Single cell transcriptomic analysis of prostate cancer cells

Area of Science:

• Molecular Biology
• Oncology
• Genomics

Background:

• Interrogating circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) is challenging due to low cell numbers.
• Prostate cancer (PCa) research requires methods to analyze single cells for transcriptomic profiling.

Purpose of the Study:

• To determine if commercially available technology can provide a transcriptomic profile from a single PCa cell.
• To assess the reliability and accuracy of single-cell transcriptomic analysis.

Main Methods:

• Clonally selected and cultured C4-2B PCa cells were synchronized for cell cycle.
• Single, 5-, and 10-cell populations were isolated using micromanipulation.
• RNA was amplified using the WT-Ovation™ One-Direct Amplification System and hybridized on a 44K Whole Human Gene Expression Microarray.
• Gene detection used a high stringency threshold (mean Alexa Fluor® 3 signal intensity > 300).
• Relative expression levels were validated using real-time PCR (RT-qPCR).

Main Results:

• Thousands of probes were positive on arrays from single-cell (17,009), 5-cell (20,423), and 10-cell (22,410) pools.
• Single-cell analysis demonstrated high sensitivity (0.814) and specificity (0.979) compared to 5-cell pools.
• Pearson correlation coefficients indicated strong agreement between single-cell and pooled samples (0.875 for 5-cell, 0.783 for 10-cell).
• DTCs from metastatic PCa patients yielded 16,039 positive probes, with cluster analysis grouping DTCs by patient.

Conclusions:

• A reliable transcriptomic profile can be obtained from a single cell using commercially available technology.
• While fewer genes are detected from single cells compared to pooled samples, the method is robust.
• This technique enables reliable transcriptomic profiling of DTCs from bone marrow in PCa patients.