# Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance

> **NIH NIH R44** · ISOPLEXIS, INC. · 2020 · $961,259

## Abstract

Although signal transduction inhibitors occasionally offer clinical benefit for cancer patients, signal flux emanating
from oncogenes is often distributed through multiple pathways, potentially underlying the resistance which
causes failure of most such inhibitors. Measuring signal flux through multiple pathways, in response to signal
transduction inhibitors, may help uncover network inter- actions that contribute to therapeutic resistance and that
are not predicted by analyzing pathways in isolation. Protein–protein interactions within signaling pathways are
often elucidated by assessing the levels of relevant pathway proteins in model and tumor-derived cell lines and
with various genetic and molecular perturbations. Such interactions, and the implied signaling networks, may
also be elucidated via quantitative measurements of multiple pathway-related proteins within single cells. At the
single-cell level, inhibitory and activating protein–protein relationships, as well as stochastic (single-cell)
fluctuations, are revealed. However, most techniques for profiling signaling pathways require large numbers of
cells, and bulk measurements have proven insufficient to detect secondary pathways post resistance. Single-
cell immunostaining is promising, and some flow cytometry techniques are relevant, yet limited in finding possible
pathways due to intracellular multiplexing limitations.
 We describe quantitative, multiplex assays of intracellular signaling proteins from single cancer cells using a
platform called the single-cell barcode chip (SCBC). The SCBC is simple in concept: A single or defined number
of cells is isolated within a microchamber that contains a sensitive antibody array specific for the capture and
detection of a panel of proteins. The SCBC design permits lysis of each individual trapped cell. Intracellular
staining flow cytometry can assay up to 11 phosphoproteins from single cells. Our SCBC can profile a
significantly larger panel (up to 90 different phosphoproteins) with ~2500 single cells per chip for a statistically
representative analysis of the sample population. This new high multi-plexed single cell phosphoproteomics
analysis tool provides an analytical approach for detecting changes in signal coordination by monitoring
phosphoproteins, on a much larger scale. This approach may identify actionable alterations in signal coordination
that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-
obvious drug combinations. SPECIFIC AIM 1: Develop a robust microchamber array flow cell that can be easily
incorporated into larger automated workflow device for analysis of intracellular protein targets. SPECIFIC AIM
2: Double multiplexing capability of high-density barcode SCBC chip by monitoring both intracellular proteins
and metabolites simultaneously. Perform single-cell 32-plex measurement for more comprehensive GBM
pathway analysis. SPECIFIC AIM 3: Improve consumable to perform “flow...

## Key facts

- **NIH application ID:** 9980309
- **Project number:** 5R44CA224505-03
- **Recipient organization:** ISOPLEXIS, INC.
- **Principal Investigator:** Timothy S McConnell
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $961,259
- **Award type:** 5
- **Project period:** 2018-09-12 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9980309

## Citation

> US National Institutes of Health, RePORTER application 9980309, Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance (5R44CA224505-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9980309. Licensed CC0.

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