Discrete microfluidics for the isolation of circulating tumor cell subpopulations targeting fibroblast activation protein alpha and epithelial cell adhesion molecule

Małgorzata A Witek,Joshua M Jackson,Paola A Gehrig,John T Soper,Rachel Aufforth ,Charles M Perou,Young E Whang,Stephanie A Montgomery ,Weiya Z Wysham,Swathi R Pullagurla,Jerry Usary,Hong Wang,Matthew I Milowsky,Mateusz L Hupert,Victoria L Bae‐Jump ,Dawud Hilliard,George George Martin ,Joyce W Kamande,Jen Jen Yeh,Lisa A Carey,Steven A Soper

doi:10.1038/s41698-017-0028-8

Abstract

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges.

Highlights

Methods relying on anti-epithelial cell adhesion molecule (EpCAM) for positive affinity-selection of circulating tumor cells (CTCs) has been cleared by the Food and Drug Administration (FDA) for metastatic breast, prostate, and colorectal cancers; enumeration of EpCAM(+) CTCs alone has demonstrated modest clinical sensitivity.[1]
PSA and prostatespecific membrane antigen (PSMA) mRNA were expressed in both CTCFAPα and CTCEpCAM, suggesting these cells originated from the prostate tumor environment as PSMA and PSA
The question arises: does the biology limit the CTC burden or is the analytical platform used for their isolation limiting? many microfluidic technologies have shown higher clinical sensitivity/ CTC test positivity compared to the FDA-approved test.[6, 38]

Summary

Introduction

Methods relying on anti-epithelial cell adhesion molecule (EpCAM) for positive affinity-selection of circulating tumor cells (CTCs) has been cleared by the Food and Drug Administration (FDA) for metastatic breast, prostate, and colorectal cancers; enumeration of EpCAM(+) CTCs alone has demonstrated modest clinical sensitivity.[1]. M-PDAC patient #68, both subpopulations showed three mutations in KRAS (Fig. 5b). CTC subpopulations were tested for their mRNA expression in five M-PDAC and two M-CRC patients (Fig. 5d, e and Supplementary Fig. S7).

Results

Conclusion