A Multi-Omic Tour: Three Nature Publications on How Cells Communicate Via IsoPlexis’ Proteomics

Multi-Omic Technology from IsoPlexis Highlighted in Several Nature Communications Publications

Three of IsoPlexis’ core functional phenotyping products were recently featured in three different Nature Communications publications: CodePlex Secretome, Single-Cell Secretome, and Single-Cell Intracellular Proteome. Each of these solutions has enabled researchers to gain insight into their samples to resolve heterogeneity, identify the functional phenotype of both tumor and immune cells, as well as analyze cells communicating in the tumor microenvironment. IsoPlexis’ platform provides multi-omic solutions with just one system, automating and simplifying workflows.

Recently, Nature Methods highlighted single-cell multimodal omics as its method of the year, “For its great impact in revealing cell functions, discovering relationships across -omes, and recording dynamic biological events.”1

While there are several single-cell high throughput assays that exist today, Nature Methods discusses the limitations of the types of omics that current technologies can measure. IsoPlexis is the only company capable of deep functional multi-omics or full functional proteomics: extracellular, intracellular, and metabolomics at single-cell and population resolution, using very small sample sizes. And, this is being done all on one fully automated system, the IsoLight.

Multi Omics Products

Understanding Cancer Cell Communication to Infer a Strategy to Inhibit Tumor Cell Migration with CodePlex Secretome

Cancer cells in the tumor microenvironment secrete cytokines that interact with other cells, such as immune cells, in a 3D extracellular matrix, which facilitates intracellular communications and jointly moderates pathophysiological processes, “including cancer-induced angiogenesis and metastasis.”2 Secretomic profiles of cancer cells advance as they multiply and become more invasive, which suggests that secreted factors may be associated with proliferation and migration. In a Nature Communications publication, researchers Jayatilaka, et al. found that the local microenvironment affects how cells migrate after studying cells in a natural, 3D environment as opposed to the traditional 2D method. They believed that the involvement of proliferation-induced local gathering and the increased local cell density of the 3D tumor microenvironment could be significant, but that there were unidentified factors directly changing tumor cell migration.2

Cytokine profiling in the tumor microenvironment, as provided by IsoPlexis’ CodePlex Secretome technology, can provide predictive data that correlate with cell behavior. After confirming that the soluble molecules were promoting cell migration, these researchers used CodePlex Secretome technology to identify what factor or factors were driving this increased mobility. This technology allowed researchers to measure and analyze the secretomic profiles of the HT1080 and MDA-MB-321 cells ingrained in 3D matrices at both low and high densities with the multiplex antibody microarray assay, which simultaneously measured the concentration of 24 soluble molecules. Because cytokines IL-6 and IL-8 were secreted at high concentrations in a specific ratio, but proteins typically associated with promoting tumor metastasis and progression were not elevated, it is likely that both of these cytokines are responsible for driving the density-dependent cell migration within 3D matrices.2 Overall, this study revealed a possible mechanism promoting tumor cell migration while inferring an approach to reduce metastatic capability of tumor cells.

Single-Cell Secretome Identifies Mechanisms of Immune Persistence in Solid Tumor to Overcome Tumor-Immune Challenges

IsoPlexis’ Single-Cell Secretome solution has been used to identify correlates to anti-tumor response, immune suppression, and persistence in various studies. Researchers Parisi, et al. published their groundbreaking findings in Nature Communications, highlighting IsoPlexis’ functional phenotyping and polyfunctionality as correlates to response.3

Researchers used IsoPlexis’ Single-Cell Secretome solution to profile translational insights between early stage development of combination PEG-IL2 (their novel kinetically engineered IL-2 receptor agonist) with ACT therapy, and clinical impact on various immune cell types. In response to PEG-IL2, mouse immune cells were found to be polyfunctional, which correlated to increased proliferation, homing, and persistence of anti-tumor T cells in vivo.3 This mechanism resulted in superior anti-tumor activity of a B16-F10 murine melanoma model as well as the promotion of polyfunctional T and NK cells in peripheral blood of patients with melanoma in a phase 1 clinical trial. In patients, the circulating NK cell response and clear mechanistic upregulation of polyfunctional cell subsets with anti-tumor associated protein secretions across melanoma patients profiled in the clinic.3

