# Spatial SILAC: Examining the Proteome in 3D Cell Cultures

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2022 · $375,728

## Abstract

Project Summary
The drug development process is inefficient and expensive, in part because most therapeutics are initially
screened with two-dimensional cell cultures. These cell cultures do not accurately reflect tissue structures in
the human body. To improve drug screening, more accurate model systems should be used. Three-
dimensional cell cultures, also known as spheroids, fill this gap. Similar to a tumor, colon cancer spheroids
contain radially symmetric nutrient and oxygen gradients. The spheroid develops distinct cellular populations
that reflect these chemical gradients. In this proposal, we are developing an analytical approach, called Spatial
SILAC, to specifically label the divergent cellular populations present within spheroids using different isotopes.
In Aim 1, we will show that these isotopes can be readily distinguished by a mass spectrometer, thus providing
an isotopic “zip code” of the cell’s origin within a spheroid. In Aim 2, we will combine imaging mass
spectrometry drug distribution studies with an evaluation of proteomic changes in response to therapeutics,
using the isotopic labels. We will evaluate irinotecan, 5-fluorouracil and Cetuximab as we have previously
examined the distribution of these three drugs in spheroids by imaging mass spectrometry. We will compare
the spatially localized proteomic changes in response to therapeutic treatment with the imaging distribution
maps. In Aim 3, we will apply our Spatial SILAC approach to screen newly developed cyclic penetrating
peptides (CPPs), designed by Co-I Professor Dehua Pei. These promising cancer drug candidates require
preclinical screening and Spatial SILAC in the spheroids provides an ideal testbed to evaluate their in vitro
pharmacokinetics and pharmacodynamics. We will first perform imaging mass spectrometry studies to
determine the distribution of the CPPs and their metabolites in the spheroids. We will then evaluate both the
targeted and off-targeted proteomic changes caused by these drugs by Spatial SILAC. The results generated
in this aim will be used to optimize the design of future CPPs. In summary, this proposal describes a novel
mass spectrometric approach that will streamline the drug development process, while also providing rich in
vitro pharmacokinetic and pharmacodynamic information on new therapeutic candidates.

## Key facts

- **NIH application ID:** 10475747
- **Project number:** 5R01GM110406-09
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Amanda B. Hummon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $375,728
- **Award type:** 5
- **Project period:** 2014-07-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10475747

## Citation

> US National Institutes of Health, RePORTER application 10475747, Spatial SILAC: Examining the Proteome in 3D Cell Cultures (5R01GM110406-09). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10475747. Licensed CC0.

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