Abstract 5760: Mass spectrometry-based proteomic investigation of patient-derived tumoroids employing fast, high-resolution accurate mass technologycatalyzes precision oncology and drug discovery
Authors: Dominique B. Figueroa, Kevin Yen-Yu Yang, Colin Paul, Pradip Shahi Thakuri, Anthony Chatman, Brett Larsen, Julian Saba, Tonya Pekar Hart, Amirmansoor Hakimi
Journal: Cancer Research (2025)

The inherent heterogeneity of cancer has spurred significant technological advancements to enhance the detailed study required to characterize this complex disease, predominantly defined by uncontrolled changes. Efforts to address and treat this multi-faceted variability are demonstrated by use of patient-derived tumoroids, which model the conditions and complexities observed in patient tumors using an in vitro cell culture system. Despite the strong correlation between patient response and that observed in matched tumoroid models, tumoroid technology has suffered from a lack of standardization in both culture methods and characterization, hindering widespread clinical translation. This work seeks to present robust and standardized methods for the generation and characterization of tumoroid models. Specifically, our methods aim to reduce variability and labor across workflows and spur the adoption of next-generation tumoroid culture and mass spectrometry methods in discovery and development research. Colorectal, lung, breast, and endometrial tumoroids were derived from donor cancer samples in OncoPro™ Tumoroid Culture Medium, a serum-free, conditioned medium-free system with broad applicability across solid cancer indications. After tumoroid cell pellets were collected, samples were digested using the AccelerOme automated sample preparation platform. Peptides were then loaded onto an IonOpticks Aurora Frontier TS 60×75 column, and separated using Vanquish Neo UHPLC system. Data was acquired on anOrbitrap Astral mass spectrometer in narrow window data-independent acquisition (DIA) mode, and the resulting raw fileswere analyzed with Proteome Discoverer using the CHIMERYS™ intelligent search algorithm or Spectronaut with 1% FDR control at the peptide-spectrum match (PSM) and protein level. Proteins identified in the breast cancer model in our experiments were linked to MAPK, ALK, and ERBB2 signaling pathways, as well as protein and RNA metabolism. The next steps in our experiments involve validating the identified proteins in the breast cancer tumoroid model, and understanding the underlying mechanisms to assess the model’s efficacy. Future directions will leverage the unique capabilities of mass spectrometry-based proteomics to investigate post-translational modifications, including phosphorylation and glycosylation, which are crucial in signaling and cancer. By integrating cancer tumoroid models, serum-free media, and the Orbitrap Astral mass spectrometer, we aim to streamline precision oncology and drug discovery by minimizing variability in these dynamic models. Citation Format: Dominique B. Figueroa, Kevin Yen-Yu Yang, Colin Paul, Pradip Shahi Thakuri, Anthony Chatman, Brett Larsen, Julian Saba, Tonya Pekar Hart, Amirmansoor Hakimi. Mass spectrometry-based proteomic investigation of patient-derived tumoroids employing fast, high-resolution accurate mass technologycatalyzes precision oncology and drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5760.