Figure of a neutrophil cell. CREDIT: Blausen.com staff (2014). "Medical Gallery of Blausen Medical 2014." WikiJournal of Medicine 1 (2). DOI:10.15347/wjm/2014.010. ISSN 2002-4436.
Neutrophils are a crucial component of the innate immune system. They initiate the body’s first response to invading microorganisms by degranulation, phagocytosis, and producing Neutrophil Extracellular Traps (NETs). Neutrophils are essential and abundant in both acute and chronic inflammation and are, therefore, pivotal in research on infectious diseases, inflammation, potential therapeutic targets, and cancer.
This blog post will explore a comparative study recently published by the Roche Innovation Center. The study evaluated single-cell RNA sequencing methods, performing transcriptome profiling of neutrophils in large-scale clinical trials. We will also discuss how single-cell sequencing is reshaping drug discovery. Additionally, you will gain insights into our capabilities for conducting large-scale clinical trials.
What is the biological significance of neutrophils?
Neutrophils have a nuanced response specific to the type of pathogen and the circumstances, displayed through a plethora of reactive phenotypes. The complexity of neutrophil types requires a closer look at their gene activity. This helps us understand how they respond to different pathogens.
Recent single-cell RNA sequencing studies reveal diverse neutrophil states with potential for clinical application:
- Wigerblad et al. (2022) have identified four distinct transcriptomic states—Nh0, Nh1, Nh2, and Nh3—during neutrophil maturation and activation. Understanding these states could provide valuable disease biomarkers.
- Montaldo et al. (2022) further explored neutrophil transcriptomes in homeostatic and stressed states, finding activation signatures predictive of organ transplant success.
- Wu et al. (2024) highlighted heterogeneity in cancer-associated neutrophils. Contrary to initial beliefs, neutrophils with an antigen-presenting program correlated with positive outcomes.
- Leclercq et al. (2022) suggest neutrophil phenotyping might help identify patients at risk for cytokine release syndrome from T-cell therapies.
Neutrophil profiling is infamously difficult because of these main factors: short lifespan, sensitivity, and low RNA content. The presence of highly active endonucleases is essential to facilitate neutrophils’ primary role. Unfortunately, it also renders their own RNA content sensitive to these enzymes.
Study overview
Traditionally, single-cell RNA sequencing methods requirement for fresh cells poses challenges for sequencing clinical samples. Researchers require on-site single-cell analysis in these cases because neutrophils often die or change during the freeze-thaw process. The need for complex immediate processing, specialized expertise, and expensive equipment limits the number of clinical sites capable of collecting samples for single-cell RNA sequencing while maintaining high processing quality and delivering high-quality data.
Given the biological significance of neutrophils and the complexity of global clinical trials, there is a pressing need for an easy-to-use stabilization protocol for single-cell RNA sequencing to enable neutrophil profiling.
To address this, the Roche Innovation Center conducted a comparative study to evaluate three methods for neutrophil profiling. They compared the following methods:
Figure of the study overview. CREDIT: Hatje et al. (2024). Comparison of Fixed Single Cell RNA-seq Methods to Enable Transcriptome Profiling of Neutrophils in Clinical Samples. bioRxiv. DOI: 10.1101/2024.08.14.607767
The study assessed on-site sample preservation methods to minimize the loss of sensitive cells and ensure the quality of downstream single-cell RNA-seq data. The researchers benchmarked these methods against FACS and antibody staining to establish a ‘gold standard’ for cell type composition.
Key Findings
All three methods yielded high-quality data and effectively identified neutrophils. Nevertheless, the researchers determined that Flex was the optimal choice for collecting and processing clinical samples. They also noted that Evercode performed commendably.
The study found that Flex outperformed the other methods in several key areas:
- Best Sensitivity: Flex detected the highest number of genes. Additionally, it could differentiate between immature and mature neutrophils.
- Low-Stress Processing: Both Flex and Evercode showed low stress levels in neutrophils.
