Optimized Preparation of Whole Murine Tumor-Bearing Lung Tissue for Flow Cytometry and Single-Cell RNA-Sequencing

Area of Science:

• Oncology
• Genomics
• Biotechnology

Background:

• Non-small cell lung cancer (NSCLC) is the most common lung cancer, often driven by KRAS mutations.
• Understanding NSCLC heterogeneity is crucial for advancing treatment, but isolating viable single cells from tumors is challenging.
• Single-cell RNA sequencing (scRNA-seq) is vital for studying cellular heterogeneity but requires high-quality single-cell suspensions.

Purpose of the Study:

• To develop and present a detailed protocol for isolating high-quality single-cell suspensions from healthy and tumor-bearing mouse lungs.
• To optimize single-cell preparation for scRNA-seq analysis of Kras-mutant lung tumors.
• To enable the study of cellular heterogeneity and plasticity in NSCLC models.

Main Methods:

• Developed a step-by-step protocol involving tissue perfusion, enzymatic digestion, mechanical dissociation, RBC lysis, and filtration.
• Utilized a Kras^G12D; tdTomato reporter mouse model for isolating tumor cells.
• Incorporated FACS for efficient isolation of tdTomato+ tumor cells prior to scRNA-seq.

Main Results:

• >85% viable cells were obtained using the optimized protocol.
• The protocol successfully generated high-quality single-cell suspensions from both healthy and tumor-bearing lung tissues.
• scRNA-seq analysis successfully detected distinct tumor cell clusters.

Conclusions:

• The presented protocol enables the isolation of high-viability single cells from mouse lung tissue, suitable for scRNA-seq.
• This method facilitates the comprehensive analysis of cellular heterogeneity in Kras-driven lung cancer models.
• The protocol is essential for advancing research into NSCLC plasticity and treatment strategies.