Team:CLSB-UK

Project BATMAN

a new way to detect cancer using toehold switches

Late presentation and non-specific symptoms are the main
reasons 1.6 million people worldwide die from lung cancer every year

Dr Sujal Desai, Consultant Chest Radiologist

We have developed a new way to detect cancer at an early stage by measuring micro-RNAs (miRNAs), biomarkers found in blood. We use toehold switches to regulate expression of GFP in response to specific miRNAs. This method could be applied to a myriad of diseases, but we have chosen to use non-small cell lung cancer (NSCLC) as a proof of concept. We hope our work in NSCLC detection demonstrates the potential that toehold switches have to offer as a cheap and effective diagnostic tool.

Late presentation of symptoms is the main reason
why 40,000 people are dying every year from lung cancer

Dr Sujal Desai, Respiratory Consultant

We developed a new way to detect cancer at an early stage by measuring micro-RNAs (miRNA), biomarkers found in blood. We used toehold switches to regulate expression of GFP in response to specific miRNAs. This method could be applied to a myriad of diseases, but we have chosen to use non-small cell lung cancer as a proof of concept.

Biomarkers in the blood

Abnormal levels of miRNAs mir-15b-5p and mir-27b-3p in blood serum are indicative of NSCLC[1]. We have designed two sequence-specific sensors that utilise synthetic riboregulators called toehold switches. These toehold switches detect mir-15b-5p and mir-27b-3p and produce fluorescent reporter proteins in their presence. We designed our sensors to work in a cell-free system, allowing them to be used safely and in a low-tech environment.

Non-small cell lung cancer

Lung cancer is the most common cause of cancer-related mortality, with 1.6million deaths in 2012. That’s 20% of all reported deaths due to cancer. Non-small cell lung cancer (NSCLC) makes up ~80% of all incidences of lung cancer.[2] 58% of all cases in 2012 were reported in less developed countries.[3]

NSCLC is characteristically aggressive and pathologically diverse.[4] Common subtypes include pulmonary adenocarcinoma (~50%) and squamous cell carcinoma (~40%). The classification of the original tumour will impact prognosis and treatment. Treatment still centres around cytotoxic chemotherapy, although new treatments show promise including immunotherapies.[5][2]

NSCLC’s high mortality rate is, in large part, down to the late stage at which the disease is normally diagnosed.[6] This often renders surgery, which can curative in early stages, pointless as the tumour has metastasised.[7][8]

About 90% of lung cancers are caused by smoking and as smoking rates have declined, there has been a corresponding reduction in incidence of lung cancers.[9] However, nearly 30% of the global population are still estimated to smoke.[10]

Late diagnosis kills

Non-small cell lung cancer 5 year survival rates by stage

Stage IA IB IIA IIB IIIA IIIB IV
Survival 49% 45% 30% 31% 14% 5% 1%

Non-small cell lung cancer (NSCLC) has low 5-year survival rates due to late presentation of symptoms, quick tumour progression and high probability of metastasis.[11] Late diagnosis renders surgery, which can curative in early stages, pointless as the tumour has metastasised.[12][13]

Micro-RNA (miRNA)

Micro-RNAs (miRNAs) can act as potent biomarkers for a myriad of diseases.

MiRNAs are short non-coding RNAs of 19-24 nucleotides in length. They are involved in regulating the post-transcriptional silencing of protein-coding genes in eukaryotes[14][15][16]. MiRNAs can be found extracellularly in various body fluids, including serum[17][1], plasma[18], saliva[19], urine[20] and breast milk[21]. It has been hypothesised that cells actively secrete miRNAs via two pathways: in microvesicles and bound to RNA binding proteins.[22]

RNA binding proteins and microvesicles shield circulating miRNAs from ribonuclease degradation in body fluids.[22] Furthermore, they’re capable of delivering the miRNAs to recipient cells.[23][24][21] These miRNAs can then trigger downstream signalling events, indicating that circulating miRNAs play a role in cell-to-cell communication.[24][21][25]

Recent studies have also shown that differential levels of miRNAs in body fluids are indicative of specific diseases, including many forms of cancer[1][18][20][26]. MiRNA stability, accessibility by non-invasive methods (liquid biopsy) and their ability to diagnose diseases in early stages provides strong evidence that circulating miRNAs are potent biomarkers[27]. Microarrays and qPCR are typically used to quantify circulating miRNAs. However, primer design for qPCR is difficult and microarrays are complicated and expensive to prepare.


