Periodic Reporting for period 1 - EVOLVE (Isolation and Analysis of Extracellular Vesicles using OptofLuidics for the eValuation of Microsatellite Instability in Endometrial Cancer)
Reporting period: 2022-09-01 to 2024-08-31
Cancer has now become a ubiquitous aspect of contemporary life and the clinical setting, and is expected to rank as the leading cause of death in upcoming years. Endometrial cancer (EC) specifically is the most common gynecological cancer, and has an estimated incidence rise of more than 50% worldwide by 2040, mainly associated with increasing obesity prevalence. In 2018 alone, there were approximately 380,000 new cases and close to 90,000 deaths attributed to EC worldwide. Moreover, it is becoming increasingly clear that EC comprises a broad range of cancer phenotypes, where distinct molecular genetic alterations blur the lines between sub-types. One of these molecular alterations is Microsatellite Instability (MSI). Microsatellites are short, repetitive nucleotide sequences in the human genome which, due to their repetitive structure, are prone to slippage or replication errors, leading to MSI. This genetic alteration is present in 20-30% of EC patients, and MSI analysis has been shown to be an effective predictive biomarker for immune checkpoint inhibitors. This makes MSI evaluation a good candidate for immunotherapy selection, which is highly effective in such patient sub-group. Thus, there is a growing need for the development of better platforms for inexpensive, early-stage EC diagnosis and molecular phenotyping. This project aimed to address these needs by creating an integrated lab-on-chip technology for EC diagnosis through the isolation of extracellular vesicles (EVs) contained in body fluids from EC patients and their molecular analysis based on surface-enhanced Raman spectroscopy (SERS). EVs were the focal point of the project due to their great potential as sources of clinically relevant biomarkers. They are cell-secreted vesicles, found in circulating blood and originating from virtually all kinds of cells (including tumor cells), that play important roles in intercellular communication. Importantly, they transport cargos of DNAs that can potentially be used for MSI determination. At its conclusion, the project made important strides in the process of isolating these valuable EVs from complex samples utilizing a microfluidic device. In this device, an aggregation agent (ExoGAG) was mixed with the EV sample in order to generate large aggregates that could then be captured out of the sample, within the device. The developed device had good capture efficiency as well as the capability to recover the DNA from within it. This DNA from the EVs could then be used for downstream analysis, such as MSI evaluation. To do this evaluation, a new SERS-based protocol was devised, utilizing gold nanoparticles called Nanostars. These Nanostars were coated with specific DNA strands that recognize the DNA sequences where MSI will be present. To capture these DNA sequences, a protocol was developed to coat gold slides with capturing DNA strands. Good functionalization and hybridization of these various DNAs was observed at the conclusion of the project. With these two different modules developed, the next steps would be to test this novel, integrated system with clinical samples from EC patients.
The project achieved some of the pre-defined objectives and milestones (with some deviations on the original plan occurring). The key objective for the project, was to develop an integrated approach for the isolation, enrichment and analysis of EVs from EC for MSI assessment using lab-on-chip, microfluidics methods in conjunction with DNA biomarkers and SERS technology. To achieve this, the work was divided in three different Work Packages (WPs).
In WP1, focused on the isolation and concentration of EVs using microfluidic device, a microfluidic herringbone mixing device was designed and successfully tested to mix the EV sample with the aggregation agent used. A microfluidic EV isolation device (“pillar chip”) was then designed and successfully tested to filter and capture EVs from complex samples. Moreover, the ExoGAG EV aggregation agent, from project partners at Nasasbiotech, was successfully integrated into the isolation and concentration protocol. Based on these results, a manuscript is under preparation, reporting on the developed EV isolation protocol and the retrieval of EV-DNA for downstream MSI assessment utilizing the finalized “evDNA chip”.
