Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems.

The analysis of circulating tumor cells (CTCs) has shown potential for detection of cancer spread, prognosis, therapeutic target selection, and monitoring of treatment response. CTCs can be obtained repeatedly by simple blood draws as so-called "liquid biopsy." Thus, they can serve as a surrogate material for primary or metastatic tissue biopsies. In addition, isolation of CTCs provides the possibility to investigate those cells which may hold the (molecular) traits responsible for metastatic progression and ultimately patient death. As such, CTCs represent a target of utmost importance in cancer research and therapy. In this chapter, we describe a workflow for the enrichment of CTCs with the FDA-cleared CellSearch® system followed by the isolation of single CTCs using the DEPArray™ technology enabling further molecular single-cell analyses.

53BP1 Accumulation in Circulating Tumor Cells Identifies Chemotherapy-Responsive Metastatic Breast Cancer Patients.

Evidence suggests that the DNA end-binding protein p53-binding protein 1 (53BP1) is down-regulated in subsets of breast cancer. Circulating tumor cells (CTCs) provide accessible "biopsy material" to track cell traits and functions and their alterations during treatment. Here, we prospectively monitored the 53BP1 status in CTCs from 67 metastatic breast cancer (MBC) patients with HER2- CTCs and known hormone receptor (HR) status of the primary tumor and/or metastases before, during, and at the end of chemotherapeutic treatment with Eribulin. Nuclear 53BP1 staining and genomic integrity were evaluated by immunocytochemical and whole-genome-amplification-based polymerase chain reaction (PCR) analysis, respectively. Comparative analysis of CTCs from patients with triple-negative and HR+ tumors revealed elevated 53BP1 levels in CTCs from patients with HR+ metastases, particularly following chemotherapeutic treatment. Differences in nuclear 53BP1 signals did not correlate with genomic integrity in CTCs at baseline or with nuclear γH2AX signals in MBC cell lines, indicating that 53BP1 detected features beyond DNA damage. Kaplan-Meier analysis revealed an increasing association between nuclear 53BP1-positivity and progression-free survival (PFS) during chemotherapy until the final visit. Our data suggest that 53BP1 detection in CTCs could be a useful marker to capture dynamic changes of chemotherapeutic responsiveness in triple-negative and HR+ MBC.

Detection of ESR1 Mutations in Single Circulating Tumor Cells on Estrogen Deprivation Therapy but Not in Primary Tumors from Metastatic Luminal Breast Cancer Patients.

Mutations in the ligand-binding domain (LBD) of the ESR1 gene result in resistance to estrogen deprivation therapy (EDT) in breast cancer. Their detection might enable optimization of therapy strategies. However, the predictive utility of the primary tumor (PT) is limited, and obtaining serial biopsies of metastatic lesions is challenging. To underline their application as a liquid biopsy, single circulating tumor cells (CTCs) were analyzed with a next-generation sequencing approach for the ESR1 coding region. CTCs from 46 metastatic luminal breast cancer patients were enriched using CellSearch system and isolated by micromanipulation. Their genomic DNA was amplified and the ESR1 gene was sequenced. Furthermore, tissue samples from corresponding PTs and/or metastatic lesions were investigated. ESR1 mutations were detected in 12 patients-exclusively in patients treated with EDT (P = 0.048). In seven cases mutations were located in the hotspot regions in the LBD. Six novel mutations were identified. ESR1 mutations were absent in PT tissue samples and were detected only in metastases obtained after CTC characterization. Single-cell CTC analysis for ESR1 mutations could be of clinical value to identify patients who progress under EDT and therefore benefit from an early switch to an alternative endocrine therapy or other treatment regimens. Furthermore, our data indicate that mutations outside the LBD's hotspot regions might also contribute to resistance to EDT.

Microfluidic enrichment, isolation and characterization of disseminated melanoma cells from lymph node samples.

For the first time in melanoma, novel therapies have recently shown efficacy in the adjuvant therapy setting, which makes companion diagnostics to guide treatment decisions a desideratum. Early spread of disseminated cancer cells (DCC) to sentinel lymph nodes (SLN) is indicative of poor prognosis in melanoma and early DCCs could therefore provide important information about the malignant seed. Here, we present a strategy for enrichment of DCCs from SLN suspensions using a microfluidic device (Parsortix™, Angle plc). This approach enables the detection and isolation of viable DCCs, followed by molecular analysis and identification of genetic changes. By optimizing the workflow, the established protocol allows a high recovery of DCC from melanoma patient-derived lymph node (LN) suspensions with harvest rates above 60%. We then assessed the integrity of the transcriptome and genome of individual, isolated DCCs. In LNs of melanoma patients, we detected the expression of melanoma-associated transcripts including MLANA (encoding for MelanA protein), analyzed the BRAF and NRAS mutational status and confirmed the malignant origin of isolated melanoma DCCs by comparative genomic hybridization. We demonstrate the feasibility of epitope-independent isolation of LN DCCs using Parsortix™ for subsequent molecular characterization of isolated single DCCs with ample application fields including the use for companion diagnostics or subsequent cellular studies in personalized medicine.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.

