Project description

Background

Lung cancer is the cancer form with the highest death toll, causing 1.69 million deaths globally in 2015 [1]. One reason for the high death toll is the late detection of the cancer, due to symptoms starting to appear first in the later stages. Three different stages are used to characterize lung cancer; I, II and III. The magnitude of the disease is based on size of the tumor and the extent of spreading to lymph nodes and other organs in the body [2]. A majority of the lung cancer cases today are diagnosed in stage III with a 5-year survival rate of 5-14%. An earlier detection at stage I or II would increase the figure to 45-49% which clearly show that early detection is a key to increase the chances of surviving lung cancer [3]. At the appearance of the first symptoms, a chest X-ray is used today to scan for tumors. The scan can indicate the position and size of the tumors. Despite being a non-invasive procedure, it is accompanied by limitations such as cost, time, expertise and sensitivity required for X-ray examinations. Due to these limitations X-ray is not suitable for mass screening of lung cancer [4]. Therefore, there is a demand for new diagnostic tools with mass screening potential. One of the most promising approaches for early diagnosis is the use of volatile organic compounds (VOCs) as biomarkers. Some specific VOCs can be detected in higher concentrations in the breath of lung cancer patients compared to healthy individuals. The cancer cells have an increased oxidative rate that results in oxidative stress products found in the breath of patients. The low concentration of VOCs, pmol/l, has provided a challenge in using this approach for detection and no efficient screening method is yet available [5]. Studies have shown that there is not only one compound that can be used as a biomarker in the breath of cancer patients [5]. An optimal diagnostic tool would therefore be able to detect multiple VOCs in order to decrease the number of false positive diagnoses. One example of a diagnostic tool is a biosensor, which is a biological-based system that can detect the presence of a target analyte and convert it into a signal. A biosensor can be developed with many different species, antibodies or enzymes. In this case, a yeast-based biosensor is a suitable approach and some of the advantages are the ease of genetic manipulation, as well as the cheap and easy cultivation [6]. To create this novel, non-invasive diagnostic tool, the budding yeast Saccharomyces cerevisiae will be used as a biosensor. The yeast cells will have two xenogeneic olfactory receptors incorporated. These can bind the biomarkers that are present in the breath of lung cancer patients, opening up for the possibility to detect the cancer before any symptoms appears.

The basic design

The aim of this project is to detect two VOCs that have been found in elevated levels in the breath of lung cancer patients, using a biosensor made of Saccharomyces cerevisiae. Two of the most commonly found VOCs; butanone and n-octanal, are chosen for detection [7, 8]. The VOCs will be detected by G-protein coupled receptors (GPCRs) with the ability to activate the pheromone pathway in yeast and thereby initiate mating between a- and α-cells. The final output will be a color change due to deletion of the ADE2 gene using gRNA and Cas9. The colony color change will indicate the presence of the target VOCs, and thereby also lung cancer. For an overview, see Figure 1.

Figure 1: Overview of the biosensor. GPCRs will sense the VOCs, activate the pheromone pathway which lead to expression of Cre recombinase as well as mating of cells. Cre recombinase will cause an inversion of loxP sites and give expression of Cas9 and gRNA. In the fused cell, the CRISPR system will delete the ADE2 gene, causing the cells to turn red.

Natively, yeast have GPCRs regulating the cell mating through activation of the pheromone pathway. These GPCRs, STE2 and STE3, are present respectively in the two different mating types of yeast, a and α. STE2 and STE3 bind pheromones from the other mating type and activate the pheromone pathway which enables mating. In our system STE2 and STE3 will be replaced with the GPCRs RatI7 and Olfr1258 which detect the two VOCs butanone and n-octanal respectively [7,8]. When the GPCRs sense the VOCs, the pheromone pathway will be activated which will result in expression of both the native mating genes and cloned Cre recombinase in both cell types. Binding of butanone and n-octanal will therefore lead to mating of cells, but solely if both VOCs are present. In each mating type, a system will be integrated consisting of a promoter in the wrong direction, surrounded by mutated LoxP sites, followed by genes expressing either gRNAs or Cas9, see Figure 2. Presence of the two VOCs will lead to, besides mating, activation of the respective genes since Cre recombinase will turn the promoters in to the right direction. The loxP sites are mutated, preventing the sites from inverting back to their original direction. This ensures that the promoter is inverted into the correct direction upon GPCR activation and remains in that position [9].

