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.
