Multiparameter Evaluation of the Heterogeneity of Circulating Tumor Cells Using Integrated RNA In Situ Hybridization and Immunocytochemical Analysis

Abstract

Circulating tumor cells (CTCs) are routinely identified as cytokeratin (CK)-positive, epithelial cell adhesion molecule (EpCAM)-positive, and CD45-negative and are enriched based on EpCAM. However, there are a number of methodological challenges regarding both isolation and characterization of these rare CTCs including downregulation or absence of EpCAM in a variety of solid tumors leading to the omission of subpopulations of CTCs, difficulties in analyzing RNA and protein targets in CTCs due to the rarity of these cells, and low levels of targets and technological limitations of visualizing the targets of interest on each individual cell. Building on our previous CTC research on CD45-based negative magnetic separation and four-color fluorescent immunocytochemical (ICC) staining, RNA in situ hybridization (ISH) was applied to fluorescently target mRNA sequences corresponding to tumor-related genes at the single CTC level. Multiple categories of markers are targeted including CK, human epidermal growth factor receptor family markers, Hedgehog pathway markers, human papillomavirus markers, and protein arginine methyltransferase 5. In addition, an integrated method of RNA ISH and fluorescent ICC staining was developed to visualize CTCs on both mRNA and protein levels. The robustness of the integrated co-ICC and RNA ISH staining was demonstrated by a series of tests on representative tumor markers of different categories. The integrated staining can incorporate the advantages of both RNA ISH and fluorescent ICC staining and provide more intense signals and more specific bindings. With this integrated staining methodology, distinct staining patterns were applied in this report to facilitate the searching and characterization of rare subgroups of CTCs. These results support the existence of diverse groups of CTCs at both protein and mRNA transcript levels and provide an analytical tool for the research on CTCs of rare subgroups.

Circulating epithelial cell counts for monitoring the therapeutic outcome of patients with papillary thyroid carcinoma

“…Hence, it is likely that some of the PDPN + and TSHR + cells loss the expression of EpCAM and results in the EpCAM - PDPN + and EpCAM - TSHR + cell populations. These observations are consistent with the observed heterogeneity of CTCs in patients with other cancer types, such as breast cancer, colorectal cancer, and lung cancer [ 58 – 60 ]. It remains to investigate whether or not the PDPN- or TSHR-specific CECs is related to the degree of malignancy and the poor prognosis of patients with PTC.…”

Cancer metastasis through the prism of epithelial‐to‐mesenchymal transition in circulating tumor cells

“…In addition to the significant advances in the ability to isolate CTCs, a second important advance in the study of CTCs is the development of analytical tools including single‐cell RNA‐Seq (Saliba et al ., ; Tang et al ., ), multicolor RNA in situ hybridization (Wu et al ., ), and single‐cell DNA methylation analysis available to characterize CTCs (Pixberg et al ., ). While still in the development stages, the potential rapid advancement of single‐cell proteomics will likely also prove to be a critical tool (Macaulay et al ., ).…”

Hedgehog Signaling, a Critical Pathway Governing the Development and Progression of Hepatocellular Carcinoma

“…It is more convincing that the circulating tumor cells (CTCs) from an HCC were found to have a high level of Hh signaling activity. SMO and GLI1 were detected in CTCs from breast cancer patients by immunohistochemistry and RNA in situ hybridization and used as markers of the heterogeneity of the CTCs [ 107 ]. At the same time, CTCs express cancer stem cell markers, such as CD90, CD133, EpCAM, CK19 and Nanog, etc.…”