- High-Quality Data: Flex provided data that closely matched FACS sorting, making it the most reliable method.
- Rapid Fixation: Flex provided an optimal workflow for sample collection at clinical sites. After finishing this study, 10x Genomics updated this protocol. These changes further enhance its compatibility for whole blood fixation at collection sites.
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Clinical trials
The Flex method improves the ability to implement single-cell RNA sequencing in large clinical trials. This enhances drug discovery and the creation of new diagnostic tools and therapies. By comprehensively profiling all cell types within blood samples, we can generate invaluable data for FDA dossiers.
The increased sensitivity and accuracy of the Flex method enable a more detailed understanding of cellular heterogeneity and its implications for disease processes. These findings lay the groundwork for the emergence of personalized medicine and targeted interventions.
Our expertise in the successful execution of large-scale clinical trials
Single Cell Discoveries offers a robust platform for large-scale clinical trials by leveraging the Flex method's unique capabilities. Our automated workflows, rapid turnaround times, and experienced data analysts guarantee reliable, high-quality neutrophil profiling data. We also offer profiling of cryopreserved PBMCs and other immune cell types, ensuring comprehensive evaluation for concluded clinical trials. We invite you to collaborate with us on your clinical research projects.
Our services
This is what you can expect if you run a Flex project with us:
- Obtain single-cell transcriptomics data from FFPE, OCT, flash-frozen, and paraformaldehyde-fixed samples.
- Aim to target up to 1,024,000 fixed single cells simultaneously. Use a cost-effective flexible multiplexing workflow that fits your schedule.
- Choose the number of reads per cell for the Single Cell Gene Expression Flex Library: with as few as 10,000 reads per cell. Discuss with our specialists to find your option.
- We can run Flex projects for mice and human tissues. For other species, please refer to our services. We have experience in sequencing over 40 different organisms.
Click here to read a case study that shows how we implemented Flex to handle complex clinical samples.
References
1. Hatje, K., Schneider, K., Danilin, S., Koechl, F., Giroud, N., Juglair, L., Marbach, D., Knuckles, P., Bergauer, T., Metruccio, M., Garrido, A., Zhang, J.D., Sultan, M., Bell, E. (2024). Comparison of Fixed Single Cell RNA-seq Methods to Enable Transcriptome Profiling of Neutrophils in Clinical Samples. bioRxiv, 607767.
2. Leclercq, G., Servera, L.A., Danilin, S., Challier, J., Steinhoff, N., Bossen, C., Odermatt, A., Nicolini, V., Umana, P., Klein, C., et al. (2022). Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils. Oncoimmunology, 11, 2039432.
3. Montaldo, E., Lusito, E., Bianchessi, V., Caronni, N., Scala, S., Basso-Ricci, L., Cantaffa, C., Masserdotti, A., Barilaro, M., Barresi, S., et al. (2022). Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress. Nature Immunology, 23, 1470-1483.
4. Wigerblad, G., Cao, Q., Brooks, S., Naz, F., Gadkari, M., Jiang, K., Gupta, S., O'Neil, L., Dell'Orso, S., Kaplan, M.J., et al. (2022). Single-Cell Analysis Reveals the Range of Transcriptional States of Circulating Human Neutrophils. Journal of Immunology, 209, 772-782.
5. Wu, Y., Ma, J., Yang, X., Nan, F., Zhang, T., Ji, S., Rao, D., Feng, H., Gao, K., Gu, X., Jiang, S., Song, G., Pan, J., Zhang, M., Xu, Y., Zhang, S., Fan, Y., Wang, X., Zhou, J., Yang, L., Fan, J., Zhang, X., Gao, Q. (2024). Neutrophil profiling illuminates anti-tumor antigen-presenting potency. Cell, 187(6), 10.1016/j.cell.2024.02.005.
More information
Find more information on 10x Genomics, and Multiome, and our approach in our information guide.
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