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Published on: February 2, 2013
Nested Nanowell Arrays for High-Throughput Quantitative Analysis of Cytokines from Single Macrophages
Claudius L Dietsche1, Lucien R Stöcklin1, Robert Strutt1
1Department of Biosystems and Engineering, ETH Zurich, Schanzenstrasse 44, CH-4056 Basel, Switzerland.
Insights
Researchers developed nested nanowell arrays to analyze single-cell protein secretion from tumor microenvironment (TME) macrophages. This technology reveals cytokine profiles and drug effects, aiding cancer research and personalized medicine.
Area of Science:
- Immunology and Cancer Biology
- Biotechnology and Bioengineering
Background:
- Macrophages are key regulators of the tumor microenvironment (TME) and exhibit diverse phenotypes.
- Understanding macrophage heterogeneity and their secreted cytokines is crucial for cancer research.
Purpose of the Study:
- To develop a high-throughput, single-cell analysis tool for multiplexed cytokine profiling of macrophages.
- To investigate the impact of drug treatments and stimuli on macrophage cytokine secretion.
Main Methods:
- Nested nanowell arrays with over 100,000 wells on a cyclic olefin copolymer (COC) substrate were fabricated.
- Functionalized, barcoded beads were used for capturing and quantifying 10 secreted proteins via sandwich immunoassays.
- An image analysis tool was developed for data processing and analysis of single-cell cytokine secretion.
Main Results:
- High secretion of interleukin (IL)-1β, IL-8, and macrophage inflammatory protein 1α (MIP-1α) was observed in over 43% of macrophages.
- Chemotherapeutic drugs paclitaxel and docetaxel increased MIP-1α secretion.
- Cosecretion of IL-1β and IL-6 was confirmed in IL-4/IL-13 stimulated macrophages, linked to multiple myeloma pathways.
Conclusions:
- Nested nanowell arrays provide an accessible platform for single-cell, multiplexed protein analysis.
- This technology has potential applications in basic research, diagnostics, and personalized medicine for analyzing cancer and immune cells.
Abstract:
Macrophages play a critical role in the development of the tumor microenvironment (TME). Recruited macrophages in the TME differentiate into various phenotypes, each with a distinct profile of secreted cytokines. To describe and understand this large heterogeneity, we developed nested nanowell arrays for the multiplexed analysis of secreted proteins at the single cell level. The array consists of more than 100,000 wells on a cyclic olefin copolymer (COC) substrate. Each well contains seven smaller indents for cocapturing functionalized beads and can be operated with standard laboratory equipment such as pipettes and microscopes. The barcoded beads capture cytokines of interest and allow their quantification via sandwich immunoassays. We developed an image analysis tool and quantified 10 proteins secreted from single macrophages and investigated the effects of stimulation and drug treatment. We found that interleukin (IL)-1β, IL-8, and macrophage inflammatory protein 1α (MIP-1α) were highly secreted by more than 43% of the macrophages, with an increase of MIP-1α secretion under treatment with the chemotherapeutic drugs paclitaxel or docetaxel. Pairwise protein analysis confirmed cosecretion of IL-1β and IL-6 in macrophages stimulated with IL-4/IL-13, which were identified as part of a critical pathway in multiple myeloma before. We also demonstrate further multiplexing with three and four cosecreted proteins, together with assessing the cell viability as an additional parameter important for drug testing. In summary, we have shown that our nested nanowell arrays are an easy-to-use analytical tool for basic research, and we believe that it can be employed for diagnostics and personalized medicine, e.g., for investigation of cancer and immune cells from a biopsy or circulating tumor cells obtained from a liquid biopsy.
