Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Patient-derived tumor organoids (PDOs) are rapidly emerging as a robust model system to study cancer development and therapeutic responses. PDOs are three-dimensional (3D) cell culture systems that recapitulate the complex genomic profile and architecture of the primary tumor^1,2. Unlike traditional two-dimensional (2D) cultures of immortalized cancer cell lines, PDOs capture and maintain intratumoral heterogeneity^3,4, making them a valuable tool for both mechanistic and translational research. Although PDOs are becoming an increasingly popular model sy....

Studies using human tumor specimens were reviewed and approved by the University of Iowa Institutional Review Board (IRB), protocol #201809807, and performed in accordance with the ethical standards as laid down in the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all subjects participating in the study. Inclusion criteria include a diagnosis of cancer and the availability of tumor specimens.

1. Plating intact PDOs in a 96-well plate

Our objective was to demonstrate the feasibility of using multiplexed live-cell imaging to assess PDO therapeutic response. Proof of concept experiments were performed in two separate PDO models of endometrial cancer: ONC-10817 and ONC-10811 (see Supplementary Figure 1 and Supplementary Figure 2 for ONC-10811 data). Apoptosis (annexin V staining) and cytotoxicity (Cytotox Green uptake) were kinetically monitored in response to the apoptosis-inducing agent, staurosporine. Specifically, PD.......

PDO cultures are becoming an increasingly popular in vitro model system due to their ability to reflect cellular responses and behaviors². Significant advances have been made in PDO generation, culture, and expansion techniques, yet methods to analyze therapeutic responses have lagged. Commercially available 3D viability kits are lytic endpoint assays, missing out on potentially valuable kinetic response data and limiting subsequent analyses by other methods⁸. Emerging stud.......

We are grateful to the Tissue Procurement Core and Dr. Kristen Coleman at the University of Iowa for providing patient tumor specimens and to Dr. Sofia Gabrilovich in the Department of Obstetrics and Gynecology for assisting with PDO model generation. We also thank Dr. Valerie Salvatico (Agilent, USA) for critical analysis of the manuscript. We acknowledge the following funding sources: NIH/NCI CA263783 and DOD CDMRP CA220729P1 to KWT; Cancer Research UK, Prostate Cancer UK, Prostate Cancer Foundation and Medical Research Council to JSdB. The funders had no role in the design or analysis of experiments or decision to publish.