In the mouse model, mice treated with PEG-IL2 recruited adoptively transferred cells into tumors, which were found to be highly polyfunctional, delivering high concentrations of functional cytokines per cell. Reported in clinical trials, these highly functional immune cells were found to be highly persistent and sustained a longer anti-tumor response.3

Single-Cell Intracellular Proteome Used for Multi-Omic Analysis of Signaling, Phenotypic, and Metabolic Regulators to Overcome Drug Resistance in Solid Tumor

A major challenge in treating many types of cancer is dealing with the cancer cell’s ability to develop drug resistance. While there have been many studies focused on gene expression in cancer cells, those changes can take time and research has shown that some cells can become resistant in a matter of days.

In order to gain true insight, tumor cells must be analyzed at the single-cell level to detect all possible paths to resistance. Previous studies have not been able to look at single cells or connect the two cell states. The first changes during drug adaptation are metabolic, and because of the importance of phosphorylated protein signaling in this process, it is critical to analyze single-cell functional phenotypic data on the intracellular proteome.

Researchers Su, et al. used IsoPlexis’ Single-Cell Intracellular Proteome technology in their recently published paper in Nature Communications to measure proteins and metabolites inside single cells, for the first time. Su, et al. used IsoPlexis’ proteomic barcoding technology to analyze mutant melanoma cancer cells (BRAFV600E M397) to gain further information about this transition from drug responsive to drug tolerant, as these cancer cells have demonstrated the ability to quickly become resistant to targeted inhibitors.4

M397 cell cultures were analyzed each day after BRAFi treatment for five days. IsoPlexis’ panel was designed to simultaneously capture phosphoproteins and metabolites from single cells to get a snapshot of how these cells were functioning each day. A panel consisting of phenotypic markers and markers of oncogenic signaling, cell proliferation, and metabolic activity, which are all transformed in the preliminary drug response, was used to analyze single cells to resolve “the complex cell-state space traversed by the cells during the first few days of BRAFi treatment.”4

Single-cell functional phenotyping revealed that many markers show a rise in variation at day 3 (D3), which suggests that there are one or possibly more cell state changes at this point in time.4 By D5, most of the cells go into a state of senescence, but do not experience any increase in apoptotic cell death. At this same time, there is an increase in the variation and abundance of Slug, the epithelial-mesenchymal transition-related transcription factor. This shows that some of the cells are beginning to trend toward the mesenchymal phenotype at this stage. Also, the factors generally associated with drug resistance, such as AXL, N-cadherin, NGFR, and TNFR, were elevated on D5.

Their research showed that cancer cells can take multiple paths to drug resistance, which makes targeting the resistance mechanism even more difficult. However, drug susceptibilities were identified for both courses, one responded to inhibiting the glycolysis enzyme PKM2, and the other responded to the inhibition of NFκB-p65. Single-cell analysis did show that the invasive melanoma cells (MITF-Low) depend on NFκB signaling. While inhibiting both pathways showed significant tumor growth inhibition, because they are so essential to normal cells, the fear is that toxicity in non-malignant tissue will arise.4 So, researchers Su, et al. realized that an “increased dosage of BRAFi from the current cytostatic level to a cytotoxic level may eliminate the MITF-High subpopulation and its respective path.”4 These data can now enable researchers to develop combination therapies to combat this common drug-resistant cell state.

To learn more about IsoPlexis’ full product suite and how each solution can help your research programs, download our Product Catalog here.

References:

  1. Method of the Year 2019: Single-cell multimodal omics. Nature Methods 17: 1, 2020.
  2. Jayatilaka H, et al. Synergistic IL-6 and IL-8 paracrine signalling pathway infers a strategy to inhibit tumour cell migration. Nature Communications8: 15584, 2017.
  3. Parisi G, et al. Persistence of Adoptively Transferred T cells with a Kinetically Engineered IL-2 Receptor Agonist. Nature Communications 11: 660, 2020.
  4. Su Y, et al. Multi-omic single-cell snapshots reveal multiple independent trajectories to drug tolerance in a melanoma cell line. Nature Communications 11: 2345, 2020.
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