Team:Thessaloniki/Description

☰







Project Description






Overview


Inspired by the poor outcomes in many cancer types, due to the inability to diagnose the disease at a primary stage, we decided to design METIS, a cost-effective, non-invasive diagnostic tool for Pancreatic Ductal Adenocarcinoma. METIS deploys Toehold Switches, a kind of synthetic riboswitches, to detect upregulated miRNAs in urine.



Why Pancreatic Ductal Adenocarcinoma?


Pancreatic Ductal Adenocarcinoma (PDAC) is the most common type of pancreatic cancer and it is estimated that it affects 13.2 people out of 100,000 every year worldwide [1]. Although PDAC is a relatively rare cancer, it is associated with a very poor prognosis, with approximately a 10% 5-year survival rate, which is the poorest prognosis among the solid types of cancers [1],[2]. According to the World Health Organization, 466,003 deaths were attributed to pancreatic cancer in 2020 [3].

~Graph taken from International Agency for Research on Cancer, WHO~
It is considered that this dismal outcome might be improved through the earlier diagnosis of the disease, that will allow both a more efficient therapeutic intervention, before the onset of migration of cancer cells in other tissues and nodes [4], as well as the better understanding of the pathogenesis of the disease [5]. However, according to the Surveillance, Epidemiology, and End Results Program, only 11% of patients are diagnosed at a primary stage [2], mostly because of the vague symptoms at these stages and the ineffectiveness of the currently available diagnostic methods [6].

~Data taken from National Cancer Institute, SEER Program, Cancer Stat Facts: Pancreatic Cancer~
But how will the earlier diagnosis affect the prognosis of the disease? The high mortality of PDAC can be attributed to some of the characteristics of this type of cancer: The anatomical situation of the pancreas which hinders the detection of the tumor, the presence of vessels that are often surrounded by malignant cells, making the surgical resection impossible, as well as the aggressive nature and high metastatic propensity of this cancer, are some of the features that affect the prognostic factors that are taken into account for the determination of the stage and the treatment [1], [7]. The resectability of a tumor is of great importance to achieve long term survival and it can be improved through the earlier diagnosis [1]. The following diagram was based on data acquired from the SEER Program and shows the 5-year survival rate by the stage of the diagnosis. 41.6% of patients survive over five years when diagnosed with a localized tumor [2].

~Data taken from National Cancer Institute, SEER Program, Cancer Stat Facts: Pancreatic Cancer~



Current Diagnostic Methods


The lack of symptoms, during the first stages of PDAC, renders the screening even of seemingly healthy populations vital [8]. At present, the diagnosis of PDAC, depends, primarily, on imaging techniques and biopsy. Imaging techniques include methods such as Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography with Computed Tomography (PET/CT) and Endoscopic Ultrasound (EUS) [9].
Both imaging and biopsy pose several limitations for the early diagnosis of the disease. On the one hand, imaging methods are incapable of detecting the early-lesions and on the other hand, biopsy is an invasive procedure and hence, the possible benefit for the patient does not exceed the risk [6].
To overcome these issues, non-invasive biomarkers have been proposed. However, CA-19-9, the only blood biomarker approved by FDA, has been proven inefficient due to its low specificity and sensitivity [4], [8].



miRNAs: A promising cancer biomarker


microRNAs (miRNAs) are an emerging type of biomarkers that have been studied for the diagnosis of a wide range of diseases, including cancer. miRNAs are small, non-coding RNA molecules that play an essential role in the regulation of gene expression at the post-transcriptional level. More specifically, miRNAs are incorporated into the RISC complex and bind to a complementary sequence of an mRNA molecule, leading to the inhibition of translation or the degradation of the target [10], [11].
Many miRNAs contribute to the regulation of cell growth and proliferation by inhibiting the expression of genes crucial for the cell cycle, migration, or apoptotic signals [12]. Thus, the dysregulation of these molecules is proposed to be involved in the initiation and development of cancer. Indeed, in many neoplasms, several miRNAs are either downregulated or upregulated in different samples, such as blood, serum, saliva and urine. The different miRNAs profile that occurs in different cancer types holds potential as a diagnostic or prognostic biomarker and thus, can be employed in new diagnostic tools [13].



METIS: A Toehold Switch Based Diagnostic System


METIS utilizes a kind of synthetic riboswitches, called Toehold Switches; RNA molecules that consist of a hairpin-like structure and a reporter gene, expressed when a trigger molecule causes the unfolding of the hairpin structure. In the absence of the trigger, the hairpin inhibits the binding of the ribosomes and hence, the expression of the gene [14], [15].
We chose three miRNAs to function as triggers. We determined that hsa-miR-143-3p, hsa-miR-30e-5p and hsa-miR-1246 are the most suitable for this purpose, due to their high specificity and sensitivity. In addition, they are upregulated in urine of patients with PDAC at stages I and II, which allows an early and non-invasive detection [16].
These three miRNAs are excreted in urine exosomes in small quantities. Therefore, the exosomal RNA should be isolated from the sample and then, the triggers should be amplified. We decided to use an EXPAR-like amplification method, which is an isothermal technique [17], in order to reduce the cost and the equipment needed to the minimum – that is a simple incubator.
The amplified miRNAs bind to a complementary sequence, within the loop, leading to the reconfiguration of the toehold and the production of the reporter gene. As a reporter, we used eGFP, a well-characterized protein that produces fluorescence, when excitated. The fluorescence can be measured with a variety of commonly used equipment, such as a fluorometer. The miRNAs can be quantified through the intensity of the fluorescence produced by the system.

~Photograph taken in iGEM Thessaloniki's Lab~



Why a fluorometer device?


During our Integrated Human Practices activities we realized the urgency for our diagnostic tool to become available in Greece as soon as possible. Since in greek diagnostic centers there is not always specialized equipment for fluorescence measurements, we decided to design a portable fluorometer device which functions as a cost-effective tool. After conducting the necessary research in the field of entrepreneurship we concluded that the device will not only decrease the cost of diagnosis but will also increase the accessibility of it.



| How does it work?


Our device should be able to solve basic problems existing in labs given the following characteristics:
  • ~Plug-and-Play Software: in order for the device to operate, it has to be plugged into a PC. The PC allows the usage of a graphical interface which maps the locations of the biological material in the wells of the testing plate at the beginning of the process and provides the measurements to the user correspondingly to the wells.
  • ~Hardware: the device uses two computational units (Arduino and Raspberry Pi) for the control of the conditions (light and temperature) under which the reactions take place (Toehold-miRNA binding, eGFP expression).



Challenges and Future Perspectives


During our project development we came across several challenges related to the lack of sufficient literature on this specific subject, since the research about miRNAs as biomarkers is a recently developed field. As the knowledge about the profile of miRNAs increases, our project could be improved by the exploitation of biomarkers specific for different high-risk groups, such as patients with diabetes, obesity or chronic pancreatitis [4], or even expanded to include different types of cancer.

Learn More:






Summary





References