# Single-Cell Protein Activity Profiling in Breast Cancer Cells and Tissues

> **NIH NIH R33** · UNIVERSITY OF CHICAGO · 2022 · $373,510

## Abstract

Project Summary
Advances in molecular profiling technologies have enabled the identification of ever-increasing numbers of
disease biomarkers, each raising the potential for more precise, and possibly even patient-specific, diagnoses.
Current methods of quantifying protein biomarkers, however, are slow and laborious, often require large
quantities of biopsied tissues, and ultimately fail to provide a complete, intact quantification of functional protein
activity. To address these limitations, we have developed an innovative activity dependent proximity ligation
platform (ADPL), with the ultimate goal of obtaining multiplexed, ultrasensitive quantification of protein activity
directly in complex biological samples. In the context of cancer specimens, we hypothesize that defining a
molecular profile based on protein activity – the end result of biologic signaling – can more precisely and
accurately inform diagnoses and treatment regimes, allowing early intervention and improving clinical outcomes.
This proposal outlines the advanced development and validation of two reliable, high-resolution ADPL-based
platforms, with the power to alternately visualize and quantify dozens of active enzymes simultaneously from
tumor tissue, cells and biofluids. Our current knowledge and expertise in the use of this integrated platform will
be leveraged in the service of the following aims: 1) development of novel chemoproteomic probes for
multiplexed, family-wide activity mapping of cancer-relevant enzymes, including kinases; 2) advanced
development of on-tissue Exchange-ADPL and on-chip single-cell ADPL (scADPL) platforms for simultaneous,
high resolution visualization and quantification of a panel of active enzymes in situ; and 3) applying these
technologies to breast cancer tissues and cells to create accurate and precise activity signatures and identify
potential correlations between these signatures and clinical outcomes. To accomplish these aims we will develop
new chemical proteomic molecular probes, validate a panel of ADPL reagents for cancer-associated enzyme
biomarkers, and perform rigorous, metric-driven benchmarking analyses in cell lines and tumor tissues.
Completion of this project will validate ADPL platforms for use in diverse cancers, provide the first single-cell
activity profiles of breast tumors, and provide novel correlations between breast cancer tumor activity signatures,
disease severity and response to therapy.

## Key facts

- **NIH application ID:** 10413516
- **Project number:** 1R33CA269094-01
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Raymond E Moellering
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $373,510
- **Award type:** 1
- **Project period:** 2022-06-08 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10413516

## Citation

> US National Institutes of Health, RePORTER application 10413516, Single-Cell Protein Activity Profiling in Breast Cancer Cells and Tissues (1R33CA269094-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10413516. Licensed CC0.

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