Single-cell RNA sequencing in thyroid cancer: a methodological review and thyroid specific dissociation protocol

Area of Science:

• Endocrinology
• Oncology
• Genomics

Background:

• Thyroid cancer is the most common endocrine malignancy, with high-grade subtypes exhibiting aggressive behavior and poor survival.
• Current treatments like multikinase inhibitors offer limited durability for radioactive iodine-refractory disease.
• Understanding molecular differences between thyroid cancer subtypes is crucial but hindered by limited high-resolution data.

Purpose of the Study:

• To critically evaluate existing single-cell RNA sequencing (scRNA-seq) studies in thyroid cancer.
• To identify methodological gaps in current scRNA-seq applications for thyroid cancer.
• To develop and present an optimized protocol for generating high-quality single-cell suspensions from thyroid specimens for scRNA-seq.

Main Methods:

• A focused literature search of PubMed and Embase identified scRNA-seq studies of primary thyroid tumors (2020-2025).
• Methodological details of tissue dissociation were assessed in 22 selected studies.
• An optimized thyroid tissue dissociation protocol was developed, replacing RBC lysis with immunomagnetic depletion.

Main Results:

• Many published studies lacked detailed methods for tissue dissociation.
• The optimized protocol improved cell recovery and viability compared to standard lysis in limited specimens.
• The protocol yields high-viability single-cell suspensions compatible with droplet-based scRNA-seq platforms.

Conclusions:

• The optimized dissociation protocol is practical for precious patient-derived thyroid samples.
• Improved single-cell data quality and cellular representation facilitate biomarker discovery for disease progression and treatment resistance.
• This approach supports the identification of novel therapeutic targets across diverse thyroid cancer subtypes.