Proteomics

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Spatial SILAC Regorafenib Treated Bottom-up Proteomic Analysis


ABSTRACT: Developing effective model systems to evaluate new potential chemotherapeutic reagents is critical. Three-dimensional cell cultures, such as spheroids, aid in bridging the gap between commonly used monolayer cell cultures and significantly more complex and expensive animal models. Spheroid model systems contain pathophysiological and chemical gradients similar to an in vivo tumor, which ultimately form distinct cellular subpopulations. In the spatial SILAC model, labels are pulsed into the spheroid at discrete time windows during development. These media pulses result in labeled proteins in distinct regions of the spheroid, which allows for tracing of quantitative proteomic changes to be correlated to different cellular subregions. In this study, we use the Spatial SILAC model to evaluate the proteomic response of a colon carcinoma spheroid to the multikinase inhibitor Regorafenib. We determined regorafenib to be an effective kinase inhibitor which significantly altered whole spheroid proliferation and resulted in increased apoptosis when the signal was averaged for all cells in the culture. However, when regorafenib treatment is more closely examined among the cellular subpopulations, drastic differences are observed for how the drug impacted the proteome of the necrotic spheroid core and the proliferating outer regions. Whole spheroid and outer region analysis shows that regorafenib treatment inhibited critical pathways such as mTOR signaling, ERK/MAPK signaling, and colorectal cancer metastasis signaling. However, analysis of the core shows that regorafenib had an entirely different effect, including upregulation of MAPK1 and KRAS, possibly indicating drug resistance within these late apoptotic cells. Ultimately, these combinatory studies can be used to further understanding of drug metabolism is different cellular subpopulations and provide valuable information to ultimately improve the accuracy of therapeutic testing.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Nicole Beller  

LAB HEAD: Dr. Amanda Hummon

PROVIDER: PXD038958 | Pride | 2024-08-09

REPOSITORIES: Pride

Dataset's files

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051122_Sample_1.raw Raw
051122_Sample_10.raw Raw
051122_Sample_11.raw Raw
051122_Sample_12.raw Raw
051122_Sample_16.raw Raw
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Publications

Evaluating the Pharmacokinetics and Pharmacodynamics of Chemotherapeutics within a Spatial SILAC-Labeled Spheroid Model System.

Beller Nicole C NC   Wang Yijia Y   Hummon Amanda B AB  

Analytical chemistry 20230718 30


Tumors have considerable cellular heterogeneity that is impossible to explore with simple cell cultures. Spheroid cultures contain pathophysiological and chemical gradients similar to in vivo tumors and show complex responses to therapeutics, similar to a tumor. Using pulsed isotopic labels, we demonstrate the pronounced differential response of the proteome to the drug Regorafenib, a multikinase inhibitor, in HCT 116 spheroids. Regorafenib treatment of outer spheroids inhibits proteins involved  ...[more]

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