Single-Cells Isolation and Molecular Analysis: Focus on HER2-Low CTCs in Metastatic Breast Cancer

Simple Summary While the concept of HER2-low expression is gaining momentum in the scientific landscape of breast cancer research, HER2-low circulating tumor cells (CTCs) also have promising relevance as biomarkers. Unveiling the biological features behind this recently highlighted evidence on CTCs might achieve both goals of speeding up the interpretation of the general understanding of HER2-low expressing breast cancer and declaring their independent biological and predictive value. If, on the one hand, studying CTCs allows to drill down more easily to a single cell resolution, on the other hand, CTC collection still remains a challenging procedure. In order to improve and standardize this process, we developed a structured pipeline for HER2-low CTC detection and collection. We defined and validated the optimal thresholds to select this specific subtype of CTCs using breast cancer cell lines of known HER2 expression. Our study represents the technical and procedural milestone that will allow a standardized collection process for future molecular investigations. Although the detection of CTCs expressing HER2 at low intensity (HER2-low CTCs) has been shown to have a negative prognostic value in metastatic breast cancer (MBC) patients, the biological intrinsic nature of HER2-low CTCs remains unexplored. Considering the technical challenges behind the selective collection of immunophenotype-specific CTCs, we developed a pipeline to individually capture HER2-low CTCs. Four different breast cancer cell lines (MDA-MB-231, T47D, MDA-MB-453, and SKBR3), that are known to express HER2 at different immunohistochemistry levels (respectively classified as 0, 1+, 2+, and 3+), were spiked in healthy donor blood tubes (7.5 mL) and processed with the CellSearch(R) (Menarini Silicon Biosystems, Bologna, Italy) for enrichment and the DEPArray NxT((TM)) for single cell selection. The HER2 signal-intensities of each cell line was compared using the nonparametric Mann-Whitney U test. The optimal cut-offs to distinguish HER2 1+ from 0 and 2+ cells were calculated performing the Receiver operating characteristic (ROC) curve. Median HER2 signal-intensities detected with the DEPArray NxT((TM)) were: 2.59 (0), 3.58 (1+), 5.23 (2+) and 38.37 (3+). DEPArray NxT efficiently differentiated each single cell line (p < 0.001). The area under the ROC curve was 0.69 and 0.70 (respectively 0 vs. 1+ and 1+ vs. 2+) and the optimal calculated cut-offs were 2.85 (lower) and 4.64 (upper). HER2-low CTCs can be detected and separately collected using predetermined intensity cut-offs. This study will allow standardized single-cell or pooled collection of HER2-low CTCs for downstream molecular analyses.