Microscopic bowls reveal the mysteries of protein secretions

microscopic bowl

Scientists have developed a tiny test tube that can hold only one cell. The tube has allowed them to link protein secretion levels with surface markers and transcriptome data from the same cell.

Cells secrete various proteins, including antibodies and growth factors that can significantly impact human health. However, even within the same cell type, some cells secrete more protein than others. This has made it challenging for scientists to establish links between the genomes, transcriptomes, and functional outputs of cells, posing a critical problem for researchers developing cell therapies. Without identifying the cells that produce the most therapeutically relevant protein, it is difficult to create an effective therapy.

A recent study published in Nature Communications tackled this issue by developing microscale hydrogel particles with subnanoliter cavities. These nanovials, which are approximately 35-micrometer diameters and bowl-shaped, can be used to measure protein secretion, transcriptome data, and surface markers from individual cells. The team demonstrated that these nanovials can reveal molecular signatures associated with the secretion of the antibody immunoglobulin G (IgG) from B cells. By using this approach, scientists can identify subpopulations of cells that secrete more beneficial proteins and leverage them for developing therapies.

One of the most common methods for measuring cell secretions is the enzyme-linked immunosorbent spot (ELISpot) assay, which is an enzyme-based approach developed 25 years ago. However, this approach does not allow researchers to isolate the cells and analyze them further. According to Dino Di Carlo, a bioengineer at the University of California, Los Angeles, and author of the paper, microfluidics technologies were not suitable for in-depth exploration of the biology of single cells. Instead, his nanovial cavities can be lined with antibodies specific to any protein of interest to measure secretions from individual cells, which are loaded into the nanovials.

“The real advantage is that these little particles, you can make millions of them. You can put them in a tube, and they can be transferred and easily used by others and be analyzed,” said Di Carlo. “It’s like a test tube for a single cell.”

Nanovials are small enough to fit through the channels of a single-cell RNA-seq platform without interfering with the normal sequencing process. This enables the transcriptomes of the cells inside to be analyzed. Nanovials can also work with methods for analyzing cell surface markers like flow cytometry. Researchers have linked the data on protein secretion, cell surface marker levels, and the transcriptomes of individual cells together using the same cell-loaded nanovial. The researchers named this method secretion-encoded single-cell sequencing (SEC-seq).

“This technology is exciting. It basically allows one to identify a function of a cell and then select it,” commented Frances Eun-Hyung Lee, a pulmonary immunologist at Emory University who was not involved in the study.

Once the nanovials were prepared, Di Carlo decided to test them on therapeutic samples. He contacted Richard James, an immunologist at the University of Washington and a co-author of the study, who develops B cell therapies. James’s team had been interested in identifying the attributes of B cells that make them most effective for therapeutic purposes, but they lacked the necessary technology to do so. The collaboration with Di Carlo proved to be exactly what James had hoped for.

“When Dino contacted me and I started reading a little bit about his nanovial technology, I got really excited about the idea of applying it to doing screens to find the right sequences to put in B cells to make them very productive and to really understand more about the biology of the cells in a way that couldn’t be done previously,” commented James.

James and his team coated the nanovials with antibodies targeting IgG, the most abundant class of antibody produced by human B cells. After capturing the secretions of the cells, they subjected the samples to flow cytometry and single-cell transcriptome sequencing.

Using SEC-seq, the team found exactly what they were looking for: surface markers associated with B cell subpopulations that secreted high levels of IgG and genes with upregulated expression in those same subpopulations. However, some of the results were surprising. The amount of IgG protein secreted by the cells did not correlate with transcription of the IgG gene but rather correlated with some other genes.

“It seems like there are pathways related to reactive oxygen species and oxidative phosphorylation that seem to be upregulated in the cells that are making more protein,” James remarked. “It’s not what we’d expect.”

James and Di Carlo are currently exploring these unexpected results in more depth to better understand the relationships between secreted proteins and their associated molecular signatures. They are also inspired to apply SEC-seq to other therapeutically relevant cell types. “This is the start of opening up this whole book… from the genome, all the way to function,” Di Carlo said. “That’s what’s exciting.”

This news is a creative derivative product from articles published in famous peer-reviewed journals and Govt reports:

1. Miwa H, et al. Single-cell sorting based on secreted products for functionally defined cell therapies. Microsystems Nanoeng. 2022;8(1):84.
2. Cheng RY-H, et al. SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells. Nat Commun. 2023;14(1):3567.
3. Slota M, et al. ELISpot for measuring human immune responses to vaccines. Expert Rev Vaccines. 2011;10(3):299-306.
4. Vogel, C. & Marcotte, E. M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 13, 227–232 (2012).
5. Peterson, V. M. et al. Multiplexed quantification of proteins and transcripts in single cells. Nat. Biotechnol. 35, 936–939 (2017).

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