Figure 2: An illustrative figure of the system. In two yeast cells of different mating types, the GPCRs RatI7 and Olfr1258 will replace the native GPCRs respectively. When sensing the VOCs, the pheromone pathway will be activated, leading to both mating and production of Cre recombinase. The Cre recombinase will turn the promoters, surrounded by loxP sites, in the right direction, which results in a production of gRNAs and the protein Cas9. When mated, the gRNAs guide Cas9 to two different sites in the ADE2 gene causing a partial disruption, eventually leading to a colony color change.

For expression of the gRNAs, the promoter pSNR52 will be used. Together with polymerase III, the pSNR52 promoter will not translate the gRNAs into proteins [10]. Two different gRNAs will be expressed by the same promoter and the two gRNAs will be linked together with two ribozymes; HDV and Hammerhead. After expression, the ribozymes will cut themselves out and leave the two gRNAs as separate sequences [11]. For the other mating type, the strong constitutive promoter pTEF1 will be used to express the Cas9 protein. Since the gRNAs and the Cas9 protein are expressed in cells of different mating types, the lack of combination will will not disrupt the adenine synthesis in haploid cells. After mating, the gRNAs and Cas9 will both be present in the newly formed diploid cell and assemble into a functional unit. The gRNAs will guide the Cas9 protein to two sites in the ADE2 gene, creating two different double strand breaks in the gene. The double stranded breaks will lead to a partial disruption of the ADE2 gene and an accumulation of a red intermediate during adenine synthesis [12]. The color shift of the cells from white to red will function as a diagnostic response.

Human practices

The focus is on putting the project into a societal context where the potential use of the developed model has been a central subject. Initially, the intention is to contact physicians and researchers specialized in lung cancer in order to receive more information about the disease, diagnostic tools and treatments available today. It is of importance to establish an open dialog where the project idea can be discussed and new ideas formed. The ease of use of the biosensor makes it suitable as a diagnostic tool in a screening program for lung cancer. For developing the screening program, ideas will be discussed with a physician currently works on implementing a lung cancer screening program in the Nordic countries. In order to show reflections from the public for a potential program, a survey has been distributed. The aim is to investigate if both non-smokers and smokers of all ages show interest and what the public would be willing to pay. Another important part of human practices is designing and constructing a prototype. At this point in the process, the thought is to collect the exhaled air in breath collection bags that will be connected to a vessel containing the modified yeast.

Modeling

Mathematical models in MATLAB will be used to estimate the sensitivity of GPCRs, the concentrations of VOCs needed for a signal transduction to occur, as well as the time the VOCs need to be in presence of the yeast. The modeling is focused on the pheromone pathway and the mating process. Mathematical models of the pheromone pathway will be based on an article by Bente Kofahl from 2004. The expression of the mating gene FUS1 can be estimated over time and the concentration dependence of the VOCs can be investigated [13]. The mating process is simulated as a culture of yeast cells containing the two mating types. The cells are illustrated as color coded circles in a 3D grid, moving in a random pattern in a defined space. When two cells of different mating type are in close proximity to each other, mating will occur. The model will incorporate the detection of VOCs by the GPCRs, the activation of the pheromone pathway and the partial deletion of ADE2. As a step in the modelling of GPCR sensitivity and VOCs kinetics, the molecular dynamics of the Olfr1258 receptor is of interest. Since the receptor has not previously been successfully incorporated in yeast, a simulation of the protein structure and the binding of the ligand could provide useful information.

