Isolation of circulating tumor cells

Abstract

Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

Keywords: Biotechnology; Cell biology; Technical aspects of cell biology.

Graphical abstract

Figure 1

The setting and challenges of CTC isolation technology Left: alongside tumor-derived proteins, exosomes and ctDNA, cancers shed cells and clusters sporadically into the blood. Provided they are not removed from the circulation too quickly, CTCs can be isolated from peripheral circulation by venous blood draw, but their extreme rarity requires high-yield enrichment from large volumes of blood, and thus multi-log₁₀ removal of normal cells (top right). Isolation is further complicated by an overlap in size of patient-derived CTCs with WBCs (sizes from blood smear) and by the variability (or lack) of available EpCAM for positive selection (lower right).

Figure 2

Label-free CTC enrichment Isolation strategies are constructed along multiple dimensions of separation for each population of normal blood cells and all or a subset of CTCs. This necessarily includes removing RBCs and platelets by size or other physical properties. Label-free isolation either accepts loss of CTCs with sizes or biophysical properties that overlap with WBCs, or leaves CTCs mixed with WBCs, relying purely on the above methods.

Figure 3

Predicted CTC yield with enrichment by size or positive selection We found as in (A) that patient-derived CTCs (melanoma, lung, prostate, and breast) are on average smaller than cultured cancer cells (from Sk-Mel28 to SkBR), overlapping with WBCs. Two waterfall plots predict CTC yield for (B) size- or (C) EpCAM-based isolation. Graphs from primary CTC-derived data are adapted without modification or warranty from Figures 2A and SI Figure 7 of (Fachin et al., 2017) under the Creative Commons Attribution 4.0 International License.

Figure 4

Antigen-dependent CTC enrichment To remove WBCs from CTCs, negative selection (AB) targets specific surface antigens (e.g. CD45). Alternately, positive selection (DE) purifies CTCs if they present surface-available EpCAM. (C) FACS can sort by multiple antigen-based or label-free metrics simultaneously.

Figure 5

CTC-iChip 2 As depicted in (A), CTC-iChip 2 combines two modules: size sorting to separate nucleated cells from blood and negative selection to remove bead-labeled WBCs from untouched CTCs. Shaded area is laser-welded macro-micro interface that connects injection-molded microchannel devices (52 μm tall) to 5 tubing ports labeled in (A) and visible in image of chip in processor (B). CTC-iChip 2 has bilateral symmetry, and each numbered stage is detailed in (D1) for size sorting and in (D2) for negative selection, from inlet to outlet. Inset images trace the path of untouched nucleated cells at key locations. After a final stage of magnetic-activated cell sorting, CTCs exit the chip, enriched for downstream analysis as tabulated in (C) for platelets, RBCs, and WBCs (whiskers span the full range of data; box encloses the interquartile range and the 50^th percentile line). Size sorting is as in (Mutlu et al., 2017). Magnets are arranged as in (Fachin et al., 2017). Blood samples were collected from healthy volunteers following experimental protocols reviewed and approved by the Massachusetts General Hospital IRB, where protocol numbers 2009-P-000295 and 2015-P-000656 were used to obtain informed consent from internal donors and donors at the MGH Blood Bank, respectively. Some healthy samples were also ordered from Research Blood Components, LLC (Brighton, MA). All samples were obtained in accordance with the applicable federal guidelines and regulations.