Team:SDU-Denmark/Poster

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PROSTATUS poster

Find all the essential information about our project here!
PROSTATUS
- Your FRIEND (Fast, Reliable, Intuitive, Easy and Non-invasive Diagnosis)

Project abstract:
The current procedures to test for prostate cancer (PCa) in Denmark are all either based on invasive tests or based on the presence of the prostate-specific antigen (PSA). The doctors must take a small biopsy of the patient’s prostate or let the patient undergo a digital rectal exam. Our team decided to develop a risk assessment for prostate cancer, that makes use of the CRISPR-Cas13a system. We focused on three mRNA biomarkers found to be present in the urine of PCa patients, specifically TMPRSS2:ERG, AMACR and PCA3. We also conducted interviews with end-users and initiated an email correspondence with experts from different fields to discuss and gain insight on how to improve our project. Apart from this, we designed a set of playing cards and a card game that takes early detection as a theme and was distributed to different social areas to spread PCa awareness.

Our team
SDU-Denmark 2020 team collage

Team members (from top left, row-by-row, to bottom right):

Aicha Robgo, Anne Mette Kristensen, Baldur Vilhjálmsson, Cecilie Andersen, Flora Fuglsang, Frederik Tolberg, Gyula Kimpán, Héléne Skou, Jakob Larsen, Jens Hillers, Katharina Carstens, Kristine de Leon, Maja Andersen, Marco Härringer, Marie-Louise Merser, Martin Soelberg, Nikolaj Juul, Rikke Andersen, Suyasha Koirala and Thomas Hegelund.

Idea

There is room for improvement regarding the current diagnostic procedures for prostate cancer. The current PSA-test only accounts for a single protein found in the blood, with an accuracy rate of only 60% [2]. Increased PSA-levels also correlate with an enlarged prostate, which 70% of males over the age of 70 possess. If the result of this test is positive, a digital rectal exam and a prostate biopsy is performed. This can lead to both incontinence, impotence, and sepsis, leaving patients worse off than if they were not tested in the first place [3]. Instead of focusing on PSA, we thought of testing malignant prostate cancer through a non-invasive measuring of biomarkers in urine.

Our system utilizes the CRISPR/Cas system to detect RNA or DNA biomarkers found in urine. When our CRISPR/Cas system recognizes the biomarkers, it is activated and begins collateral cleavage of single-stranded nucleotides (DNA or RNA). By introducing a nucleotide reporter, the activation of our CRISPR/Cas is detected.

Through our journey, we came up with two tests: the prostate malignancy test (PMT) and the congenital risk assessment test (CRAT). PMT detects RNA biomarkers for already established malignant prostate cancer, while CRAT detects DNA mutations in saliva that increase one's risk for prostate cancer.

SDU-Denmark 2020 thereby presents PROSTATUS, your fast, reliable, intuitive, easy, non-invasive diagnosis for malignant prostate cancer.



[2] Benecchi, L. PSA velocity and PSA slope. Prostate Cancer Prostatic Dis 9, 169–172 (2006). https://doi.org/10.1038/sj.pcan.4500866

[3] Loeb, Stacy et al. “Systematic review of complications of prostate biopsy.” European urology vol. 64,6 (2013): 876-92. doi:10.1016/j.eururo.2013.05.049

Considerations

The basic idea was to improve the status quo: the PSA-test. Being a diagnostics tool for people, all the possible consequences for our product were to be considered. We considered the pros and cons, points for improvement, and risk factors. Among these were:

  • Considerations on biomarkers: which biomarkers would be best to diagnose prostate cancer with? What kinds of cancer do these biomarkers indicate? How do we use these biomarkers in the test? Would we have to amplify them first, and would they always be present in each cancer test? Also, does the biomarker only present as a sign of disease at a high concentration or if present at all?
  • Considerations on Human Practice: what kind of ethical considerations should be made? On what grounds would this test be responsible and good for the world? What could be the negative outcomes of implementing our test? What kind of consequences does a diagnosis have for the individual? How do we make awareness of dispositions for prostate cancer as well as living with the disease?
  • Design Considerations: who are going to use the product? How big is the market? What components are to be manufactured? What safety precautions should be made in order to make the product as user-friendly as possible?

All these factors played a part in developing the final ideas and prospects of our project, and they all, to some degree, interrelate to each other.

For example, how the reagents could be integrated into the prototype was mostly a task for the design subgroup, while how much of each reagent was to be used as a consideration that would have to be done with the lab team.

Thoughts on the text to be written in the manual were a task that required cooperation with the Human Practice team as well. Therefore, the considerations in general required inter-scientific practice.

In addition, we made an expert correspondence about what we should consider when trying to develop a diagnosis. This led to key insights, both into what the major problems are in the field, as well as what the demands of a new prostate cancer test are. Raising awareness is key to any disease-handling, and therefore we also made qualitative interviews with the end-users of our product.

