Difference between revisions of "Team:ETH Zurich/Project Description"

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<p>Overall, the integration of two general markers allows for the detection of various types of cancers with high reliability and a low false positive rate, while control of signal expression by the bacteria should result in a considerable increase in the signal to noise ratio compared to assay kit-based tests. The direct binding of our MicroBeacons to cancer cells allow for the detection of lactate and AHL in a very controlled microenvironment with minimal crosstalk from other cells in the sample or unbound MicroBeacons. Finally, the use of a fluorescent signal as our output signal will allow for easy measurement from the microfluidic chip through microscopy.</p>
 
<p>Overall, the integration of two general markers allows for the detection of various types of cancers with high reliability and a low false positive rate, while control of signal expression by the bacteria should result in a considerable increase in the signal to noise ratio compared to assay kit-based tests. The direct binding of our MicroBeacons to cancer cells allow for the detection of lactate and AHL in a very controlled microenvironment with minimal crosstalk from other cells in the sample or unbound MicroBeacons. Finally, the use of a fluorescent signal as our output signal will allow for easy measurement from the microfluidic chip through microscopy.</p>
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<h3> References </h3>
 
<h3> References </h3>

Revision as of 11:28, 14 July 2015

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

The light at the end of the tunnel...

With cancer diagnosis being one of modern medicine’s major and challenging problems1, designing a novel method to test for cancer cells quickly caught our attention as a potential project idea. After researching current testing methods and brainstorming how a test could be implemented in a genetically-modified organism, we came up with a system for the assessment of metastasis risk in cancer patients and high-risk groups using a genetically-engineered strain of Escherichia coli which we call MicroBeacon E. coli.

Circulating tumour cells (CTCs) can be used as an indicator of the initial stages of metastasis2. In our project we aim to develop a novel method that is fast, economical, and reliable for the identification of CTCs by using MicroBeacons to detect two general cancer markers and indicate this through fluorescence. By design, this makes our system applicable to many types of cancer. To facilitate handling the cells, our detection system will be embedded in a microfluidic chip built especially for this purpose. In contrast to existing methods which rely on detection of very specific cancer markers3,4,5, a single general test would be more economical if it can be mass produced and deployed in a cost-effective manner.

The first marker we are planning to detect is the increased production rate of lactate by cancer cells due to the Warburg effect, a metabolic shift that occurs in tumour cells that is characterised by a much higher glycolysis rate and subsequent lactic acid fermentation6,7. An important part of our method to detect elevated lactate concentration around CTCs is the constitutive expression of LldR, a repressor of the lldPRD operon in E. coli. Binding of lactate to LldR causes a conformational change inhibiting its ability to bind to its target operators and activating the operon8. Due to the accumulation of lactate over time in the environment, we designed our sensor to detect fold-changes instead of a concentration threshold using a gene expression system with an incoherent feedforward loop topology9. Overall, a newly designed lldR-lacO fusion promoter acts as a filter which will only allow the production of quorum sensing molecules (AHL) if an elevated lactate production rate is detected. This is where the second cancer marker comes into play.

For the second signal, apoptosis is induced only in cancer cells by incubating the sample in a solution containing soluble TNF-related apoptosis-inducing ligand (sTRAIL) prior to the introduction of MicroBeacons to the environment10. The initiation of apoptosis can then be detected by the strong binding of Annexin V to phosphatidylserine (PS) that appears on the outer membrane of the target CTCs11. Although it is normally an intracellular protein12, we plan to engineer it to be expressed on the surface of our MicroBeacons, allowing them to bind to the target CTCs. Co-localisation of several MicroBeacons on the surface of an apoptotic CTC will be detected via quorum sensing signals which are only initiated upon detection of elevated lactate levels. The final output of the system is the expression of reporter green fluorescent protein (GFP) coupled to this quorum sensing system, which should only be significantly expressed near a target if it is a cancer cell.

Overall, the integration of two general markers allows for the detection of various types of cancers with high reliability and a low false positive rate, while control of signal expression by the bacteria should result in a considerable increase in the signal to noise ratio compared to assay kit-based tests. The direct binding of our MicroBeacons to cancer cells allow for the detection of lactate and AHL in a very controlled microenvironment with minimal crosstalk from other cells in the sample or unbound MicroBeacons. Finally, the use of a fluorescent signal as our output signal will allow for easy measurement from the microfluidic chip through microscopy.

image/svg+xml sTRAIL cancercell Normalcell MicroBeacon coli 1. Apoptosis is induced only in cancer cellsby sTRAIL and causes phosphotadylserine (PS)to flip to the outer membrane. 2. Mammalian cells and MicroBeacons are sentthrough the microfluidic chip. 3. MicroBeacons with membrane-expressed annexin V bind to exposedPS on apoptotic cancer cells and not tonormal cells. 4. Higher lactate production rates in theapoptotic cancer cell initiates quorum sensing between the MicroBeacons to amplify the signal. 5. A green fluorescent signal isproduced by MicroBeacons that havedetected the quorum sensing signals. lactate AHL

References

  1. International Agency for Research on Cancer. (2014). World cancer report 2014. Geneva: WHO.
  2. Sleijfer, S., Gratama, J. W., Sieuwerts, A. M., Kraan, J., Martens, J. W., & Foekens, J. A. (2007). Circulating tumour cell detection on its way to routine diagnostic implementation?. European Journal of Cancer, 43(18), 2645-2650.
  3. Cooke, T., Reeves, J., Lanigan, A., & Stanton, P. (2001). HER2 as a prognostic and predictive marker for breast cancer. Annals of oncology, 12(suppl 1), S23-S28.
  4. Garcea, G., Neal, C. P., Pattenden, C. J., Steward, W. P., & Berry, D. P. (2005). Molecular prognostic markers in pancreatic cancer: a systematic review. European Journal of Cancer, 41(15), 2213-2236.
  5. Singhal, S., Vachani, A., Antin-Ozerkis, D., Kaiser, L. R., & Albelda, S. M. (2005). Prognostic implications of cell cycle, apoptosis, and angiogenesis biomarkers in non–small cell lung cancer: a review. Clinical Cancer Research,11(11), 3974-3986.
  6. Chen, Z., Lu, W., Garcia-Prieto, C., & Huang, P. (2007). The Warburg effect and its cancer therapeutic implications. Journal of bioenergetics and biomembranes, 39(3), 267-274.
  7. Vander Heiden, M. G., Cantley, L. C., & Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 324(5930), 1029-1033.
  8. Aguilera, L., Campos, E., Giménez, R., Badía, J., Aguilar, J., & Baldoma, L. (2008). Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. Journal of bacteriology, 190(8), 2997-3005.
  9. Goentoro, L., Shoval, O., Kirschner, M. W., & Alon, U. (2009). The incoherent feedforward loop can provide fold-change detection in gene regulation. Molecular cell, 36(5), 894-899.
  10. Srivastava, R. K. (2001). TRAIL/Apo-2L: mechanisms and clinical applications in cancer. Neoplasia, 3(6), 535-546.
  11. van Engeland, M., Nieland, L. J., Ramaekers, F. C., Schutte, B., & Reutelingsperger, C. P. (1998). Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry,31(1), 1-9.
  12. Hawkins, T. E., Das, D., Young, B., & Moss, S. E. (2002). DT40 cells lacking the Ca2+-binding protein annexin 5 are resistant to Ca2+-dependent apoptosis. Proceedings of the National Academy of Sciences, 99(12), 8054-8059.