December 27, 2019.
Cell-based liquid biopsy has, as we see it, great potential to improve current diagnostics, but also to evolve and replace diagnostic procedures in many diseases and otherwise relevant health conditions with respect to early phase detection, prevention or monitoring disease evolution. Cancer is certainly the most concerned aspect of cell-based liquid biopsy, yet admittedly exists for quite a while and has not lived up to its potential with clinicians already turning their backs on cell-based liquid biopsy. We may argue that the actual bottleneck may be simply a technological problem that comes with the rarity of desired cells and boils down to inadequate lower limits of detection. However, this may not be the entire truth for having to acknowledge biological hurdles, which in short are represented by a greater heterogeneity and plasticity of circulating tumor cells than initially anticipated. Consequently, cell-based liquid biopsy technology failed to comply with the physiology of cancer. Herein, technology was focused on the detection of epithelial circulating tumor cells by means of positive selection using magnetic cell separation technology (Figure 1) or microfluidic capture (CTC-Chip).
Fig.1 The CELLSEARCH® System is the result of a commitment to innovation that began in 1999 and led to the first standardized, FDA-cleared, semi-automatic system that can capture and quantify circulating tumor cells from a 7.5-mL blood sample with a high level of sensitivity and specificity.
It was found that not all solid tumors egress epithelial cells into the circulation with sufficient expression of epithelial surface marker thus, failed to capture the rare cells and leading to insensitivity in particular when testing on early-stage cancer patients and those having completed treatment. The new insight supported the emergence of new and likewise old “niche technology” with respect to isolation, purification, enrichment and analysis that was unbiased or non-selective in nature and finally opened the door for the awareness of greater complexity of peripheral blood rare cell types. In this regard, biophysics techniques by utilizing the nanoparticles comes to play role in cell separation as shown and explained in Figure 2.
Fig.2 Summary of magnetic separation method. Step 1 Mixing the beads with the cell suspension. Step 2 Subjecting the incubation container to continued axial rotation at around 5–30 rpm in direct vicinity of a strong permanent magnet for 1 min. Step 3 Mixing of the incubated suspension by continued pipetting and dispensing or vortexing. Steps 2 and 3 have been repeated 5 times. Step 4 Washing by diluting the incubated solution
with cell-friendly buffer solution 1:1 and intensive mixing. Step 5 Placing the incubation tube into the vicinity of a strong permanent magnet for magnetic capture and keep at rest for at least 4 min .
The next generation of CTC enrichment technology was often referred to as “EpCam-independent“ as to allude to the former problem of EpCam dependent capture. EpCam is the only epithelial antigen expressed at the outer surface membrane that has been successfully targeted. Such EpCam-independent technology relied on isolation methods based on physical distinctions between a white blood cell and a tumor cell including size, membrane charge, deformability, density or buoyancy. The alleged physical differences originated from the analysis of cultivated cell lines. However, it was found that “wild” circulating tumor cells often have little in common with the few stable model cancer cell lines and consequently also failed to advance the field. Nevertheless, upon the use of this technology, the complexity of “abnormal” cells became more evident comprising a spectrum of non-hematopoietic somatic or bone-marrow-derived cell groups that still await further exploration. Furthermore, the association with tumor growth has been shown for some of the cell types. Therefore, cell-based liquid may take new turns in view of the rich spectrum of new biomarkers. Such biomarkers include but are not limited to endothelial cells, megakaryocytes, erythroblasts and all kinds of stromal cells. In any case, the wealth in information originating from the circulating rare cell spectrum has already been recognized. This had led to the adoption of circulating endothelial and circulating endothelial progenitor cells to cancer cell-based liquid biopsy. Now, cell-based liquid biopsy stands to take its chances of advancement by exploring and exploiting the entirety of the spectrum of circulating rare cells for diagnostic interpretation, but also therapeutic use thereof. However, biological awareness is more advanced than the available technology. Only two analytical strategies remain as to include the diverse circulating rare cells into cancer diagnostics in parallel. One is the analysis without prior isolation or enrichment using sophisticated analytical procedures in microscopy or flow cytometry. The other is enrichment prior to analysis (by common means) based on negative selection. The latter method depletes all undesired cells mostly being leukocytes by magnetic cell separation technology, therein mostly targeting the CD45 receptor with anti-CD45 reactive magnetic particles. Other or additional antigens included CD11b, CD15, and CD66b, all being antigens of the hematopoietic lineage. Technological solutions of the latter are spares and far from routine. Technological problems include loss of desired cells as well as high costs per sample. Therefore, it has been understood by others and our group that technology was and is the bottleneck in cell-based liquid biopsy, and deserve greater attention to realize the potential of cell-based liquid biopsy as to firstly advance into early and earliest stage cancer, but also to expand application from cancer into other big-killer diseases, such as myocardial infraction or hypertension.
1. Stefan Schreier, Piamsiri Sawaisorn, Rachanee Udomsangpetch and Wannapong Triampo, “Advances in rare cell isolation: an optimization and evaluation study”, Journal of Translational Medicine 15, Article number: 6 (January 2017).
Dr. Stefan Schreier* and Assoc. Prof. Dr. Wannapong Triampo+
*School of Bioinnovation and Bio-based Product Intelligenc., Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand
+ R&D Group for Biological and Medical Physics, Department of Physics, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 10400, Thailand