Negative enrichment of circulating tumor cells from unmanipulated whole blood with a 3D printed device

Abstract

Reliable and routine isolation of circulating tumor cells (CTCs) from peripheral blood would allow effective monitoring of the disease and guide the development of personalized treatments. Negative enrichment of CTCs by depleting normal blood cells ensures against a biased selection of a subpopulation and allows the assay to be applied on different tumor types. Here, we report an additively manufactured microfluidic device that can negatively enrich viable CTCs from clinically-relevant volumes of unmanipulated whole blood samples. Our device depletes nucleated blood cells based on their surface antigens and the smaller anucleated cells based on their size. Enriched CTCs are made available off the device in suspension making our technique compatible with standard immunocytochemical, molecular and functional assays. Our device could achieve a ~ 2.34-log depletion by capturing > 99.5% of white blood cells from 10 mL of whole blood while recovering > 90% of spiked tumor cells. Furthermore, we demonstrated the capability of the device to isolate CTCs from blood samples collected from patients (n = 15) with prostate and pancreatic cancers in a pilot study. A universal CTC assay that can differentiate tumor cells from normal blood cells with the specificity of clinically established membrane antigens yet require no label has the potential to enable routine blood-based tumor biopsies at the point-of-care.

The integrated on-chip isolation and detection of circulating tumour cells

“…98,99 A key advantage of negative enrichment is that intact (label-free) CTCs are obtained independent of their specific antigen expression, and thus, a heterogenous population of CTCs can be isolated (i.e., including low-EpCAM-expressing CTCs). 100,101 This approach does not, however, afford a tumour-specific selection and thus typically results in a low purity (as exemplified by the low and inconsistent purities across different systems varying between 0.97-10%, going up to a maximum of 34.8 ± 14%). [102][103][104][105][106][107][108][109][110][111] 2.1.1 Applying antigen-dependent isolation in fully integrated platforms.…”

“…, including low-EpCAM-expressing CTCs). 100,101 This approach does not, however, afford a tumour-specific selection and thus typically results in a low purity (as exemplified by the low and inconsistent purities across different systems varying between 0.97–10%, going up to a maximum of 34.8 ± 14%). 102–111…”

Clinical Applications of Liquid Biopsy in Colorectal Cancer Screening: Current Challenges and Future Perspectives

“…However, CTCs are described by phenotypical heterogeneity, with some CTCs failing the selection process due to a lack of marker expression [ 29 ]. In these conditions, negative selection methods could successfully isolate CTCs by identifying and excluding from analysis various non-malignant cells using antibodies recognizing cell surface markers expressed by these circulating blood cells [ 25 , 30 ]. In comparison, label-independent CTC enrichment methods subject CTCs to separation based on biophysical features such as density, size, deformability, electrical characteristics and invasiveness [ 31 ].…”