Enrichment, Isolation and Molecular Characterization of EpCAM-Negative Circulating Tumor Cells.

The presence of EpCAM-positive circulating tumor cells (CTCs) in the peripheral blood is associated with poor clinical outcomes in breast, colorectal and prostate cancer, as well as the prognosis of other tumor types. In addition, recent studies have suggested that the presence of CTCs undergoing epithelial-to-mesenchymal transition and, as such, may exhibit reduced or no expression of epithelial proteins e.g. EpCAM, might be related to disease progression in metastatic breast cancer (MBC) patients. Analyzing the neoplastic nature of this EpCAM-low/negative (EpCAM-neg) subpopulation remains an open issue as the current standard detection methods for CTCs are not efficient at identifying this subpopulation of cells. The possible association of EpCAM-neg CTCs with EpCAM-positive (EpCAM-pos) CTCs and role in the clinicopathological features and prognosis of MBC patients has still to be demonstrated. Several technologies have been developed and are currently being tested for the identification and the downstream analyses of EpCAM-pos CTCs. These technologies can be adapted and implemented into workflows to isolate and investigate EpCAM-neg cells to understand their biology and clinical relevance. This chapter will endeavour to explain the rationale behind the identification and analyses of all CTC subgroups, as well as to review the current strategies employed to enrich, isolate and characterize EpCAM-negative CTCs. Finally, the latest findings in the field will briefly be discussed with regard to their clinical relevance.

Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™.

Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch® and the CellCelector™ micromanipulation system. CTCs were isolated from CellSearch® cartridges using the CellCelector™ system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector™ screen we reidentified 97% of CellSearch® SKBR-3 cells. Furthermore, we isolated 97% of CellSearch® -proven patient CTCs using the CellCelector™ system. Therein, we found an almost perfect correlation of R2  = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch® CTC count (n = 271) and the CellCelector™ detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF-7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector™ system ensures a comprehensive detection of CTCs preidentified by the CellSearch® system. Moreover, the isolation of CTCs after CellSearch® using the CellCelector™ system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125-132, 2017.

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients.

The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients. From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch(®). Genomic DNA of enriched CTCs was isolated, amplified and analyzed for PIK3CA mutations in exons 9 and 20 which lead to E542K, E545K or H1047R amino acid changes and result in increased PI3K activity. The mutations were detected by using SNaPshot-methodology comprising PCR amplification and single nucleotide primer extension. SNaPshot analysis was established using genomic DNA from different breast cancer cell lines and then successfully transferred to investigate blood samples and single cells. Overall, twelve hotspot mutations in either exon 9/E545K (6/12, 50%) or exon 20/H1047R (6/12, 50%) could be determined within 9 out of 57 (15.8%) blood samples from 7 out of 44 (15.9%) patients; CTC counts ranged from 1 to 9748. PIK3CA variants E542K, E545G and E545A were not detected. Analysing the PIK3CA genotype of CTCs has clinical relevance with respect to drug resistance, e.g. against HER2-targeted therapy. The herein described approach including SNaPshot technology provides a simple method to characterize hotspot mutations within CTCs enriched from peripheral blood and can be easily adopted for analysing further therapeutically relevant SNPs.

Overexpression of progesterone receptor membrane component 1: possible mechanism for increased breast cancer risk with norethisterone in hormone therapy.

OBJECTIVE: Clinical trials have demonstrated an increased risk of breast cancer during estrogen/norethisterone (NET) therapy. With this in mind, the effects of estrogen/NET combination on the proliferation of breast cancer cells overexpressing the progesterone receptor membrane component 1 (PGRMC1) were examined. The same combination was used for the first time in a mouse xenograft model to determine its effects on tumor development. METHODS: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12 cells) or empty vector control (pcDNA-3HA). NET, medroxyprogesterone acetate (MPA), and progesterone were tested alone and sequentially and continuously combined with estradiol (E2). Six-week-old nude mice were inoculated with E2 pellets 24 hours before the injection of tumor cells into both flanks (n = 5-6 mice per group). After 8 days, animals were inoculated with a NET pellet or with placebo pellets, and tumor volumes were recorded twice a week. RESULTS: NET alone significantly increased the proliferation of WT-12 cells, MPA was effective only at the two highest concentrations, and progesterone had no effect. The twofold to threefold E2-induced increase (10 M) was not significantly influenced by the addition of the various progestogens. In contrast, 10 M E2 had no effect; however, addition of MPA and NET triggered a significant proliferative response. In vivo, a sequential combination of NET and E2 also significantly increased the tumor growth of WT-12 cells; empty vector cells did not respond to NET. CONCLUSIONS: We have demonstrated for the first time that an E2/NET combination increases the proliferation of PGRMC1-overexpressing breast cancer cells, both in vivo and in vitro. Our results suggest that undetected tumor cells overexpressing PGRMC1 may be more likely to develop into frank tumor cells in women undergoing E2/NET hormone therapy.

