Isolation of circulating epithelial and tumor progenitor cells with an invasive phenotype from breast cancer patients

Abstract

Recent research advances show that tumor cell intravasation (entry into the circulation) and metastasis occur very early in breast cancer progression. Clinical studies also illustrate the potential importance of detection of circulating tumor cells (CTCs) in outcomes of patients with metastatic breast cancer. Whether these cells exhibit the invasiveness and express tumor stem or progenitor markers, hallmark of the metastatic phenotype, is less well characterized. To detect CTCs with the invasive phenotype and to explore their molecular features, we applied a functional cell separation method, called collagen adhesion matrix (CAM) assay, as enrichment and identification steps. The CAM-coated device successfully recovered tumor cells spiked in 1 ml of blood with a 54% 6 9% (n 5 18) recovery rate and 0.5-35% purity, and detected invasive tumor cells in 10/10 blood samples (100% yield) from patients with metastatic breast cancer with a range of 18-256 CTCs/ml and average of 126 6 25 (mean 6 SD) CTCs/ml. CTCs were detected in blood samples of 28/54 (52%) Stage I-III breast cancer patients with a mean count of 61 CTCs/ml. Furthermore, the relative frequency of these cells correlated to the staging, lymph node-status and survival of patients with early stage breast cancer. CAM-captured cells were capable of propagation in culture. Gene expression and multiplex flow cytometric analyses on CAM-captured cells demonstrated the existence of distinct populations of CTCs including these of epithelial lineage and stem or progenitor cells. Thus, CAM-initiated CTC detection provides advantages for examining invasiveness and tumor progenitor phenotypes.Recent studies show that entry of tumor cells into the circulation and subsequent metastasis occur early in breast cancer progression.

Molecular mechanisms of metastasis

“…EPISPOT has been applied to blood and BM samples of breast, prostate, and colon cancer patients, providing initial clinical data. Another functional cell separation method called the collagen adhesion matrix (CAM) assay has been recently reported in breast, prostate, and ovarian cancer: CAM ingestion and epithelial immunostaining identifies CTC based on their invasive properties in vitro [89].…”

Circulating Tumor Cells: A Review of Non–EpCAM-Based Approaches for Cell Enrichment and Isolation

“…Nonadherent, supposedly nontarget cells are washed off, leaving the CTCs attached to the surface. On the basis of this approach, the cell adhesion matrix has been used to detect and isolate the most invasive CTCs from patients with metastatic and local carcinomas of different origins (64,65 ). Microfluidic adhesion-based devices consist of microchannels coated with an antibody against CTCs.…”

The Promise of Circulating Tumor Cells for Precision Cancer Therapy

“…The Vita-Assay™ (Vitatex, NY, USA) method identifies CTCs based on preferential adhesion of invasive CTCs to a tissue or tumor micro environment mimic (cell adhesion matrix or CAM), which has been shown to enrich viable CTCs from blood up to 1,000,000-fold [82,83]. Moreover, CAM-captured CTCs can ingest the CAM itself such that the use of fluorescent-labeled CAM allows for direct visualization of cancer cell invasion (Figure 1).…”