In short, we started to consider and investigate how society would be influenced by our test, as well as testing the PROSTATUS systems in the wet lab in a prove of concept.

Proof of concept

With all the considerations on prostate cancer diagnosis in mind we developed PROSTATUS. PROSTATUS was shown to work by two proof of concept set-ups. The first set-up shows that our CRISPR-Cas system is activated and begins collateral cleavage in the presence of the sgRNA-matching biomarker sequence. Ribosomal RNA (rRNA) was used as a reporter, as it is collaterally cleaved when our Cas proteins are activated. To visualize the cleavage of rRNA, the samples were run on agarose gel electrophoresis. Degraded rRNA is seen as a smear while non-degraded rRNA is seen as two bands.
As shown in Figure 1, the rRNA in lane 5 and 6 is the most degraded. These two lanes are the only ones with both Cas, sgRNA, and biomarker targets. In lane 4, CRISPR-Cas complexes without targets are not activated and do not cleave rRNA to the same extent. This experiment is done with Cas13a and sgRNA and target for the risk-allele rs6983267-G.

Figure 1: Proof that Cas13a is activated by the sgRNA and target sequence for rs6983267-G. All samples contain rRNA as a reporter and then Cas13a, sgRNA and target unless other is specified. Lane 1 is a positive control containing RNase A and Tris-HCl. Lane 2, 3, 4 and 7 are negative controls containing Cas13a only, sgRNA and target (no Cas13a), Cas13a and sgRNA (no target), and only water, respectively. Lane 5 and 6 contain Cas13a, 0,043 ng/µL or 0,0043 ng/µl sgRNA and target. It is observed that Cas13a is only fully activated and cleaves the rRNA when target and sgRNA are also present (Lane 5-6).

Further proof of concept was made by testing our system with flow strips and by testing the possibilities of inhibiting already existing RNases in the urine. This is important for the real test, as RNases would degrade our RNA reporter and give a false positive.
In Figure 2, it is possible to see the difference between a positive and a negative test (strip 1 and 2). It is also seen that RNases are present in untreated urine (strip 3) but that 3-hour treatment with proteinase K inhibits the RNases, thus leading to a negative result (strip 4). Before introducing our CRISPR-Cas complexes to the test, it is important to inhibit proteinase K, otherwise, proteinase K would inactivate the Cas-proteins leading to a false negative. This is possible by adding PMSF and it is seen that when urine (placeholder for CRISPR-Cas) is reintroduced, the test is positive again (strip 6).

Figure 2: Flow strips after treatment with proteinase K and PMSF. All samples contain either nuclease-free water or urine together with RNA-reporters and HybridDetect Assay Buffer. Strip 1 is a positive control containing RNase A and Tris HCl while strip 2 is the negative control containing water. Strip 3-6 show the procedure of inhibiting RNases in urine with strip 3 being the urine, then it is treated with proteinase K for 3-hours (strip 4), then PMSF is added (strip 5) and lastly urine is reintroduced (strip 6) to visualize inhibition of proteinase K. As seen, the RNases in urine are efficiently inhibited by proteinase K, which is again inhibited by PMSF allowing for reintroduced RNases to be active.

Hereby, we demonstrate that our CRISPR-Cas complexes are activated in the presence of a biomarker and thus begin collateral cleavage. Secondly, we confirm that the flow strip system works to detect collateral cleavage and that RNases already present in urine will not be a problem for our final test as we can inhibit them without disturbing the CRISPR-Cas complexes. These results were then integrated into the design of the final test.

Design

We created three hardware components:

  1. The PMT-container
  2. The test strip holder
  3. The CRAT-container

The design and construction of the hardware components was based on problem-driven engineering, there agile design thinking was utilized through iterative workshops, rapid pre-to-typing and end-user communication.

The PMT container consists of three parts: the cup, the lid and a mechanic valve. The separated bottom of the cup is divided into four containers with reagents. The proteinase K should already be in the bottom part – this again ensures safety by avoiding any direct skin contact with the reagents. After tightening the lid, the user can push a button on top to activate the valve and release a small amount of urine into the bottom chambers – approx. 2ml per chamber. After the reaction time the user can put the strips through the pill holes and wait for the result.

The CRAT test is very similar to the PMT in the testing process. The CRAT is, however, a little simpler than the PMT, which is reflected in the design.

Implementation

Our combination of the CRISPR/Cas systems and flow-strip technology has been implemented into a testing cup and lid. These are specifically designed to be safe, intuitive and ergonomic for our intended users outside the laboratory.

This unique malignant prostate cancer diagnostic system can then be implemented into society, following the learnings gathered through the interviews that were conducted. This led us to define two potential directions of approaching the implementation process. These are:

  • The private model, which involves commercial sales directly to individuals.
  • The public model, which distributes our solution through the public healthcare system and medical experts.

