Abstract
The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, α-EpCAM+, α-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0–5% as compared with CTC counts obtained with the CellSearch® system. Viability of the detected CTC ranged from 0–36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses.
Highlights
Assessment of the molecular characteristics of tumor cells from patients is essential for treatment decision making and for research
A cell suspension of MCF-7 cells was stained with Calcein AM (Calc AM) to identify live cells and Ethidium homodimer-1 (EthD1) to identify dead cells and seeded into the microwells
In this study we aimed to develop a workflow where self-sorting microwells are used as a tool to isolate and grow single circulating tumor cells (CTC) from blood
Summary
Assessment of the molecular characteristics of tumor cells from patients is essential for treatment decision making and for research. Tissue from solid tumors for such characterization is obtained through invasive surgical procedures such as taking a biopsy [1,2] These procedures cannot be performed on a regular basis and for patients with metastatic disease one tumor site may not be representative for all the metastatic sites. The presence of tumor cells and tumor nucleic acids in the blood of cancer patients is being investigated for its potential as a non-invasive real time biopsy process. These non-invasive methods can yield information about the genetic profile of cancers and track genomic transformations [1,4,5]
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