Abstract
Conventional strategy of anti-EpCAM capture and immunostaining of cytokeratins (CKs) to detect circulating tumor cells (CTCs) is limited by highly heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. In this study, a novel integrated cellular and molecular approach of subtraction enrichment (SE) and immunostaining-FISH (iFISH) was successfully developed. Both large or small size CTCs and circulating tumor microemboli (CTM) in various biofluid samples including cerebrospinal fluid (CSF) of cancer patients and patient-derived-xenograft (PDX) mouse models were efficiently enriched and comprehensively identified and characterized by SE-iFISH. Non-hematopoietic CTCs with heteroploid chromosome 8 were detected in 87-92% of lung, esophageal and gastric cancer patients. Characterization of CTCs performed by CK18-iFISH showed that CK18, the dual epithelial marker and tumor biomarker, was strong positive in only 14% of lung and 24% of esophageal CTCs, respectively. Unlike conventional methodologies restricted only to the large and/or both EpCAM and CK positive CTCs, SE-iFISH enables efficient enrichment and performing in situ phenotypic and karyotypic identification and characterization of the highly heterogeneous CTC subtypes classified by both chromosome ploidy and the expression of various tumor biomarkers. Each CTC subtype may possess distinct clinical significance relative to tumor metastasis, relapse, therapeutic drug sensitivity or resistance, etc.
Highlights
The clinical implications of circulating tumor cells (CTCs) have been reported elsewhere [1, 2]
Our results indicate that the absence of cytokeratin 18 (CK18) in CTCs was not a consequence of the CK18-iFISH methodology itself, which is in agreement with the concept that tumor cells may regulate CK18 expression under certain circumstances, and such post-translational modulation of CK18 protein in CTCs revealed and quantified by phenotypic immunostaining is of particular biological and clinical significance
EGFR mutation analysis performed on lung cancer CTCs was reported to be more sensitive than conventional serum nucleic acid analysis [34]
Summary
The clinical implications of circulating tumor cells (CTCs) have been reported elsewhere [1, 2]. Anti-EpCAM-dependent antibody capture [1] and tumor cell size-based filtration [4] currently constitute common strategies for isolating CTCs [5,6,7]. Increasing evidence has emerged that besides existence of significant amount of small size CTCs, clinical application of anti-EpCAM strategy is significantly limited due to inherent methodological deficiencies and intrinsic heterogeneity of cell biomarkers in cancer patients. It has been reported that only 70% of the examined 134 epithelial solid tumors express EpCAM [11], and there is a significant phenotypic heterogeneity of dynamically expressed EpCAM even among the individual CTC within the same sample [8, 9]. It has been recently published that www.impactjournals.com/oncotarget
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