A CRISPR-based sensitive liquid biopsy for endometrial cancer using tumor-derived hallmarks (CRISPR SLEUTH)

Challenges. Endometrial cancer (EC) exhibits increasing incidence and mortality rates, contrasting with trends observed in most other cancers. Notably, uterine serous carcinoma (USC) is a distinct subtype typically presenting at advanced stages with an associated poor 5-year survival (<20% for stages III and IV) and high recurrence rates (up to 80%) even when caught early. This prognostic disparity and clinical peril highlight the critical need for a new diagnostic and tracking strategy that can i) detect early lesions and perhaps minimal residual disease and ii) identify aggressive subtypes, particularly USC, to inform optimal treatment and its timing. A preferred test format would be minimally invasive liquid biopsies to facilitate broad implementation and patient access. Extracellular vesicles (EVs) are promising analytes in liquid biopsy and could serve as significant sources of USC biomarkers, offering a less invasive and cost-effective alternative to current modalities such as transvaginal ultrasound and hysteroscopy. Innovations. We recently achieved a technical breakthrough in EV assays through the novel use of a CRISPR approach. CRISPR-associated proteins first recognize their specific mRNA target, triggering RNA replication and signal amplification. This unique dual- function mechanism enables the assay to differentiate between single-nucleotide polymorphisms while achieving a detection limit in the sub-attomolar range. Goals. Building upon the promise of our CRISPR assay, we aim to advance the next generation of USC diagnostics, termed SLEUTH (Sensitive Liquid biopsy for Endometrial cancer Using Tumor-derived Hallmarks). We seek two primary objectives: i) implementing a SLEUTH platform for multimodal EV analysis, and ii) rigorously evaluating EVs' clinical utility for USC diagnostics. In Aim 1, we will develop a fully automated SLEUTH assay system. The core component will be a disc-based cartridge to streamline EV lysis and RNA extraction, CRISPR reaction, and fluorescent signal detection. In Aim 2, we will apply the SLEUTH assay to analyze EVs collected from patient-derived organoids (PDOs). This preclinical study will identify a panel of USC-specific EV markers. In Aim 3, we will evaluate SLEUTH's clinical utility for USC detection by analyzing prospectively collected urine samples from women with benign or malignant endometrial conditions. EV profiling results will be used to construct a diagnostic model to differentiate USC from benign conditions and other EC subtypes. Team. We have assembled a unique team with expertise in EV detection technology, gynecologic oncology, cancer biology, and biostatistics. We also have access to outstanding resources (a well-annotated biorepository of gynecologic specimens and a collection of PDOs) for the identification of clinically relevant EV markers. Impact. The new SLEUTH platform will be a transformative solution for EV molecular profiling in urine and eventually other biofluids. Its capabilities for automation, high throughput, and multimodal detection surpass existing technologies. This proposal is thus highly responsive to PAR-25-336 and its emphasis on scalable nano-based translational testing. Project Number: 1R01CA310785-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Cesar Castro (+1 co-PI) | Institution: MASSACHUSETTS GENERAL HOSPITAL, BOSTON, MA | Award Amount: $650,513 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 MCST-U (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11329854