Colorectal cancer (CRC) is the third most prevalent cancer type and the second leading cause of cancer deaths worldwide [1]. In CRC, early diagnosis and treatment are crucial to improve survival. If CRC is detected early, there is a 91% survival rate. However, late-stage detection drops this number to 14% [2].
Different countries have screening programmes to facilitate early detection of CRC.
In the Netherlands, anyone above the age of 55 is invited once every two years to participate in the screening programme [3]. In the first step, participants are tested using a Faecal Immunochemical Test (FIT), which checks for occult blood in the stool as a marker for bowel disease [4]. Based on the results of this test, patients are referred to a follow-up colonoscopy to determine if CRC is actually present. Recent analysis by the National Institute for Public Health and Environment (RIVM) showed that roughly 5% of FIT participants were referred for colonoscopy. In 30% of these colonoscopies no polyps or tumours were found [5]. This means a false-positive FIT will lead to an unnecessary colonoscopy. Moreover, the FIT is less susceptible to detect early-stage cancers than neoplasms [6].
Figure 1: Colorectal cancer is the cancer that causes the second most deaths in the world.
Figure 2: Colorectal five-year survival rate decreases from 91% to 14% when caught in late stage.
This is why we created Colourectal. Colourectal strives to create a new living diagnostic tool to allow for more convenient and more frequent testing for those at risk of CRC. To achieve this, we are engineering Escherichia coli Nissle 1917 (EcN) to screen the users gut for CRC, and if detected produces a coloured signal observable in the stool. We are basing this project on four subprojects: colonisation, detection, signalling and biosafety. These four pillars come together to form our living diagnostic tool.
Interaction
Our first step in finding colorectal cancer is the binding of potential cancer cells. This allows our living diagnostic tool to stay close to the cancer, giving it a higher chance of detecting our biomarkers. Our living diagnostic tool will bind to cancer cells by binding CEACAM6, a glycoprotein which is overexpressed in tumour cells and by binding to integrin α5β1 present in invasive colon cancer in humans [3]. Additionally, we were interested in the interactions EcN may have with cancer cells. For this we performed RNA sequencing on EcN and Caco-2 cells. Curious to learn more? Check out our fimbriae results page.
In order to target CRC cells, we try to detect one of its biomarkers, lactate [7]. We have investigated how we can make a lactate inducible system sensitive to lactate levels associated with cancer. For this we have characterised the lactate inducible ALPaGA promoter which is well suited to the anoxic and glucose rich colon environment. Additionally we built and tested several constructs based on inhibition of the promoter to tune the lactate threshold response. Curious to learn more? Check out our threshold system results page!
In our project, chromoproteins serve as the way of colouring the stool and alerting the user colorectal cancer has been detected. In order to do so succesfully, we investigated how the chromoproteins could be secreted from EcN. Additionally, the chromoproteins are modified in such a way that they are only visible when the second cancer biomarker, Matrix Metalloproteinase 9 (MMP-9), is present. This is done by inserting two small regions in the chromoprotein. Curious to learn more? Check out our chromoprotein results page!
Throughout our project we have also considered safety-by-design. To achieve this we have implemented both a biocontainment circuit, as well as an inducible kill-switch for EcN. The biocontainment is based on a temperature sensitive system regulating a toxin-antitoxin system as well as a chimeric mucin sensor which we have engineered. The inducible kill-switch is based on a CRISPR-Cas system, with spacers designed for EcN. Curious to learn more? Check out our safety results page!
To make these systems work together in our tool and increase the modularity, we needed to induce several systems with external metabolites. Induction of promoters and genetic constructs in the lab is easy, but when working in the human gut suddenly choosing a suitable inducer becomes a lot tougher. Therefore, we experimented with creating a three-step inducer system, where one inducer can induce three separate responses based on the amount of inducer present. Curious to learn more? Check out our three step inducer results page!
In short, our EcN will localise around CRC cells using its modified ‘bacterial tentacles’, or pilli. Subsequently an engineered threshold circuit detects elevated lactate levels indicative of CRC. This triggers the production of modified chromoproteins, the proteins which will colour the stool and give a signal to the user. In order to ensure specificity for CRC, the chromoproteins are modified so that they only show colour when they are cleaved by MMP-9, another CRC biomarker. Finally, use of a living diagnostic tool in the gut environment requires it to be safe, both for humans and the environment. To ensure this, we introduced a biocontainment circuit. This circuit limits our living diagnostic tool to an environment containing mucin and with a temperature of 37 oC, typical for the large-intestine. Additionally, a kill-switch can be induced with an external metabolite.
All these aspects combined form a non-invasive, simple-to-use self-test which will lead to more effective detection of colorectal cancer and fewer unneeded colonoscopies.
Colorectal cancer (CRC) is the third most common cancer type and the second-leading cause of cancer deaths worldwide [1]. Early diagnosis and treatment are crucial for improving these numbers. If detected early, there is a 91% survival rate. But if detected late, the survival rate drops to 14% [2].
Different countries have screening programmes to help detect CRC early on. In the Netherlands, anyone over 55 gets screened every two years. First, patients are given a test (called FIT) that checks for hidden blood in the stool, which can indicate bowel disease [3]. If blood is found, patients are referred to a follow-up colonoscopy to confirm if CRC is present. Research found that roughly 5% of patients were referred for a colonoscopy. In 30% of these colonoscopies, no polyps or tumours were found. This means a significant portion of patients had to take an unnecessary colonoscopy [4].
Colourectal is a new diagnostic tool that allows for more convenient and frequent testing for people at risk of CRC. It works like this:
Figure 1: Colorectal cancer is the cancer that causes the second most deaths in the world.
Figure 2: Colorectal five-year survival rate decreases from 91% to 14% when caught in late stage.
- The modified bacterial tentacles (or ‘pili’) of the probiotic strain Escherichia coli Nissle 1917 attach to CRC cells.
- The strain detects if there are high levels of lactate, which is the first indicator of CRC.
- This triggers the production of modified chromoproteins, which will colour the stool to give a signal to the user. The chromoproteins will only show colour when they are split by Matrix Metalloproteinase 9 (MMP-9), the second indicator of CRC.
To ensure Colourectal is safe for both humans and the environment, we limited its functionality to an environment that contains mucin and has a temperature of 37°C, which is typical for the large intestine. Additionally, an external metabolite can stop the diagnostic.
All of these aspects combine to form a non-invasive, simple-to-use self-test that allows people at risk of CRC to screen themselves easily.