Isolation of Circulating Tumor Cells from Multiple Epithelial Cancers with ApoStream(®) for Detecting (or Monitoring) the Expression of Folate Receptor Alpha

Abstract

This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream(®) technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα(+) CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα(+) CTCs. We believe that the developed methodology will have applications in both the diagnosis and the monitoring of FRα-expressing cancers. Folate receptor alpha (FRα) expression may have utility as a potential diagnostic and therapeutic target in solid tumors. As tissue samples are not always available for patient screening, this study evaluated a noninvasive assay in CTCs from blood samples to detect FRα expression. The presence of FRα(+) CTCs enriched using ApoStream(®) and detected using laser capture cytometry was evaluated in blood samples from cancer patients [NSCLC adenocarcinoma (n = 14), breast cancer (n = 20), ovarian cancer (n = 6), and squamous lung cancer patients (n = 6)] and healthy subjects (n = 20). The data demonstrated that FRα(+) CTCs were detected in blood from NSCLC adenocarcinoma, breast, and ovarian cancer patients, whereas squamous cell lung cancer patients and normal healthy controls lacked FRα(+) CTCs as previously known. We demonstrate that CTCs captured using ApoStream(®) can be used to detect FRα(+) CTCs and may have clinical utility as a real-time liquid biopsy for assessing FRα levels in cancer patients.

Keywords: ApoStream®; CTC; folate receptor alpha; liquid biopsy; solid tumors.

Figure 1

Flow cytometry of FRα-stained cells: histograms of flow cytometric analysis of SK-OV-3 cells (A and B) and HCT-116 cells (C and D) stained by anti-FRα antibodies LK26 (A and C) and 26B3 (B and D) where the antibodies were diluted to 0.1 μg/mL (magenta), 1 μg/mL (green), and 10 μg/mL (orange). Controls included autofluorescence (gray), Dylight-649-conjugated anti-mouse secondary antibody only (black line) and mouse IgG plus secondary antibody (green shading).

Figure 2

Secondary antibody detection of FRα: histograms from flow cytometric analysis of HCT-116 cells stained by anti-FRα antibodies LK26 and 26B3 where the antibodies were diluted to 0.1 μg/mL (magenta), 1 μg/mL (green), and 10 μg/mL (orange) and detected by either FITC- or DyLight-649-conjugated anti-mouse antibodies as secondary antibody. Controls included autofluorescence (gray), Dylight-649-conjugated anti-mouse secondary antibody only (black line), and irrelevant mouse IgG plus secondary antibody (green shading).

Figure 3

Detection of FRα using LSC: histograms obtained using LSC analysis of SK-OV-3 cells (A and B), HCT-116 cells (C and D), and PBMCs from a healthy individual (E and F) stained with anti-FRα antibody LK26 (A, C, and E) and 26B3 (B, D, and F). Antibodies were diluted to 0.1 μg/mL (magenta), 1 μg/mL (green), and 10 μg/mL (red). For controls, irrelevant mouse IgG plus secondary antibody is shown in blue.

Figure 4

Effect of fixation on FRα staining: unfixed and 2% PFA fixed SK-OV-3 cells were stained for FRα using either MAb LK26 or MAb 26B3 and Dylight-649 anti-mouse and immunofluorescence measured using LSC. This analysis demonstrated that there is little or no effect due to fixation on the intensity of the FRα signal.

Figure 5

FRα-positive CTCs isolated using ApoStream^®: ApoStream^® enriched samples were stained using a multiplexed assay for CK, CD45, and FRα (MAb 26B3) and imaged using LSC. Images are gated CTCs (DAPI⁺/CK⁺/CD45^–) from breast, ovarian, and NSCLC (adenocarcinoma and squamous cell carcinoma) patients. Images show a coincidence of staining for DAPI, CK, and FRα (NSCLC adenocarcinoma, breast cancer, and ovarian cancer) while being negative for CD45, whereas CTCs isolated from NSCLC squamous cell carcinoma are FRα negative. In addition, staining is shown for cells isolated from a normal donor using ApoStream and demonstrate that while some CD45⁺ cells are isolated, they are neither CK⁺ nor FRα⁺.