Optimized protocol for processing murine tumor-bearing lung tissue for flow cytometry and single cell RNA-sequencing.

Area of Science:

• Molecular Biology
• Oncology
• Genomics

Background:

• Non-small cell lung cancer (NSCLC) is the most common lung cancer, with KRAS mutations frequent.
• Tumor heterogeneity and plasticity in NSCLC remain poorly understood.
• Single-cell RNA sequencing (scRNA-seq) is vital for studying cellular heterogeneity.

Purpose of the Study:

• To develop a robust protocol for isolating high-quality single-cell suspensions from healthy and tumor-bearing mouse lungs.
• To enable effective scRNA-seq analysis of lung cancer models.
• To facilitate the study of NSCLC heterogeneity and Kras-driven tumorigenesis.

Main Methods:

• Detailed protocol for single-cell suspension preparation from mouse lungs.
• Includes tissue perfusion, enzymatic digestion, mechanical dissociation, RBC lysis, and filtration.
• Utilizes a Kras^G12D; tdTomato reporter mouse model for tumor cell identification.

Main Results:

• Achieved >85% viable single cells suitable for scRNA-seq.
• Successfully isolated tdTomato+ tumor cells using Fluorescence-Activated Cell Sorting (FACS).
• Demonstrated distinct tumor cell clusters in scRNA-seq analysis.

Conclusions:

• The developed protocol yields high-quality single-cell suspensions from complex lung tissues.
• This method is essential for advancing scRNA-seq studies in NSCLC research.
• Enables detailed investigation of Kras-mutant lung cancer biology at the single-cell level.