Pathway Mapping

Whether it is for biomarker discovery, readout selection, or the characterization of disease models, a full profile of the intracellular signaling landscape can rapidly provide full characterization of cellular signaling responses.

Using this information, cell models can be compared to primary cells, disease samples can be compared to healthy donors, and costimulations can be compared to mono stimulations in the most all-inclusive, biologically relevant systems available.

Our Pathway Mapping platform surveys 100+ different signaling nodes to create high-resolution maps of cellular responses, disease states, and compound activity.

Our Pathway Mapping platform combined with traditional surface marker staining measures all readouts in each cell population at the single-cell level.

This unique capability allows different populations to be compared and heterogeneous responses to be identified across a wide range of readouts.

With years of expertise in phosphoflow, we extended our services to include the investigation of other important cell signaling pathway modulations such as acetylation and methylation.

Such single-cell analysis can be performed in various sample types, after treatment with small molecules, antibody therapeutics, or extracellular ligands to:

• Assess the phosphorylation, acetylation, and methylation status across a diverse range of signaling pathways
• Gain new insight into on- and off-target compound activities
• Benchmark competitor and tool compounds for efficacy and side effects
• Measure basal signaling profiles in cancer cells and healthy samples

We work with all types of samples (live, frozen, or fixed) and offer in-house sample procurement, preparation, stimulation, or stabilization if needed.

• PBMC (fresh or cryopreserved)
• Whole blood
• Dissociated Tumors/Tissue
• In vitro expanded primary cells
• Suspension or adherent cell lines
• Small molecule compounds
• Synthetic proteins/peptides
• Antibody therapeutics

Our Pathway Mapping platform is designed to provide comprehensive data across a wide range of stimulations.

To provide the most complete analysis possible, a full panel is available for applications that require a broad survey of signaling molecules, such as

• phenotypic screening deconvolution
• off-target screening
• pathway profiling
• and compound synergy studies.

Custom panels can be generated to fully characterize the responding populations with a select set of readouts.

These are designed from our full panel or using our focused panels set to investigate well-known signaling pathways (cytokine signaling, JAK/STAT, T cells, cell stress/apoptosis).

This makes for a completely customizable solution ideal for routine profiling, inhibitor studies, clinical studies, and medicinal chemistry programs.

Compound profiling information obtained can further be used to evaluate structure/activity relationships (SAR) and mechanisms of action (MoA).

The readouts encompassed by the Pathway Mapping platform have been hand-selected based on years of experience in intracellular flow cytometry to provide the richest and most robust data sets possible.

The readouts are focused on intracellular signaling nodes that integrate multiple signaling pathways and cover the broadest set of signaling responses.

These readouts have been selected and characterized for their responsiveness, assay performance, biological relevance, and specificity.

Generating a clustering analysis of specific signaling pathways following various cytokine or polyclonal stimulations is possible with our Pathway Mapping services.

Through various stimulations, we can trigger as few or as many pathways as desired. Specific or broad stimulations are used to determine the ability of a drug to inhibit pathways.

The resulting mapping generates an easy-to-interpret and visual assessment of changes in cells.

Readouts obtained from mapping analysis can be quantified. The changes in the frequency of intracellular responses given an input stimulation can help understand selective inhibition and off-target activity of compounds.

From this analysis, potency of compounds against select targets can be evaluated.

Figure legend: Various stimulation methods have been evaluated to understand the modulation of pathway-specific readouts using flow cytometry. A selective stimulation defines modulations of pathway-specific readouts (e.g. IFN signaling) and a broad stimulation leads to pleiotropic readouts (e.g. PMA).

Our Pathway Mapping service can provide insights into specific signaling pathways targeted by drug treatment.

Mapping drug activity across cellular signaling networks and identifying heterogeneous responses across a wide range of readouts increases chances of identifying a new drug and its mechanism of action.

Creating a map of drug activity across cellular signaling networks can be used for:

• Competitor benchmarking
• Tool compound validation
• Phenotyping screening deconvolution
• Detailed compound characterization

For instance, in the image below, PI3K inhibitors and BTK inhibitors exhibit marked similarities in their pattern of inhibition.

However, the MEK 1/2 inhibitor (AZD-6244) exhibited a completely different pattern of inhibition downstream of MAPK signaling.

Figure legend: BTK, PI3K and MEK1/2 inhibitors were evaluated across the Pathway Mapping analysis tool on B cells. The ability of a compound to modulate signaling pathways in B cells was evaluated on isolated PBMC followed by stimulation with anti-IgM. Readouts were clustered based on profiles of readouts.

Multiple readouts are obtained from our Pathway Mapping platform and allow for detailed compound characterization including pharmacokinetic profiling (e.i. EC50).

Using various amounts of stimulation, the readouts of each specific pathways can be assessed.

Figure Legend: Various treatment doses (X axis) used to stimulation different signaling pathways (colored lines).

Our technology platform is empowered by proprietary sample processing techniques designed to reduce sample variability and dramatically speed up data acquisition.

By miniaturizing the reaction volumes and automating the sample processing, high-content data can be delivered in a fraction of the time and cost of traditional systems.

This allows for exploring variables and dose responses to be compared quantitatively and reproducibly.