A novel method for the isolation of single cells mimicking circulating tumour cells adhered on Smart Bio Surface slides by Laser Capture Microdissection

Abstract

In recent years, the importance of isolating single cells from blood circulation for several applications, such as non-invasive tumour diagnosis, the monitoring of minimal residual disease, and the analysis of circulating fetal cells for prenatal diagnosis, urged the need to set up innovative methods. For such applications, different methods were developed. All show some weaknesses, especially a limited sensitivity, and specificity. Here we present a new method for isolating a single or a limited number of cells adhered to SBS slides (Tethis S.p.a.) (a glass slide coated with Nanostructured Titanium Dioxide) by Laser Capture Microdissection (LCM) and subsequent Whole Genome Amplification. SBS slides have been shown to have an optimal performance in immobilizing circulating tumour cells (CTCs) from early breast cancer patients. In this work, we spiked cancer cells in blood samples to mimic CTCs. By defining laser parameters to cut intact samples, we were able to isolate genetically intact single cells. We demonstrate that SBS slides are optimally suited for isolating cells using LCM and that this method provides high-quality DNA, ideal for gene-specific assays such as PCR and Sanger sequencing for mutation analysis.

Fig 1. Single-cell laser microdissection capture procedure from SBS slides.

After visualizing the cell of interest at a 63X magnification (black arrow) (A), and discarding the other cells around it in a ‘trash’ cap (B, C, D), the laser microdissection of the sample is performed by moving the laser cut line close to the cell of interest until it falls into the collection cap (E, F). In B-F, the dark circle is due to the laser engraving mark on the slide.

Fig 2. Visualization of SBS-microdissected cells in the tube collection cap.

A Giemsa-stained microdissected nucleus cell, visualized at 63X magnification, is recovered in the 0.2 mL PCR tube collection cap.

Fig 3. Examples of single-cell WGA QC analysis results.

The gel electrophoresis shows examples of the QC analysis (consisting of multiplex PCR reactions, see paragraph 2.6) performed in cells microdissected from mimic cancer blood samples obtained with the PANC-1 (A) and SW-620 (B) tumour cell lines. WGA+ and WGA-: positive (30 pg of BEAS-2B genomic DNA) and negative controls of the WGA step, respectively; QC+ and QC-: positive (100 ng of BEAS-2B genomic DNA) and negative controls of the QC, respectively; 5PFAC: five BEAS-2B cells fixed in PFA 4%; M (molecular weight marker): 2-Log DNA ladder (New England Biolabs); NTC: no-template control. The black arrows pinpoint the size of the expected products, as reported in the Ampli1™ QC kit manufacturer’s instructions. All the samples showed at least one band except for sample 6 in (A).

Fig 4. Detection of TP53 and KRAS mutations in PANC-1 and SW-620 single-cell WGA products, respectively.

PCR and Sanger sequencing chromatograms corresponding to the positive PCR products obtained using primers specific for TP53 exon 4 (A) and KRAS exon 2 (B). WGA+ and WGA-: positive (30 pg of BEAS-2B genomic DNA) and negative controls of the WGA step, respectively; NTC: no-template control; M: 2-Log DNA ladder used as a DNA molecular weight marker.