Circulating Tumor Cell Composition in Renal Cell Carcinoma

Abstract

Purpose: Due to their minimal-invasive yet potentially current character circulating tumor cells (CTC) might be useful as a "liquid biopsy" in solid tumors. However, successful application in metastatic renal cell carcinoma (mRCC) has been very limited so far. High plasticity and heterogeneity of CTC morphology challenges currently available enrichment and detection techniques with EpCAM as the usual surface marker being underrepresented in mRCC. We recently described a method that enables us to identify and characterize non-hematopoietic cells in the peripheral blood stream with varying characteristics and define CTC subgroups that distinctly associate to clinical parameters. With this pilot study we wanted to scrutinize feasibility of this approach and its potential usage in clinical studies.

Experimental design: Peripheral blood was drawn from 14 consecutive mRCC patients at the West German Cancer Center and CTC profiles were analyzed by Multi-Parameter Immunofluorescence Microscopy (MPIM). Additionally angiogenesis-related genes were measured by quantitative RT-PCR analysis.

Results: We detected CTC with epithelial, mesenchymal, stem cell-like or mixed-cell characteristics at different time-points during anti-angiogenic therapy. The presence and quantity of N-cadherin-positive or CD133-positive CTC was associated with inferior PFS. There was an inverse correlation between high expression of HIF1A, VEGFA, VEGFR and FGFR and the presence of N-cadherin-positive and CD133-positive CTC.

Conclusions: Patients with mRCC exhibit distinct CTC profiles that may implicate differences in therapeutic outcome. Prospective evaluation of phenotypic and genetic CTC profiling as prognostic and predictive biomarker in mRCC is warranted.

Trial registration: ClinicalTrials.gov NCT01731158 NCT01521715.

Conflict of interest statement

Competing Interests: Dr. Nel, shared first author, declares that she has no significant competing financial, professional or personal interests that might have influenced the performance or presentation of the work described in this manuscript. The lab work was completed and the manuscript was written while she was employed at M.O.R.E.—Molecular Oncology Risk-Profile Evaluation, Department of Medical Oncology, West German Cancer Center, University Duisburg-Essen. Final changes, submission and revision of the manuscript was processed during her employment at ABA GmbH & Co. KG, where she is currently working on a co-operative project. In accordance with her ethical obligation as a researcher, she is reporting that she receives salaries from the company ABA GmbH & Co. KG that may be affected by the research reported in the enclosed paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Basic principle of CTC isolation.

1. Leucosep tube: 2. Separation media; 3. Whole blood/PBMNC mixture; 4. Plasma; 5. Separation media after centrifugation; 6. Erythocytes; 7. Buffy coat incl. CTCs; 8. Anti-CD45 beads; 9. Anti-EpCAM beads (positive isolation) or anti-CD15 beads (negative isolation); 10. Washing buffer; 11. EpCAM-bead bound CTC suspension for qRT-PCR; 12i. Depleted bead free cell suspension containing CTCs for Cellspin and immunofluorescence staining. 12 ii. Depleted bead free cell suspension containing CD45-/EpCAM- CTC for qRT-PCR.

Fig 2. CTC detection.

A) Positive control consisting of PBMNC mixed with CD133-expressing K562 cells which were stably transduced with lentiviral vectors endocoding an internal ribosomal entry site (IRES)-mediated co-expression cassette of CD133 and enhanced green fluorescent protein (eGFP) (K562-CD133:IEG). Cells were stained with DAPI (nucleus; blue) and for CD133 (pseudo-color red). The antibodies used for anti-CD133 immunostaining specifically bound to K562-CD133:IEG cells, visualized by Cy3 (red) counterstaining (40x magnification). B) CTC isolated from mRCC patients stained with DAPI (blue), for pan-CK (epithelial; red) and for CD45 (hematopoietic; green; 20x magnification). The cell marked with a white arrow shows a DAPI-positive (blue)/CD45-negative staining and was positive for pan-CK (red) and subsequently considered as CTC.

Fig 3. Detection of CTC subtypes.

CTC isolated from mRCC patients were stained with DAPI (nucleus; blue) and for CD45 (hematopoietic; green), pan-CK (epithelial; red) and N-cadherin (mesenchymal; yellow) on one slide and with DAPI and for CD45, pan-CK and CD133 (stem cell; yellow) on a second slide. Cells marked with a white arrow were considered as CTC. The image displays various CTC subtypes with epithelial, mesenchymal and/or stem cell-like features such as N-cadherin+/CK-/CD45-; N-cadherin-/CK+/CD45-; CD133+/CK+/CD45+ and CD133-/CK+/CD45 (low) cells.

Fig 4. CTC subtypes were associated to clinical outcome.

A) Kaplan-Meier test showed that the number of N-cadherin+/CK- cells was significantly associated to progression free survival (PFS) of mRCC patients during first-line treatment with anti-angiogenesis therapy (PFS; 7 vs. 15 months; p = 0.03; [HR] = 0.31; CI: 0.06–1.59). B) Mann-Whitney test revealed a significantly increased number of CD133+ cells in the presence of N-cadherin+ cells (p = 0.05).

Fig 5. Gene expression analysis.

A) Visualization of significant interrelationships between gene expressions. B and C) Mann-Whitney test showed that mRNA expression levels of HIF1A (B) and KDR1 (VEGFR) (C) were significantly decreased (p = 0.05) in the EpCAM-/CD45- fraction when CD133+ cells were present in the blood sample.