Sample preparation strategies for high-throughput mass spectrometry imaging of primary tumor organoids

Abstract

Patient-derived 3D organoids show great promise for understanding patient heterogeneity and chemotherapy response in human-derived tissue. The combination of organoid culture techniques with mass spectrometry imaging provides a label-free methodology for characterizing drug penetration, patient-specific response, and drug biotransformation. However, current methods used to grow tumor organoids employ extracellular matrices that can produce small molecule background signal during mass spectrometry imaging analysis. Here, we develop a method to isolate 3D human tumor organoids out of a Matrigel extracellular matrix into gelatin mass spectrometry compatible microarrays for high-throughput mass spectrometry imaging analysis. The alignment of multiple organoids in the same z-axis is essential for sectioning organoids together and for maintaining reproducible sample preparation on a single glass slide for up to hundreds of organoids. This method successfully removes organoids from extracellular matrix interference and provides an organized array for high-throughput imaging analysis to easily identify organoids by eye for area selection and further analysis. With this method, mass spectrometry imaging can be readily applied to organoid systems for preclinical drug development and personalized medicine research initiatives.

Keywords: MALDI MSI; drug discovery; high throughput; microwells; organoids.

Figure 1.. Organoids in Matrigel:

m/z 184.0722 is a peak resulting from a phosphatidyl choline head group which indicates where organoid cells are located in the Matrigel material. The white dotted line outlines Matrigel. m/z values 285.0634, 239.1049, 214.9708, 175.1189, and 240.0840 represent examples of small molecule background signal in the Matrigel. This background interference can complicate MSI analysis.

Figure 2.. Organoid Microarray Workflow:

Primary organoids are first grown in Matrigel. Media is removed, and organoids embedded in Matrigel are washed with 1X PBS three times and transferred to a microcentrifuge tube. The microcentrifuge tube is then centrifuged at 1,000 rpm at 4°C for 5 min to pellet out the organoids in the bottom of the microcentrifuge tube. The Matrigel is removed from the top of the tube, and then using a wide-bore pipette, organoids are transferred to the gelatin microwell, which is the nest in a cryomold sitting on top of a 50mL conical tube. Centrifugation is then applied at 1,000 rpm at 4°C for min to ensure that organoids lie evenly within the microwells. Excess PBS is removed following centrifugation, and the gelatin-embedded organoids are flash frozen on dry ice and stored at −80°C prior to sectioning.

Figure 3.. Gelatin Organoid Microarray:

(Right) High-throughput MSI analysis of organoids can be performed in a gelatin microarray as a direct result of alignment in the same z-axis prior to sectioning compared to direct sectioning in Matrigel (left).

Figure 4.. Chemotherapy Treatment Alters Organoid Metabolites:

A. m/z 348.0688, tentatively identified as AMP, is significantly increased in 5-FU+GEM treated organoids versus controls B. m/z 428.0352, tentatively identified as ADP, is also significantly increased in 5-FU+GEM treated organoids versus controls.

Figure 5.. 200μm Microwell Organoid Microarray:

m/z 184.0722, a phosphatidyl choline head group, is shown in red to demonstrate the location of the small pancreatic cancer organoids. m/z 434.3803, shown in blue, is a background signal from gelatin microarray only. This overlay analysis of the organoids and the microarray demonstrates that organoids are aligned on similar z-axis for sectioning and hundreds of organoids can be imaged in a microarray by MSI.