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Development of Spheroid-FPOP: An In-Cell Protein Footprinting Method for 3D Tumor Spheroids
Raquel L Shortt1, Yijia Wang2, Amanda B Hummon2
1Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, United States.
Abstract:
Many cancer drugs fail at treating solid epithelial tumors with hypoxia and insufficient drug penetration thought to be contributing factors to the observed chemoresistance. Owing to this, it is imperative to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address this, we developed a mass spectrometry-based protein footprinting method for exploring the impact of hypoxia on protein in 3D colorectal cancer cells. Our group has previously extended the protein footprinting method fast photochemical oxidation of proteins (FPOP) for live cell analysis (IC-FPOP); however, this is the first application of IC-FPOP in a 3D cancer model. In this study, we perform IC-FPOP on intact spheroids (Spheroid-FPOP) using a modified version of the static platform incubator with an XY movable stage (PIXY) FPOP platform. We detected modification in each of three spheroid layers, even the hypoxic core. Pathway analysis revealed protein modifications in over 10 distinct protein pathways, including some involved in protein ubiquitination; a process modulated in cancer pathologies. These results demonstrate the feasibility of Spheroid-FPOP to be utilized as a tool to interrogate protein interactions within a native tumor microenvironment.
Insights
We developed Spheroid-FPOP, a novel mass spectrometry method to study protein changes in 3D cancer models under hypoxia. This technique reveals how tumor microenvironments impact protein interactions, aiding drug development for chemoresistant solid tumors.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Biology
Background:
- Solid epithelial tumors often exhibit hypoxia and poor drug penetration, contributing to chemoresistance.
- Evaluating cancer drugs in physiologically relevant conditions is crucial but challenging.
- Existing methods struggle to assess molecular changes within the complex tumor microenvironment.
Purpose of the Study:
- To develop and validate a mass spectrometry-based method for analyzing protein modifications in 3D colorectal cancer models under hypoxic conditions.
- To investigate the impact of hypoxia on protein interactions within intact tumor spheroids.
Main Methods:
- Adaptation of the fast photochemical oxidation of proteins (FPOP) technique for live cell analysis (IC-FPOP) in a 3D cancer model.
- Application of intact spheroid FPOP (Spheroid-FPOP) using a modified PIXY FPOP platform.
- Detection of protein modifications across multiple layers of tumor spheroids, including the hypoxic core.
Main Results:
- Successfully detected protein modifications in all analyzed layers of the 3D colorectal cancer spheroids, including the hypoxic core.
- Pathway analysis identified over 10 distinct protein pathways affected by hypoxia, including those involved in protein ubiquitination.
- Demonstrated the feasibility of Spheroid-FPOP to probe protein interactions within a native tumor microenvironment.
Conclusions:
- Spheroid-FPOP is a viable tool for interrogating protein dynamics in complex, native tumor microenvironments.
- This method can provide insights into chemoresistance mechanisms driven by tumor hypoxia.
- Facilitates the evaluation of potential cancer drugs under more realistic *in vivo*-like conditions.