The overview of the two implementation pathways is shown in the figure below.

Possible implementation directions for PROSTATUS

The main goal of implementing PROSTATUS into society is to increase the willingness of men to get the health of their prostate examined, thereby increasing the chance of early detection of malignant prostate cancer.

The expected consequence of combining our CRAT and PMT, and the presented screening models, is the socio-economic benefit of avoiding unnecessary ultrasound examinations, rectal examinations, and biopsies, while improving survival rate of men affected by malignant prostate cancer. We make this assumption on the ground of the expert correspondence and qualitative interviews that we made. The tests would have a beneficial effect on both the healthcare system and the patients’ lives not only in Denmark, but anywhere in the world.

Gamification

Our team placed a large focus on how our project can impact the community. This was the main purpose behind our gamification concept. We wanted to create a platform to not only spread awareness about prostate cancer but also empower others to talk openly about the subject. We also believe that behavioural change requires a light and fun approach instead of a raised finger as we have so often seen with health campaigns.

All this was achieved through our Early Detection campaign, where we promoted the detection of significant health issues earlier by changing health-seeking behaviour among men. The campaign consisted of rolls of toilet paper inscribed with the question: “How is your prostate today?” which were distributed around Odense, Denmark along with a brochure about andropause. We also designed a set of playing cards containing facts, quotes, and conversation starters. This was accompanied by instructions to a game that our team designed. The card game takes early detection as a theme and is designed for everyone to play. These cards were distributed to different pubs and gameboard cafés around Denmark to reach a wide audience and hopefully spark up a conversation about men’s health.

Concluding Remarks

In the end, PROSTATUS has prevailed in both laboratory, hardware and human practice work. We have successfully created PMT, a fast reliable, intuitive, easy and non-invasive diagnosis for malignant prostate cancer, while also spreading awareness and proposing different ways to initiate and normalize conversations about male reproductive health. In our journey towards this success, we have included both expert and end-users in our product development, to ensure that both personal and clinical needs are met. Furthermore, we have used the knowledge gained from our expert conversations to develop CRAT, a risk assessment for the congenital risk of developing prostate cancer. Our goal with CRAT is to shine a light on the importance of early detection. We also tried to embody the importance of this, through our Early Detection campaign.

We hope that future iGEM teams, or others, will be able to pick up this project, and have the possibility to fully develop the PROSTATUS systems to not only be applicable for prostate cancer but also other diseases. Thereby PROSTATUS greatly contributes to the future of diagnostics.

Slippery Slope - A PROSTATUS music video

One of the goals of the PROSTATUS project has been to raise more awareness and break taboos around prostate cancer. Therefore, we have produced a song and created a music video that accommodates some of the feelings and worries one might experience when diagnosed with prostate cancer. The full video can be watched on our Wiki page under "Videos".

Below we present the lyrics for “Slippery Slope,” so you can sing along to the music video wherever and whenever. 😊

Slippery Slope – by PROSTATUS

[intro]
We got an offer for you: PROSTATUS

[verse]
We don’t want to see you hurt, but we can tell you got a secret deep down and it burns
Pushing down won’t help you cope but let me tell you that denial is a slippery slope

[chorus]
You might experience some symptoms we know, that can make you feel lonesome
Oh, don’t numb your feelings with this alcohol, it won’t change the outcome of your Gleason score
the pain in your gut needs a clean cut, because, your gland is on the size of a coconut
You need to understand and read a little more, this is too important for you to ignore

[verse]
You don’t know why you hurt, so you just go ahead and hide u-u-n-der your dress shirt
You think people are judging you, but if you try and open up, you’ll find that it is untrue

[chorus]
You might experience some symptoms we know, that can make you feel lonesome
Oh, don’t numb your feelings with this alcohol, it won’t change the outcome of your Gleason score
the pain in your gut needs a clean cut, because, your gland is on the size of a coconut
You need to understand and read a little more, this is too important for you to ignore
Acknowledgements

We would like to give special thanks to our supervisors Jens Pettersen, Joel Nielsen, Tina Kronborg, Simon Rose, Søren Jensen and our PI Mikkel Jørgensen for supporting us throughout our iGEM journey!
Without them PROSTATUS could not have succeeded.

Mikkel Girke Jørgensen
Mikkel Girke Jørgensen
Joel Mario Vej-Nielsen
Joel Mario Vej-Nielsen
Jens Sivkær Pettersen
Jens Sivkær Pettersen
Tina Kronborg
Tina Kronborg
Simon Rose
Simon Rose

We would also like to thank everyone from the Research Unit for Molecular Microbiology (RUMM) from SDU for their patience and assistance regarding laboratory work.


Our human practices were greatly aided by the medical experts and the interviewees, so we would like to give them a big thank you as well!



We would like to bring special attention to our sponsors: