Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Summary

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Abstract

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

Introduction

Imaging mass spectrometry (IMS) has been optimized to characterize the spatial distribution of molecular features in three widely used applications: tissue slices, spheroids, and microbial colonies^1,2,3. Tissue slices can be used to evaluate the localization of metabolites in the context of biological conditions in a host, either targeted or untargeted within a specific mass range. However, differences between molecular features are the most significant and obvious when a healthy tissue is compared to a diseased condition, for example, a tumor. This IMS approach is particular....

Protocol

All animal procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use and Care (IACUC) committee at the University of Illinois at Chicago.

1. Preparation of Reagents

1. Maintain murine oviductal epithelium (MOE) cells at 37°C with 5% CO₂ in a humidified incubator in alpha Minimum Essential Medium (αMEM) media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mg/mL insulin, transferrin, selenium (ITS), 1.8 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin-streptomycin, 1 mg/mL ge....

Results

An optimally dried ITO slide will result in a flat desiccated sample with minimal to no wrinkles across the surface of the agarose and agarose pieces that maintain spatial separation on the slide (Figure 3). Figure 3A shows optimal drying, while Figure 3B shows the wrinkles that should be avoided. This optimization requires careful monitoring of the slide in the oven, as exact times can vary based on.......

Discussion

There is a growing body of evidence that implicates norepinephrine’s role in HGSOC^12,17,18, and this technique has contributed more mechanistic information. With at least eight biological conditions present on the same slide, the method can account for biological controls such as gene and cell specificity as well as media controls in a single IMS run. While the method was optimized to evaluate exchanged small molecules in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL Falcon tubes	Denville	C1017-O	To collect cells
8-well chamber (Millipore EZ-slide chamber)	Millipore	PEZGS0816	Repurposed from Millipore Millicell EZ-slide chamber slide
Acetonitrile	Sigma-Aldrich	34998-4L	Solvent for sprayed matrix
Alpha Minimum Essential Medium (αMEM)	Fisher	10-022-CV	Cell culture media
Autoflex speed MALDI-TOF LRF	Bruker		For IMS data analysis
Centrifuge	Eppendorf	5810 R	To collect cells and remove supernatant
CHCA Matrix	Bruker Daltonic	8201344	Matrix sprayed onto dried slide
DHB Matrix	Bruker Daltonic	8201346	Matrix sprayed onto dried slide
Disposable Scalpels	Fisher	22-079-707	For removal of the ovaries
Dissecting Scissors	Fisher	13-804-6	For removal of the ovaries
DMEM Media	Gibco	11995-065	Media mixed with agarose
epidermal growth factor	Peprotech Inc.	100-15	Cell culture media supplement
Eppendorf tubes	Genesee Scientific	22-282	For agarose aliquots
Estradiol-17β	Simga-Aldrich	E2758	Cell culture media supplement
Fetal Bovine Serum	Denville	fb5001	Cell culture media supplement
FlexControl 3.4	Bruker Daltonic		IMS data acquisition software
FlexImaging 4.1	Bruker Daltonic		IMS data analysis software
Forceps (fine)	Fsiher	22-327379	For removal of the ovaries
Gentamycin	Cellgro	30-005-CR	Cell culture media supplement
Insulin, Transferrin, Selenium (ITS)	Sigma-Aldrich	11074547001	Cell culture media supplement
ITO-coated slide	Bruker	8237001	Platform for co-culture incubation
Leibovitz's L-15 Medium	Gibco	11415064	Media used during tissue dissection
L-glutamine	Gibco	25030-081	Cell culture media supplement
Low-melting agarose	Sigma-Aldrich	A9414-10G	Mixed with media for plating
Media basin	Corning	4870	Used to cut plastic dividers for divided chambers
Penicillin-streptomycin	Gibco	15140-122	Cell culture media supplement
Peptide Calibration Standard	Bruker Daltonic	8206195	Calibrant for medium mass range
Phophorus red	Sigma-Aldrich	343-242-5G	Calibrant for low mass range
SCiLS Lab 2015	Bruker Daltonic		IMS data statistical analysis
Surgical Forceps (blunt)	Fisher	08-875-8B	For removal of the ovaries
TFA	Fisher Technologies	A116-50	Added to matrix solution
TM Sprayer	HTX Technologies		For applying matrix