Short-Term Ex Vivo Culture of CTCs from Advance Breast Cancer Patients: Clinical Implications

Abstract

Background: Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. Thus, it is essential to identify these surviving cells capable of forming such metastases.

Methods: We isolated viable CTCs from 50 peripheral blood samples obtained from 35 patients with advanced metastatic breast cancer using RosetteSep^TM for ex vivo culture. The CTCs were seeded and monitored on plates under low adherence conditions and with media supplemented with growth factors and Nanoemulsions. Phenotypic analysis was performed by immunofluorescence and gene expression analysis using RT-PCR and CTCs counting by the Cellsearch^® system.

Results: We found that in 75% of samples the CTC cultures lasted more than 23 days, predicting a shorter Progression-Free Survival in these patients, independently of having ≥5 CTC by Cellsearch^®. We also observed that CTCs before and after culture showed a different gene expression profile.

Conclusions: the cultivability of CTCs is a predictive factor. Furthermore, the subset of cells capable of growing ex vivo show stem or mesenchymal features and may represent the CTC population with metastatic potential in vivo.

Keywords: CTC; breast cancer; cell culture; liquid biopsy.

Figure 1

Schematic workflow of Circulating Tumor Cells (CTCs) samples isolation, culture and analyses. (1) Methodology followed for the isolation and ex vivo cell culture of CTCs from metastatic Breast Cancer (mBC) patients. Cells were isolated using: CellSearch^® System (determination of EpCAM⁺ cells) and RosetteSep^TM (enrichment of CTCs for cell culture). Cells were cultured under ultra-low attachment plates to support 3D culture and every two days cells were supplemented with fresh supplemented medium and Nanoemulsions (NEs). (2) Immunofluorescence characterization by confocal microscopy and fluorescence microscopy of cell cultures. (3) Gene expression analysis was performed using paired samples before and after cell culture. (4) Survival analysis was carried out to determine the relation between time (days) of culture or presence of Red Blood Cells (RBCs) as a predictive factor in the patient’s outcome.

Figure 2

(A) Representative image of cultured Circulating Tumor Cells (CTCs) in ultra-low attachment 96-well plates at 7 and 15 days (t7, t15), left and right panel, respectively. Two growth behaviors were observed: (i) cells that grow in suspension (Susp.) (upper panel); (ii) cells growing in suspension (Susp.) co-existing with adherent cells (Adh.) (bottompanel). Blue arrows represent cells in suspension while orange arrows point to cells growing in adherence. Both samples had not receive treatment (basal). The scales bar represent 75 µm. (B) Number of CTCs measured by CellSearch^® with respect to their ability to establish a successful culture in 34 metastatic breast cancer samples. Negative culture, in blue (n = 11) and positive culture in red (n = 25).

Figure 3

Representative images by confocal microscopy of Immunofluorescence characterization of Circulating Tumor Cells (CTCs) after culture. Immunofluorescent staining was performed using a combination of anti-human Epithelial markers (Epith: EpCAM, E-Cadh, and PanCK) (in green), anti-Vimentin (VIM, in yellow), and anti-CD45 (in red). Scale bar represents 25 µm.

Figure 4

CTC gene expression analysis of six samples. Of these samples, four of them were not included in the previous immunofluorescence characterization. Five samples were collected after therapy initiation. (A) Relative gene expression for epithelial markers (E-Cadh, EpCAM and KRT5) of CTC-fraction isolated by Rosettesep and normalized with paired Peripheral Blood Mononuclear Cells (PBMCs) (before culture). Comparison with Cellsearch^® data is shown. (B) Clustered heatmap depicting CTCs gene expression levels of the listed genes. Light red indicates no expression. Relative gene expression (to B2M) was ranged and coded from 1 to 7 (dark red). (C) Principal component analysis using gene expression of the listed genes (see Heatmap).

Figure 5

Kaplan–Meier plots for Progression free Survival (PFS) and Overall Survival (OS) for: cultivability (Culture negative (black) or positive (red)) (A,C) and CTCs enumeration by Cellsearch^® system (≥5 CTCs (red) or <5 CTCs (black) (B,D). p-values were calculated using the log-rank test.

Figure 6

Kaplan–Meier plots for PFS (A) and OS (B) for presence of RBCs (red) or absence (black) in the culture. p-values were calculated using the log-rank test.