Project progress

Lab work has been started. All the main constructs have been designed and ordered. The GPCRs have been amplified and the work is now proceeding towards transforming yeast cells with the GPCRs and the gene constructs containing Cas9 and gRNAs. The models of the pheromone pathway and the mating process are under development. Interesting physicians and organizations have been contacted for human practices to get more information about today’s diagnostic tools, symptoms, treatments and how a future screening program could be organized. The finished survey will be further spread to reach as broad a crowd as possible. The designing of a potential prototype has started.

Collaborations

We are interested in all sorts of collaborations, just contact us. We have short list of what we are specifically interested in and which specific competences we have, see below. Specific interests Screening program. If there is a team working on screening program for any type of disease, we would like to discuss the topic with them to exchange knowledge and thoughts. Design of device. We would like to do a prototype of our device. Parts of the prototype are potentially suitable for 3D printing. Help with designing these parts in CAD would be much appreciated. Molecular dynamics of Olfr1258. The GPCR Olfr1258 has not been incorporated in yeast before and thus we would gladly receive help with its molecular dynamics. Competences Parameter estimation. We can help with parameter estimation for reaction networks (coupled ODEs). Yeast tests and verification. We have a wide knowledge in working with yeast and can help your team to test systems in yeast.

References

[1]

Cancer [Internet]. World Health Organization; 2017 [Cited 2017-06-13]. Available at: http://www.who.int/mediacentre/factsheets/fs297/en/

[2]

Non-Small Cell Lung Cancer Stages [Internet]. American Cancer Society; 2016 [Updated 2017-03-02; Cited 2017-06-26]. Available at: https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/staging.html

[3]

Non-Small Lung Cancer Survival Rates, by Stage [Internet]. American Cancer Society; 2016 [Updated 2016-05-16; Cited 2017-06-13]. Available at: https://www.cancer.org/cancer/non-small-cell-lung-cancer/detection-diagnosis-staging/survival-rates.htm

[4]

Lungcancer [internet]. Cancerfonden; 2015 [Cited 2017-06-07] Availiable at: https://www.cancerfonden.se/om-cancer/lungcancer

[5]

D’Amico A, Pennazza G, Santonico M, Martinelli E, Roscioni C, Galluccio G, Paolesse R, and Di Natale C. An investigation on electronic nose diagnosis of lung cancer. Lung Cancer, 68(2):170–176, 2010.

[6]

French C, de Mora K, Joshi N, Elfick A, Haseloff J, and Ajioka J. Synthetic biology and the art of biosensor design. The Science and Applications of Synthetic and Systems Biology:Workshop Summary, 2011.

[7]

Minic J, Persuy M-A, Godel E, Aioun J, Connerton I, Salesse R, and Pajot-Augy E. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS Journal, 272(2):524-537, 2005.

[8]

Suzuki Y and Shimono K. Deciphering the receptor repertoire encoding specific odorants by time-lapse single-cell array cytometry. Scientic Reports, 6(19934):1-9, 2015.

[9]

Carter Z and Delneri D. New generation of loxp-mutated deletion cassettes for the genetic manipulation of yeast natural isolates. Yeast, 27(9):765-775, 2010.

[10]

DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, and Church GM. Genome engineering in saccharomyces cerevisiae using crispr-cas systems. Nucleic Acids Research, 41(7):4336-4343, 2013.

[11]

Fujita T and Fujii H. Applications of engineered dna-binding molecules such as tal proteins and the crispr/cas system in biology research. International Journal of Molecular Sciences, 16(10):23143-23164, 2015.

[12]

Ugolini S and Bruschi CV. The red/white colony color assay in the yeast saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Current Genetics, 30(6):485-492, 1996.

[13]

Kofahl, B. and Klipp, E., Modelling the dynamics of the yeast pheromone pathway. Yeast, 21: 831–850, 2004