PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells
Scientific Reports. 2019;9(1):17470. DOI 10.1038/s41598-019-53899-4. PMID 31767951. PMCID PMC6877641.
How to cite
AMA
Kamal M, Saremi S, Klotz R, Iriondo O, Amzaleg Y, Chairez Y, et al. PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells. Sci Rep. 2019;9(1):17470. doi:10.1038/s41598-019-53899-4APA
Kamal, M., Saremi, S., Klotz, R., Iriondo, O., Amzaleg, Y., Chairez, Y., et al. (2019). PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells. Scientific Reports, 9(1), 17470. https://doi.org/10.1038/s41598-019-53899-4BibTeX
@article{kamal2019picrun,
title = {PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells},
author = {Kamal, Mohamed and Saremi, Shahin and Klotz, Remi and Iriondo, Oihana and Amzaleg, Yonatan and Chairez, Yvonne and others},
journal = {Scientific Reports},
volume = {9},
number = {1},
pages = {17470},
year = {2019},
doi = {10.1038/s41598-019-53899-4}
}Circulating tumor cells break away from a solid tumor and travel in the blood, where they carry clues about how a cancer spreads and resists treatment. They are vanishingly rare, and most ways of finding them either fix and stain the cells, which kills them, or need several rounds of purification to pull out clean, living cells.
The researchers built an assay called PIC&RUN on the AccuCyte–CyteFinder system, which spreads every nucleated cell from a blood tube onto slides without an up-front capture step. Tumor cells were flagged either by cancer surface markers or by the absence of immune markers, then lifted out one at a time with a fine needle.
The single cells came out intact enough for single-cell RNA sequencing or for weeks of culture, so one workflow both counts rare tumor cells and returns living cells to study.
Key findings
- AccuCyte captured live tumor cells at high efficiency. Tumor cells stained with a live dye (165 to 1,209 per sample) and spiked into 7.5 mL of donor blood were recovered at an average of 91.6%, matching the more than 90% previously reported for fixed cells.
- A two-step needle-picking protocol returned ultra-pure single living cells. Single picks succeeded in over 80% of attempts but carried roughly 100 to 150 contaminating white blood cells; a second pick removed them, delivering one viable cell in a 0.5 µL final volume.
- The assay detected a single tumor cell in 7.5 mL of blood. Individually picked cells spiked into donor blood were recovered across three independent experiments with no false-positive or false-negative calls.
- Retrieved cells supported both single-cell sequencing and live culture. Positive-selection picks separated cleanly from immune cells by RNA-sequencing principal-component analysis and were compatible with single-cell transcriptomics, while negative-selection cells were expanded in ex vivo culture for 3 weeks.
The AccuCyte–CyteFinder workflow in the methods
“Blood samples were processed using AccuCyte as previously described 41 . Briefly, 7.5 ml of blood was added to each AccuCyte Separation Tube (RareCyte) and tubes were centrifuged at 3000 g for 25 minutes (min). After centrifugation, a brass ring clamp (CyteSeal) was applied using a CyteSealer (RareCyte) to each tube. Plasma was then aspirated and 4 ml of displacement solution (RareCyte) was added to each tube. An EpiCollector (RareCyte) with an isolation tube pre-filled with 160 µL of isolation buffer (RareCyte) was placed on the top of each separation tube. The whole system was centrifuged for 20 min at 1000 g (ThermoFisher Scientific) and buffy coats were then collected into either 800 µl transfer fluid containing a non-formalin fixative (RareCyte) or 1 ml of CTC media and transferred to 1.8 ml eppendorf tubes for further investigation. Buffy coats resuspended in transfer fluid are kept at RT for 10 min then were spread on glass slides (8 slides per blood sample). Prepared slides were air dried for 30 minutes and stored in −20 °C until the day of staining. Detection of CTCs using AccuCyte-CyteFinder system Slides were fluorescently stained with the 4D staining kit (RareCyte) which contains antibodies against cytokeratin, EpCAM, and CD45 plus DAPI using the automated staining instrument (BOND RXm, Leica Biosystem). Stained slides were automatically scanned using the fluorescence platform of the RareCyte and images were captured at 10x objective magnification. Slide images were analyzed with integrated image analysis software that automatically analyzes the images to find cytokeratin and/or EpCAM positive cells which are CD45 negative.”
— Kamal et al., Scientific Reports (2019), Methods, “CTC enrichment using AccuCyte”
Why it matters for The AccuCyte–CyteFinder workflow users
If you are weighing an enrichment-free route to circulating tumor cells, this study puts the AccuCyte–CyteFinder workflow through the full job and then extends it to living cells. Blood went into an AccuCyte separation tube, and instead of selecting cells by an epithelial marker first, the sample-preparation step harvested every nucleated cell by density and spread it across slides. The CyteFinder fluorescence scanner then imaged the slides, its integrated analysis flagged cytokeratin- or EpCAM-positive, CD45-negative candidates, and a ceramic-tipped needle retrieved each one individually. What the authors added is a live-cell path: the same platform recovered viable single cells that fed single-cell RNA sequencing and three weeks of culture. For your own work, the point is that one workflow can enumerate rare cells and hand back intact, living single cells for downstream analysis, rather than forcing a choice between counting cells and keeping them alive. Because the harvest is enrichment-free, cells that fall outside a fixed antigen window are not discarded before you ever see them.
