MINDEC-An Enhanced Negative Depletion Strategy for Circulating Tumour Cell Enrichment

Abstract

Most current methods of circulating tumour cell (CTC) enrichment target the epithelial protein EpCAM, which is commonly expressed in adenocarcinoma cells. However, such methods will not recover the fraction of CTCs that have a non-epithelial phenotype due to epithelial–mesenchymal transition. For phenotype-independent CTC enrichment, we developed a new enhanced negative depletion strategy—termed MINDEC—that is based on multi-marker (CD45, CD16, CD19, CD163, and CD235a/GYPA) depletion of blood cells rather than targeted enrichment of CTCs. Here we validated the performance of MINDEC using epithelial and mesenchymal cancer cell lines, demonstrating a mean recovery of 82 ± 10%, high depletion (437 ± 350 residual white blood cells (WBCs)/mL peripheral blood), linearity between spiked and recovered cells (correlation coefficient: r = 0.995), and a low detection limit (≥1 cell recovered in all four replicates spiked with 3 cells). For clinical validation of this method, we enumerated CTCs in peripheral blood samples from patients with metastatic pancreatic cancer, detecting CTCs in 15 of 21 blood samples (71%) from 9 patients. The promising performance of the MINDEC enrichment strategy in our study encourages validation in larger clinical trials.

Genetic Heterogeneity of Single Circulating Tumour Cells in Colorectal Carcinoma

“…To date, several studies have been published on the molecular characterisation of single CTCs from different cancers, such as prostate, pancreatic and colorectal carcinomas [ 9 , 20 , 21 ]. In the current study, the single-cell quantitative PCR performance was validated by gene expression of GAPDH from the colon cancer cell lines.…”

“…Many groups have confirmed the presence of heterogeneous CTC phenotypes (such as epithelial, epithelium–mesenchymal transition (EMT) and mesenchymal/stem) in patients with different cancers, including CRC [ 6 , 7 ]. Interestingly, immunoaffinity-dependant negative selection techniques demonstrated higher efficacy in isolating heterogeneous CTCs from the blood of patients with cancer [ 8 , 9 ]. Molecular characterisation of the heterogeneous CTCs showed that they were more concordant with metastatic tumours than primary tumours in patients with colorectal cancers, suggesting the acquired characteristics of the CTCs in the blood microenvironment [ 10 ].…”

Circulating tumour cells and DNA as liquid biopsies in gastrointestinal cancer

“…CTCs are extremely rare compared with other cells in the blood of patients with cancer ( Fig . ), so extensive enrichment is required to detect them ( Table ). In general, enrichment is based on physical (such as size or density) or biological (for example presence of specific proteins) properties.…”

Perspective on Cancer Therapeutics Utilizing Analysis of Circulating Tumor Cells

“…The simple concept of CTC analysis is to gather or detect small numbers of CTCs from among larger numbers of white blood cells. Existing techniques for CTC isolation can be divided into three types: antibody-, physical property-, and function-based approaches [ 22 , 23 , 24 ]. A recently established microfluidic method is based on CTC isolation techniques that divided into three types as above.…”

“…Cell surface proteins are often used as targets in antibody-based CTC isolation methods. Because of the lack of a strictly tumor-specific antigen, epithelial-specific proteins—such as epithelial cell adhesion molecule (EpCAM)—are used for CTC-positive selection and antibodies against leukocyte-specific surface antigens, such as CD45, are used to deplete leukocytes [ 24 , 25 ]. Technologies based on physical properties dominate the field of CTC separation.…”