EGFR-Based Immunoisolation as a Recovery Target for Low-EpCAM CTC Subpopulation

Abstract

Circulating tumour cells (CTCs) play a key role in the metastasis process, as they are responsible for micrometastasis and are a valuable tool for monitoring patients in real-time. Moreover, efforts to develop new strategies for CTCs isolation and characterisation, and the translation of CTCs into clinical practice needs to overcome the limitation associated with the sole use of Epithelial Cell Adhesion Molecule (EpCAM) expression to purify this tumour cell subpopulation. CTCs are rare events in the blood of patients and are believed to represent the epithelial population from a primary tumour of epithelial origin, thus EpCAM immunoisolation is considered an appropriate strategy. The controversy stems from the impact that the more aggressive mesenchymal tumour phenotypes might have on the whole CTC population. In this work, we first characterised a panel of cell lines representative of tumour heterogeneity, confirming the existence of tumour cell subpopulations with restricted epithelial features and supporting the limitations of EpCAM-based technologies. We next developed customised polystyrene magnetic beads coated with antibodies to efficiently isolate the phenotypically different subpopulations of CTCs from the peripheral blood mononuclear cells (PBMCs) of patients with metastatic cancer. Besides EpCAM, we propose Epidermal Growth Factor Receptor (EGFR) as an additional isolation marker for efficient CTCs detection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

A) SEM image. B) Yield and initial Fe3O4 content versus actual Fe3O4 content (wt%). C) Plot of magnetisation versus applied magnetic field for beads.

Fig 2

A) Df/Di (final diameter/initial diameter) ratio after several washing steps of magnetic beads. B) Df/Di ratio of beads after serial 4 months storage period.

Fig 3. Cell recovery using magnetic beads.

A) Flow cytometry analysis of SW480 cells incubated with anti-EpCAM antibody, beads coated with anti-EpCAM or magnetic beads alone. B) Capture efficiency (%) of SW480 cells with magnetic beads functionalised with anti-EpCAM (40 μg/μL) at different cell numbers. C) Capture efficiency (%) of MDA-MB-231 cells (10000) using anti-EpCAM beads or anti-EGFR beads (40 μg). Isolation was performed sequentially: EpCAM-EGFR or EGFR-EpCAM (1 or 2 indicates first or second isolation step, respectively). D) Capture efficiency (%) of SW480 cells (23.25±5.08) spiked in donor control PBMCs (9 or 18 million) for different amounts of anti-EpCAM beads (24 and 48 μg). Independent isolation assays, n≥3, (duplicates per condition). E) Capture efficiency (%) of SW480 cells (22.81±15.40) spiked in donor control PBMCs using 5.8 μg anti-EpCAM beads per million. Three independent isolation assays (n≥3 replicates per assay).

Fig 4. Viability of captured tumour cells.

A, A´) Microscopic images of spiked SW480 cells isolated from PBS (A) or healthy donor PBMCs (A´). B) Cell proliferation was measured by Alamar Blue assay after 24 h in culture. Each point represents the mean ± S.E.M. of 6 replicates per group.

Fig 5. CTCs isolated with magnetic beads from blood from patients with metastatic cancer.

Representative immunofluorescence for Hoechst (blue, for nuclei staining), pan-cytokeratins (green), CD45 (red) and merge of CTC isolated from a patient with metastatic breast cancer (upper panel). Merge for indicated staining of CTCs isolated with anti-EpCAM, anti-EGFR or anti-FGFR magnetic beads. The size of each CTC is indicated in μm.

Fig 6. EGFR-positive CTCs correlates with worse prognosis.

A) EGFR-positive CTC enumeration from patients with colon, prostate and breast metastatic cancer (from Table 3); (Spearman rank correlation coefficient = 0.575, p<0.05). B) Kaplan–Meier plot of overall survival or progression free survival based on EGFR-positive CTCs isolation.

Fig 7. EGFR isolated CTCs depict differential subpopulation.

Gene expression profiling of indicated genes in isolated CTCs from patients with metastatic prostate cancer using coated magnetic beads for the three fractions: EpCAM (inblack); EGFR (in green) and FGFR (in grey); (n = 8). CellSearch data regarding CTC number for each patient is summed in the box. Wilcoxon signed rank test, *p