Membrane-receptor initiated proliferative effects of dienogest in human breast cancer cells.

OBJECTIVES: Dienogest (DNG) is already used in hormone therapy, since recently being also the progestogenic component of the first estradiol based contraceptive pill. Data on breast cancer risk are currently not available. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in tissues of breast cancer patients and has already been proposed as a predictor for breast cancer risk. METHODS: MCF-7 cells overexpressing PGRMC1 were stimulated with DNG, medroxyprogesterone acetate (MPA), norethisterone (NET) and progesterone (P) as well as sequentially and continuously combined with estradiol (E2). RESULTS: DNG and MPA alone elicited a significant proliferation at 10⁻6 and 10⁻5 M. NET increased cell proliferation at all concentrations tested whereas P showed no effect. E2 alone elicited a significant increase at 10⁻¹° M, no effect was seen at 10⁻¹² M. Addition of the progestins (10⁻6 M) to E2 at 10⁻¹° M had, compared to E2 only, no additional proliferating effect. However, at the low E2 concentration, DNG, MPA and NET significantly increased the E2-stimulated cell proliferation. CONCLUSION: DNG increased proliferation alone and in combination with low E2 concentrations. Thus a progestogen-derived breast cancer risk in the presence of low E2 concentrations cannot be excluded at least in women overexpressing PGRMC1.

Nomegestrol acetate sequentially or continuously combined to estradiol did not negatively affect membrane-receptor associated progestogenic effects in human breast cancer cells.

OBJECTIVES: Recently the first monophasic contraceptive pill containing estradiol has been developed which is thought to be a milestone in contraception. Nomegestrol acetate (NOM) is the progestogenic component. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the tissue of breast cancer patients, and can predict a progestogen dependent risk of breast cancer. METHODS: MCF-7 cells were transfected with PGRMC1 expression plasmid, and were stimulated with estradiol (E2, 10(-12) and 10(-10) M). NOM, progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) (each 10(-7) M) were added sequentially or continuously. RESULTS: E2 at 10(-10) M elicited a significant increase of cell proliferation from 150 to 200%. No effect was seen at 10(-12) M. Addition of the progestogens to E2 at 10(-10) M had no significant effect. However, at an E2 10(-12) M, NET significantly stimulated cell proliferation more pronounced in the continuous combined model. No effect was seen for NOM, P and MPA. The E2/NET combined effect could be abrogated by the addition of an estrogen receptor (ER) antagonist. CONCLUSION: Since NOM did not increase proliferation it may be concluded that it will be neutral in terms of breast cancer risk when combined with E2 at least in women overexpressing PGRMC1.

New insight on a possible mechanism of progestogens in terms of breast cancer risk.

OBJECTIVES: Progestogens influence mammary gland development and probably breast cancer tumorigenesis by regulating a broad spectrum of physiological processes. We investigated receptor membrane-initiated actions of progestogens in MCF-7 breast cancer cells overexpressing progesterone receptor membrane component 1 (PGRMC1). DESIGN: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (MCF-7/PGRMC1-3HA) and overexpression of PGRMC1 was verified by immune fluorescent analysis and Western blot. To test the effects of progestogens on cell proliferation, MCF-7 and MCF-7/PGRMC1-3HA cells were stimulated with a membrane-impermeable progesterone: BSA-fluorescein-isothiocyanate conjugate (P4-BSA-FITC), unconjugated progesterone (P4), medroxyprogesterone acetate (MPA), norethisterone (NET) and drospirenone (DRSP). Furthermore, reverse phase protein technology was applied to identify modified downstream signaling. RESULTS: Progesterone did not elicit any proliferative effect on MCF-7/PGRMC1-3HA cells. By contrast, P4-BSA-FITC, DRSP, MPA and NET significantly triggered proliferation of MCF-7/PGRMC1-3HA cells, the effect being more pronounced for NET. Almost no effect of progestogens on proliferation was observed in MCF-7 cells. In MCF-7/PGRMC1-3HA cells, expression of Erk1/2 was significantly reduced by 40% compared to MCF-7 cells. CONCLUSIONS: Our data indicate that PGRMC1 mediates a progestogen-dependent proliferative signal in MCF-7 cells. Of significant interest is that progesterone and synthetic progestins that are used for hormone therapy are different in their proliferative effects on MCF-7 and MCF-7/PGRMC1-3HA cells. Progesterone appears to act neutrally, whereas MPA, NET and DRSP trigger proliferation and thus might increase breast cancer risk. The data presented are very important in terms of the positive results of progestogens and breast cancer risk in clinical studies so far.