Single-cell analysis of CTCs with diagnostic precision: opportunities and challenges for personalized medicine.

The generation of variant cancer cells is the major cause of acquired resistance against systemic therapies and consequently, of our inability to cure advanced cancer patients. Circulating tumor cells are gaining increasing clinical attention because they may enable the monitoring cancer progression and adjustment of treatment. In recent years multiple technologies for enrichment, isolation as well as molecular and functional analysis of circulating tumor cells have been developed. Implementation of these technologies in standardized and automated workflows in clinical diagnostics could provide valuable information for real-time monitoring of cancer and eventually new therapeutic strategies for the benefit of patients.

Molecular profiling of single circulating tumor cells with diagnostic intention.

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

The insulator protein CTCF binding sites in the orf73/LANA promoter region of herpesvirus saimiri are involved in conferring episomal stability in latently infected human T cells.

Herpesviruses establish latency in suitable cells of the host organism after a primary lytic infection. Subgroup C strains of herpesvirus saimiri (HVS), a primate gamma-2 herpesvirus, are able to transform human and other primate T lymphocytes to stable growth in vitro. The viral genomes persist as nonintegrated, circular, and histone-associated episomes in the nuclei of those latently infected T cells. Epigenetic modifications of episomes are essential to restrict the transcription during latency to selected viral genes, such as the viral oncogenes stpC/tip and the orf73/LANA. In this study, we describe a genome-wide chromatin immunoprecipitation-on-chip (ChIP-on-chip) analysis to profile the occupancy of CTCF on the latent HVS genome. We then focused on two distinct, conserved CTCF binding sites (CBS) within the orf73/LANA promoter region. Analysis of recombinant viruses harboring deletions or mutations within the CBS indicated that the lytic replication of such viruses is not substantially influenced by CTCF. However, T cells latently infected with CBS mutants were impaired in their proliferation abilities and showed a significantly reduced episomal maintenance. We detected a reduced transcription of the orf73/LANA gene in the T cells, corresponding to the reduced viral genomes; this might contribute to the loss of HVS episomes, as LANA is central in the maintenance of viral episomes in the dividing T cell populations. These data demonstrate that the episomal stability of HVS genomes in latently infected human T cells is dependent on CTCF.

Genome-wide histone acetylation profiling of Herpesvirus saimiri in human T cells upon induction with a histone deacetylase inhibitor.

Herpesviruses establish latency in suitable host cells after primary infection and persist in their host organisms for life. Most of the viral genes are silenced during latency, also enabling the virus to escape from an immune response. This study addresses the control of viral gene silencing by epigenetic mechanisms, using Herpesvirus saimiri (HVS) as a model system. Strain C488 of this gamma-2-herpesvirus can transform human T cells to stable growth in vitro, and it persists in the nuclei of those latently infected T cells as a nonintegrating, circular, and histone-associated episome. The whole viral genome was probed for histone acetylation at high resolution by chromatin immunoprecipitation-on-chip (ChIP-on-chip) with a custom tiling microarray. Corresponding to their inactive status in human T cells, the lytic promoters consistently revealed a heterochromatic phenotype. In contrast, the left terminal region of the genome, which encodes the stably expressed oncogenes stpC and tip as well as the herpesvirus U RNAs, was associated with euchromatic histone acetylation marks representing "open" chromatin. Although HVS latency in human T lymphocytes is considered a stable and irreversible state, incubation with the histone deacetylase inhibitor trichostatin A resulted in changes reminiscent of the induction of early lytic replication. However, infectious viral particles were not produced, as the majority of cells went into apoptosis. These data show that epigenetic mechanisms are involved in both rhadinoviral latency and transition into lytic replication.

Episomal replication timing of gamma-herpesviruses in latently infected cells.

This study addresses the timing of gammaherpesviral episomal DNA replication with respect to the cell cycle. For the first time we analyzed a rhadinovirus, the prototype Herpesvirus saimiri (HVS), and compared it to the lymphocryptovirus Epstein-Barr virus (EBV). Newly synthesized DNA of latently infected B- or T-cells was first BrdU-labeled; then we sorted the cells corresponding to cell cycle phases G(0/1), G(2/M), and S (4 fractions S(1)-S(4)) and performed anti-BrdU chromatin immunoprecipitation. Next, DNA of different viral gene loci was quantitatively detected together with cellular control genes of known replication time. The sensitive technique is further enhanced by an internal coprecipitation standard for increased precision. Both gammaherpesviruses replicated very early in S-phase, together with cellular euchromatin. Our work suggests that early S-phase DNA replication is a general characteristic of episomal herpesviral genomes.

Histone modification pattern of the T-cellular Herpesvirus saimiri genome in latency.

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.