References

  1. ↑ 1.0 1.1 1.2 Hennessey, P. T., Sanford, T., Choudhary, A., Mydlarz, W. W., Brown, D., Adai, A. T., & Califano, J. A. (2012). Serum microRNA biomarkers for detection of non-small cell lung cancer. PloS one, 7(2), e32307.
  2. ↑ 2.0 2.1 Chan, B. A., & Hughes, B. G. (2015). Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Translational lung cancer research, 4(1), 36.
  3. ↑ Ferlay, J., Soerjomataram, I., & Ervik, M. (2012). GLOBOCAN, cancer incidence and mortality worldwide: IARC cancer base no. 11 [Internet]. Lyon, France: International Agency for Research on Cancer; 2013.
  4. ↑ Board, P. A. T. E. (2017). Non-Small Cell Lung Cancer Treatment (PDQ®).
  5. ↑ Chen, Z., Fillmore, C. M., Hammerman, P. S., Kim, C. F., & Wong, K. K. (2014). Non-small-cell lung cancers: a heterogeneous set of diseases. Nature reviews. Cancer, 14(8), 535.
  6. ↑ Dajac, J., Kamdar, J., Moats, A., & Nguyen, B. (2016). To Screen or not to Screen: Low Dose Computed Tomography in Comparison to Chest Radiography or Usual Care in Reducing Morbidity and Mortality from Lung Cancer. Cureus, 8(4).
  7. ↑ Uramoto, H., & Tanaka, F. (2014). Recurrence after surgery in patients with NSCLC. Translational lung cancer research, 3(4), 242.
  8. ↑ Molina, J. R., Yang, P., Cassivi, S. D., Schild, S. E., & Adjei, A. A. (2008, May). Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. In Mayo Clinic Proceedings(Vol. 83, No. 5, pp. 584-594). Elsevier.
  9. ↑ (n.d.). Lung cancer: diagnosis and management - NICE. Retrieved October 7, 2017, from https://www.nice.org.uk/guidance/cg121/chapter/introduction
  10. ↑ Gopal, M., Abdullah, S. E., Grady, J. J., & Goodwin, J. S. (2010). Screening for lung cancer with low-dose computed tomography: a systematic review and meta-analysis of the baseline findings of randomized controlled trials. Journal of thoracic oncology, 5(8), 1233-1239.
  11. ↑ Jemal, A., Bray, F., Center, M. M., Ferlay, J., Ward, E., & Forman, D. (2011). Global cancer statistics. CA: a cancer journal for clinicians, 61(2), 69-90.
  12. ↑ Uramoto, H., & Tanaka, F. (2014). Recurrence after surgery in patients with NSCLC. Translational lung cancer research, 3(4), 242.
  13. ↑ Molina, J. R., Yang, P., Cassivi, S. D., Schild, S. E., & Adjei, A. A. (2008, May). Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. In Mayo Clinic Proceedings(Vol. 83, No. 5, pp. 584-594). Elsevier.
  14. ↑ Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. cell, 116(2), 281-297.
  15. ↑ Ambros, V. (2004). The functions of animal microRNAs Nature 431: 350–355. Proceedings of the National Academy of Sciences of the United States of America, 103, 3687-3692.
  16. ↑ Kim, V. N., Han, J., & Siomi, M. C. (2009). Biogenesis of small RNAs in animals. Nature reviews Molecular cell biology, 10(2), 126-139.
  17. ↑ Zen, K., & Zhang, C. Y. (2012). Circulating microRNAs: a novel class of biomarkers to diagnose and monitor human cancers. Medicinal research reviews, 32(2), 326-348.
  18. ↑ 18.0 18.1 Mitchell, P. S., Parkin, R. K., Kroh, E. M., Fritz, B. R., Wyman, S. K., Pogosova-Agadjanyan, E. L., ... & Lin, D. W. (2008). Circulating microRNAs as stable blood-based markers for cancer detection. Proceedings of the National Academy of Sciences, 105(30), 10513-10518.
  19. ↑ Park, N. J., Zhou, H., Elashoff, D., Henson, B. S., Kastratovic, D. A., Abemayor, E., & Wong, D. T. (2009). Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection. Clinical Cancer Research, 15(17), 5473-5477.
  20. ↑ 20.0 20.1 Hanke, M., Hoefig, K., Merz, H., Feller, A. C., Kausch, I., Jocham, D., ... & Sczakiel, G. (2010, December). A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. In Urologic Oncology: Seminars and Original Investigations (Vol. 28, No. 6, pp. 655-661). Elsevier.
  21. ↑ 21.0 21.1 21.2 Kosaka, N., Izumi, H., Sekine, K., & Ochiya, T. (2010). microRNA as a new immune-regulatory agent in breast milk. Silence, 1(1), 7.
  22. ↑ 22.0 22.1 Chen, X., Liang, H., Zhang, J., Zen, K., & Zhang, C. Y. (2012). Secreted microRNAs: a new form of intercellular communication. Trends in cell biology, 22(3), 125-132.
  23. ↑ Vickers, K. C., Palmisano, B. T., Shoucri, B. M., Shamburek, R. D., & Remaley, A. T. (2011). MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nature cell biology, 13(4), 423.
  24. ↑ 24.0 24.1 Valadi, H., Ekström, K., Bossios, A., Sjöstrand, M., Lee, J. J., & Lötvall, J. O. (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology, 9(6), 654.
  25. ↑ Turchinovich, A., Samatov, T. R., Tonevitsky, A. G., & Burwinkel, B. (2013). Circulating miRNAs: cell–cell communication function?. Frontiers in genetics, 4.
  26. ↑ Slater, E. P., Strauch, K., Rospleszcz, S., Ramaswamy, A., Esposito, I., Klöppel, G., ... & Bartsch, D. K. (2014). MicroRNA-196a and-196b as potential biomarkers for the early detection of familial pancreatic cancer. Translational oncology, 7(4), 464-471.
  27. ↑ Etheridge, A., Lee, I., Hood, L., Galas, D., & Wang, K. (2011). Extracellular microRNA: a new source of biomarkers. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 717(1), 85-90.