13C Tracer Analysis and Metabolomics in 3D Cultured Cancer Cells

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Scope of the method

The Method relates to

Human health

The Method is situated in

Basic Research

Type of method

In vitro - Ex vivo

This method makes use of

Human derived cells / tissues / organs

Specify the type of cells/tissues/organs

human cancer cell lines

Description

Method keywords

3D Cell culture
Cancer cells
Metabolism
Metabolic quenching
spheroids
metabolomics

Scientific area keywords

Cancer metabolism
13C tracer analysis
3D
spheroids
extracellular matrix
cancer
metastasis

Method description

Metabolomics and 13C tracer analysis are state-of-the-art techniques that allow determining the concentration of metabolites and the activity of metabolic pathways, respectively. Three dimensional (3D) cultures of cancer cells constitute an enriched in vitro environment that can be used to assay anchorage-independent growth, spheroid formation, and extracellular matrix production by (cancer) cells. Here, we describe how to perform metabolomics and 13C tracer analysis in 3D cultures of cancer cells. Intracellular metabolites are extracted from these quenched cells, and 13C-label incorporation patterns and metabolite levels are determined via MS-based analysis.

Lab equipment

- Humidified temperature and CO2-controlled cell culture incubator ;
- Vacuum aspirator ;
- Biological safety cabinet ;
- Cell counter ;
- Thermometer (-40°C to 0°C) ;
- Chemical fume hood ;
- Tissue Lyser ;
- Heating block ;
- Centrifuge fitting 15 and 50 mL tubes ;
- Vortex ;
- Refrigerated centrifugal vacuum concentrator ;
- Mass Spectrometer with gas or liquid chromatographic technique.

Method status

Published in peer reviewed journal

Pros, cons & Future potential

Advantages

A key feature of the method lies in the possibility to study metabolism in cells growing in an anchorage-independent manner which might be more representative of the physiological status of metastatic cancer cells. This allows us to recapitulate some cancer phenotypes of disseminated cancer cells during the metastatic process.

In addition, the described quenching process enables to rapidly stop the metabolism of cancer cell spheroids as well as to measure the concentration and labeling patterns of metabolites in a reliable manner.

Challenges

To obtain accurate metabolic measurements you need to have an adequate amount of cells to extract. When growing cells in this system, the number of spheroids is limited per well to avoid a group of cells to grow as a single spheroid (which will not recapitulate the metabolism of colonizing cells). Therefore, it is necessary to optimize the number of wells to pool together according to cell type and condition to test to achieve the proper amount of cell extracts.

Metabolic quenching of spheroids that grow in suspension is a more time-consuming protocol compared to the quenching of attached cells. Since metabolism is a rapidly adapting process, it is critical to work as fast as possible and maintain low temperature during the process to minimize the chance of perturbations in cellular metabolism.

Modifications

This protocol can be adapted for metabolic measurements of alternative systems of cells growing in suspension.

Future & Other applications

This protocol can be adapted for metabolic measurements of alternative systems of cells growing in suspension.