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 will enevtually reach the same levels, just within different time frames. 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. We designed this topology using natural E. coli repressory mechanisms.

Detection of apoptosis by quorum sensing

Detection of apoptosis by E. coli in our case relies on MicroBeacon bindig to the respective cells. But getting a fluorescent signal only upon binding is not a trivial task since there are no well established two-component signalling cascades available for E. coli. This is why we conceptualized a system based on apoptosis detection by quorum sensing upon colocalization of MicroBeacon bacteria on apoptotic cells. We therefore implemented the LuxR-system in our bacteria. In previous iGEM projects it was shown that leakiness of this system can pose a rather big problem. We tried to reduce leakiness 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. Our model showed that a sequential setup, filtering first for elevated lactate production and only allowing quorum sensing if the first signal is present, would be the best solution for our system. In this fashion we were able to further reduce leakiness, which is caused mainly by LuxR via an unknown mechanism. In our system, LuxR is only present if the first signal is detected, therefore preventing non-specific firing of GFP in any other situation.

We would like to thank our sponsors