In WP2, focused on sensing and characterization of EVs using optofluidic device, a protocol for MSI evaluation using gold nanoparticles (Au-NPs) was first developed. Synthetic single-stranded DNA (ssDNA) probes were then designed to match different regions of a common MSI biomarkers (MONO-27), aiming at the capture of both wild-type and MSI EV-DNA. There were delays in the manufacturing of these synthetic strands, leading to delays in their application in the project. To implement the designed protocol, substrates for MSI-SERS assessment were fabricated, specifically Au-coated Si slides and Au-NPs, with a protocol to functionalize both the Au-coated Si slides and Au-NPs with specific synthetic ssDNA probes being developed. It was possible to observe good functionalization of Au-coated Si slides with the ssDNA probes used to capture the EV-DNA.
Finally, WP3 was focused on the pre-clinical validation of isolation and characterization modules. However, since the full optimization of both the isolation and MSI-SERS modules was only concluded close to the project’s end, it was not possible to move forward with tests utilizing either patient-derived EVs or clinical plasma samples.
In WP1, focused on the isolation and concentration of EVs using microfluidic device, a microfluidic herringbone mixing device was designed and successfully tested to mix the EV sample with the aggregation agent used. A microfluidic EV isolation device (“pillar chip”) was then designed and successfully tested to filter and capture EVs from complex samples. Moreover, the ExoGAG EV aggregation agent, from project partners at Nasasbiotech, was successfully integrated into the isolation and concentration protocol. Based on these results, a manuscript is under preparation, reporting on the developed EV isolation protocol and the retrieval of EV-DNA for downstream MSI assessment utilizing the finalized “evDNA chip”.
In WP2, focused on sensing and characterization of EVs using optofluidic device, a protocol for MSI evaluation using gold nanoparticles (Au-NPs) was first developed. Synthetic single-stranded DNA (ssDNA) probes were then designed to match different regions of a common MSI biomarkers (MONO-27), aiming at the capture of both wild-type and MSI EV-DNA. There were delays in the manufacturing of these synthetic strands, leading to delays in their application in the project. To implement the designed protocol, substrates for MSI-SERS assessment were fabricated, specifically Au-coated Si slides and Au-NPs, with a protocol to functionalize both the Au-coated Si slides and Au-NPs with specific synthetic ssDNA probes being developed. It was possible to observe good functionalization of Au-coated Si slides with the ssDNA probes used to capture the EV-DNA.
Finally, WP3 was focused on the pre-clinical validation of isolation and characterization modules. However, since the full optimization of both the isolation and MSI-SERS modules was only concluded close to the project’s end, it was not possible to move forward with tests utilizing either patient-derived EVs or clinical plasma samples.
EC has high incidence both at the European (up to 20/100 thousand women) and global (400 thousand new case in 2020 alone) levels. Yet, clinical advances have focused on the identification of molecular alterations, with limited impact on the clinical management of patients. With the fast technical evolution in the fields of liquid biopsy and personalised oncology, the ‘EVOLVE’ project aimed to improve the clinical management of advanced EC by aiding therapeutic selection for each patient according to their molecular profile through liquid biopsy. Scientifically, the project explored completely unfamiliar territory by aiming to integrate microfluidic EV enrichment with SERS-based MSI detection. The work performed within the project lead to the creation of new protocols for both the isolation of EVs from complex samples and the evaluation of MSI from EV-DNA, the latter being the first time such process has been proposed and devised utilizing SERS (and the specific assay envisioned). Considering these advancements, it is made clear that the capability to merge a commercially available assay (ExoGAG) with an industrially scalable, microfluidic device for EV isolation has great potential for future implementation on clinical settings, providing EV material (e.g. DNA, RNA, proteins) for downstream analysis with an automatable system. Such implementation would have economical and societal impacts, chiefly in the form of better-informed decision-making processes by oncologists. This would result in a reduction of costs with inefficient treatments and expanding the time window for other treatments to be conducted, hopefully resulting in the reduction of avoidable discomfort, pain and, in extreme cases, mortality. Moreover, after the final implementation of the “MSI-SERS” system, the impact of the project could go even further, as MSI is an important biomarker not only in EC but also in other cancer types, making the platform developed during ‘EVOLVE’ a potentially important tool to be deployed in a variety of oncological centres.