Fluorescence Analysis of Vitamin D Receptor Status of Circulating Tumor Cells (CTCS) in Breast Cancer: From Cell Models to Metastatic Patients

Abstract

The Vitamin D receptor (VDR) expressed in normal breast tissue and breast tumors has been suggested as a new prognostic biomarker in breast cancer (BC). Besides, increasing evidence supports the view that the detection of circulating tumor cells (CTCs) predicts outcome in early and metastatic BC. Consequently, an evaluation of VDR expression in the CTCs of BC patients may allow optimization of their treatment. As an attempt to profile and subtype the CTCs of metastatic patients, we established an innovative fluorescence technique using nine BC cell lines to visualize, define, and compare their individual VDR status. Afterwards, we tested the CTC presence and VDR expression in blood samples (cytospins) collected from 23 metastatic BC patients. The results demonstrated major differences in the VDR levels among the nine cell lines, and VDR positive CTCs were detected in 46% of CTC-positive patients, with a total of 42 CTCs individually analyzed. Due to the limited number of patients in this study, no correlation between VDR expression and BC subtype classification (according to estrogen receptor (ER), progesterone receptor (PR) and HER2) could be determined, but our data support the view that VDR evaluation is a potential new prognostic biomarker to help in the optimization of therapy management for BC patients.

Keywords: breast cancer; circulating tumor cells; vitamin D receptor.

Figure 1

VDR expression detection in MCF-7 and T47D cells mixed with PBMCs. Fluorescence labeling of VDR (in red), CK (in green), and CD45 (in blue) was performed on a 10⁶ MCF-7 and T47D cells/PBMCs mix (3/1). Original magnification, ×40. Scale bar (white bar in the upper left image), 10 μm. CK: cytokeratin; VDR: vitamin D receptor; PBMCs: peripheral blood mononuclear cells.

Figure 2

VDR expression detection in MCF-7 cells. Fluorescence labeling of VDR (in red) and CK (in green) with DAPI (in blue) nuclear staining was performed on 10⁶ MCF-7 cells. Original magnification, ×40. Scale bar (white bar in the upper left image), 10 µm. DAPI: 4′-6-Diamidino-2-Phenylindole.

Figure 3

VDR expression in nine BC and one endometrial cell lines. Fluorescence labeling of VDR (in red), CK with DAPI (in blue) nuclear staining was performed on 10⁶ cells from 10 cell lines. Various levels of VDR expression were observed, including low (+), average (+~++), or high (+++) levels. Photographs presented are representative of five independent reproducible experiments. Original magnification, ×40. Scale bar (white bar in the upper left image), 10 μm. BC: breast cancer.

Figure 4

VDR status determination on CTCs of metastatic BC patients. Triple fluorescence labeling of CD45 (in blue), CK (in green), and VDR (in red) was performed on 10⁶ PBMCs, with parallel phase analysis. CTCs (with white arrows) were classified as VDR+ or VDR-. For both patients M25 (a,b) or M16 (c–f), either status was observed with superimposed VDR and CK labeling. CTCs exhibit size heterogeneity for patient M16 (“Normal” or “Tiny” CTCs). VDR staining was also seen on PBMCs (with red arrows), with superimposed VDR and CD45 labeling. Original magnification, ×40. Scale bar (white bar in the upper left image), 10 μm.

Figure 5

Particular subtypes of CK positive CD45 negative cells. Triple fluorescence labeling of CD45, CK, and VDR was performed on 10⁶ PBMCs from patients, with parallel analysis. Distinct subtypes of CTCs (with white arrow) were observed: patient M1 (a,b) with more than 500 CTCs identified and patients M9 (c,d) and M25 (e,f) with superimposed VDR and CK labeling. VDR staining was also seen in PMBCs (with red arrow), with superimposed VDR and CD45 labeling. Original magnification, ×40. Scale bar (white bar in the upper left image), 10 μm.