Comparison of Cell and Organoid-Level Analysis of Patient-Derived 3D Organoids to Evaluate Tumor Cell Growth Dynamics and Drug Response

Abstract

3D cell culture models have been developed to better mimic the physiological environments that exist in human diseases. As such, these models are advantageous over traditional 2D cultures for screening drug compounds. However, the practicalities of transitioning from 2D to 3D drug treatment studies pose challenges with respect to analysis methods. Patient-derived tumor organoids (PDTOs) possess unique features given their heterogeneity in size, shape, and growth patterns. A detailed assessment of the length scale at which PDTOs should be evaluated (i.e., individual cell or organoid-level analysis) has not been done to our knowledge. Therefore, using dynamic confocal live cell imaging and data analysis methods we examined tumor cell growth rates and drug response behaviors in colorectal cancer (CRC) PDTOs. High-resolution imaging of H2B-GFP-labeled organoids with DRAQ7 vital dye permitted tracking of cellular changes, such as cell birth and death events, in individual organoids. From these same images, we measured morphological features of the 3D objects, including volume, sphericity, and ellipticity. Sphericity and ellipticity were used to evaluate intra- and interpatient tumor organoid heterogeneity. We found a strong correlation between organoid live cell number and volume. Linear growth rate calculations based on volume or live cell counts were used to determine differential responses to therapeutic interventions. We showed that this approach can detect different types of drug effects (cytotoxic vs cytostatic) in PDTO cultures. Overall, our imaging-based quantification workflow results in multiple parameters that can provide patient- and drug-specific information for screening applications.

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

“…Thus, CellTiter-Glo may be most suitable for primary screens and cCasp3 staining as a follow up to probe effects on specific cellular subpopulations. Another common readout in 3D cell culture screens is aggregate size ( Yao et al, 2020 ; Mittler et al, 2017 ; Kim et al, 2020 ). Interestingly, the largest cross-sectional area of organoids did not correlate with G418 doses or CellTiter-Glo survival data in the conditions and time scales tested here, but remained largely constant despite escalating toxin levels (see Figure 8—figure supplement 1h ).…”

Challenges of applying multicellular tumor spheroids in preclinical phase

“…MCTs are cell aggregates typically composed of cancer cells cultured under scaffold-based or -free conditions. Unlike MCTs, organoids are comprised of organ-specific cells derived from primary tissue or stem cells capable of self-renewal, self-organization and exhibit organ functionality [ 26 , 27 ]. A scaffolding extracellular environment such as Matrigel and collagen is used to support the developing microstructure architecture of organoids.…”

Glioblastoma Organoids: Pre-Clinical Applications and Challenges in the Context of Immunotherapy

“…Proliferation of tumor cells within genetically engineered organoids is possible by detection and quantification of the fluorescence/bioluminescence signal of the genetically modified GBM-like cells ( 61 , 62 ). Viral barcoding labeling can further enable tracing of clonal lineages and proliferation capacities ( 75 ).…”