The Power of Multi-Omics at the Single-Cell Level

There are an estimated 37 trillion cells in the human body, and no two are exactly alike. This heterogeneity within each patient further complicates cell analysis as well as the development and optimization of immune therapies. Single-cell analysis is a relatively new technology that has revolutionized the ability of researchers to study variation within a population of cells (e.g. organs, tissues, and cell cultures). IsoPlexis’ single-cell proteomics, for example, has allowed researchers to identify the specific cytokines each single cell is secreting to identify highly functional subsets of cells that can accelerate the development of vaccines and therapeutics.

The latest advance in the field of single-cell analysis is single-cell multi-omics—just last year, Nature Methods chose it as their Method of the Year “for its great impact in revealing cell functions, discovering relationships across ‘omes, and recording dynamic biological events.”

What is Single-Cell Multi-Omics?

Individually, high-throughput analyses of -omes—genomes, transcriptomes, proteomes, and metabolomes—of single cells can offer key insights into gene expression, disease pathogenesis, and other cellular processes. Analyzing several -omes at once provides even more comprehensive cell profiles. Single-cell multi-omics allows researchers to obtain multiple large -omics datasets from individual cells. 

IsoPlexis has recently demonstrated the ability to provide this type of data as depicted in the image below. Single-cell secretomics was used by researchers Parisi, et al. to identify mechanisms of immune persistence in solid tumors to overcome tumor-immune challenges. IsoPlexis’ technology found that a novel kinetically engineered IL-2 receptor agonist had the potential to improve the anti-tumor activity of adoptive cell transfer (ACT) therapy in humans by increasing in vivo expansion and polyfunctionality of the adoptively transferred T cells.

In another study, researchers Su, et al. used IsoPlexis’ single-cell intracellular proteomics and single-cell metabolomics to measure proteins and metabolites inside single mutant melanoma cancer cells to gain insights into what causes the cells to transition from a drug responsive to drug resistant state. IsoPlexis’ technology revealed that cancer cells can take multiple paths to drug resistance with each path exhibiting unique susceptibilities that will help researchers develop new combination therapies to combat drug resistance.

IsoPlexis’ technology bridges the gap left by using genomics alone. IsoPlexis’ functional phenotyping technology 1) identifies and characterizes the true cytokines each single cell is secreting, 2) identifies and characterizes the intracellularly expressed proteins, and 3) measures functional energy states and adaptive resistance pathways in the metabolome.

Technologies and Challenges

Single-cell multi-omics traditionally requires a number of instruments to perform each of the separate layers of analysis. Even within a given modality, the traditional workflow for single-cell analysis is complicated with multiple instrumentation required. Several manual workflow steps are traditionally required as well—capture antibody binding, adding sample, adding detection antibody, washing the microplate, adding substrate, reading the microplate, and calculating results. Each additional -ome, such as metabolomics, lengthens the workflow steps even further.

A key limitation to most single-cell technologies is that they are not capable of performing functional analyses at the single cell level. For instance, there are several tools on the market today that are designed to analyze cellular function. Enzyme-linked immune absorbent spot (ELISpot) assays measure cytokine secretion but are limited to measuring only a few cytokines per cell. Flow cytometry detects proteins on the cell’s surface, only a few at a time, and the cells must be fixed and permeabilized and the sample blocked due to a high level of non-specific binding. While the resulting data may provide basic information on function, they do not provide full functional insights. Other technologies take more of a brute force approach with obliteration of the cell followed by mass spectrometry to identify unknown proteins.

A Hub for Functional Multi-Omic Analyses

IsoPlexis’ technology allows researchers to perform deep functional multi-omic analyses on the secretome, phosphoproteome, and metabolome at single-cell and population resolution, using very small sample sizes, on a single piece of equipment. The system that accomplishes this is the IsoLight—a hub for comprehensive functional cell profiling across a large assay menu of chip and software products. With the IsoLight system, each chip family offers several different panels of analytes with proteomic barcoding technology. Looking at any particular -ome is simply a matter of loading the appropriate chip. 

IsoPlexis’ automated proteomics workflow uses one system to measure cytokines in the secretome, phosphoproteome, and metabolome, with more solutions to come.

IMPACTS OF SINGLE-CELL MULTI-OMICS

Tackling Solid Tumors

In the realm of therapeutics, IsoPlexis’ single-cell technology has been used in the preclinical development of novel CAR-Ts for solid tumors by providing functional and direct proteomic profiling of single CAR-T cells for functional quality analytics. The potency of T cells and other immune cell subsets was found to correlate with clinical outcomes of CAR-T cell therapy.

IsoPlexis’ platform easily fits into existing cell therapy workflows. When engineering cells or editing with technologies like CRISPR, IsoPlexis’ platform can confirm the true function of the cell therapy product prior to re-introducing it to the patient, ensuring potency and efficacy pre-infusion.

While progress in immuno-oncology is improving the clinical outcomes for many patients with malignant tumors, leading to remission and long-term survival for some patients, a portion of patients experience tumor recurrence or simply do not respond to treatments. With single-cell analysis, researchers can stratify pre-clinical and clinical populations into responders and non-responders based on the level of functionality in their immune cells. Case studies show that increased levels of highly functional immune cells in responders correlate with greater success rates of treatment. Researchers can then use this information to determine out of several drug candidates which will be most effective prior to moving to clinical trials.  

Fighting Infectious Diseases 

IsoPlexis’ technology is also helping to reveal the mechanisms of immune response in infectious diseases, which has been a particularly crucial task during the current pandemic. Functional immune profiling at the single-cell level is showing researchers how different subsets of immune cells function within infected patients. Recent research has focused on the cytokine storm response to COVID-19, highlighting the correlation of high levels of circulating inflammatory cytokines and severity of illness. For example, one study revealed the association between immune cell dysfunction and the inflammation cascades and cytokine storms that occur in some patients infected with COVID-19.

In a recent publication in BMC Neurology, researchers Farhadian, et al utilized IsoPlexis’ platform to analyze plasma and CSF of a COVID-19 patient who initially presented with neurological symptoms, identifying key inflammatory signatures.

Another group of researchers is examining the role of cytokines and neuroinflammation in COVID-19 patients with neurological manifestations. IsoPlexis’ functional proteomics revealed that patients presenting with neurological complications, such as seizure and confusion, exhibited elevated levels of cytokines such as IL-6 and IL-8. A unique finding was a key inflammatory signature identified only in cerebrospinal fluid (CSF) when COVID-19 infection was only found in patient plasma, not CSF. It is significant that inflammation was identified in CSF despite absence of viral presence, further validating the importance of cellular and cytokine level monitoring for predicting toxicities in infectious diseases, such as those related to cytokine storm seen in many COVID-19 patients. Predicting which patients are likely to experience cytokine storm can help researchers formulate better therapeutics and potentially prevent the response from occurring. 

Outside of COVID-19, IsoPlexis’ technology has been instrumental in infectious disease research and vaccine development. For example, IsoPlexis’ multi-omics technology has aided in the development of malaria vaccines, understanding of the rapid innate immune response, and uncovering of immune functional states in response to pathogenic stimulation.

Conclusion

Several single-cell technologies are available on the market, but IsoPlexis sets itself apart with its deep functional single-cell multi-omics capabilities for analysis of the secretome, phosphoproteome, and metabolome, automated on the same system. As sequencing technologies continue to advance, the IsoLight proteomics hub makes it possible to profile cells in multiple modalities on a single system—an advance that is making waves in cancer, infectious disease research, and beyond.

Learn more at https://isoplexis.com/