Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products.

INTRODUCTION: Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS: DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS: Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS: The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

Immunohistochemical detection of lymph node-DTCs in patients with node-negative HNSCC.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

A new workflow for whole-genome sequencing of single human cells.

Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.

Genomic high-resolution profiling of single CKpos/CD45neg flow-sorting purified circulating tumor cells from patients with metastatic breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) are promising surrogate markers for systemic disease, and their molecular characterization might be relevant to guide more individualized cancer therapies. To enable fast and efficient purification of individual CTCs, we developed a work flow from CellSearch(TM) cartridges enabling high-resolution genomic profiling on the single-cell level. METHODS: Single CTCs were sorted from 40 CellSearch samples from patients with metastatic breast cancer using a MoFlo XDP cell sorter. Genomes of sorted single cells were amplified using an adapter-linker PCR. Amplification products were analyzed by array-based comparative genomic hybridization, a gene-specific quantitative PCR (qPCR) assay for cyclin D1 (CCND1) locus amplification, and genomic sequencing to screen for mutations in exons 1, 9, and 20 of the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) gene and exons 5, 7, and 8 of the tumor protein p53 (TP53) gene. RESULTS: One common flow-sorting protocol was appropriate for 90% of the analyzed CellSearch cartridges, and the detected CTC numbers correlated positively with those originally detected with the CellSearch system (R(2) = 0.9257). Whole genome amplification was successful in 72.9% of the sorted single CTCs. Over 95% of the cells displayed chromosomal aberrations typical for metastatic breast cancers, and amplifications at the CCND1 locus were validated by qPCR. Aberrant CTCs from 2 patients harbored mutations in exon 20 of the PIK3CA gene. CONCLUSIONS: This work flow enabled effective CTC isolation and provided insights into genomic alterations of CTCs in metastatic breast cancer. This approach might facilitate further molecular characterization of rare CTCs to increase understanding of their biology and as a basis for their molecular screening in the clinical setting.

Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System.

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

Genetic analysis of single disseminated tumor cells in the lymph nodes and bone marrow of patients with head and neck squamous cell carcinoma.

Considering the limited information on the biology and molecular characteristics of disseminated tumor cells (DTCs) in head and neck squamous cell carcinoma (HNSCC), we examined the genomic alterations in DTCs from HNSCCs and their potential clinical relevance. To analyze both the lymphatic and hematogenous routes of tumor cell dissemination, we investigated samples from lymph nodes (LNs) and bone marrow (BM) of 49 patients using immunofluorescence double staining for epithelial cells expressing cytokeratin 18 (KRT18) and/or epithelial cell adhesion molecules (EpCAM, CD326). The identified marker-positive cells were isolated by micromanipulation followed by single-cell whole-genome amplification and metaphase-based comparative genomic hybridization (mCGH) to determine genome-wide copy number alterations. The findings were correlated with clinical parameters and follow-up data. We detected chromosomal aberrations in KRT18- and EpCAM-positive cells from both compartments; BM-derived cells showed a significantly higher percentage of aberrant genome (PAG) per cell than cells detected in LNs. No significant association was found between DTC data and clinical follow-up. Genomic profiling of BM-DTCs revealed genomic alterations typical for HNSCC, suggesting hematogenous dissemination of subclones around the time of surgery. In contrast, DTC data in LNs revealed that several marker-positive cells were not of malignant origin, indicating the presence of epithelial glandular inclusions in parts of the processed neck LN samples. Therefore, DTC detection of LNs in the neck based only on epithelial markers is not advisable and requires detection of chromosomal instability (CIN), gene mutations, or additional markers, which have yet to be identified. Nevertheless, our investigation paves the way for larger studies to focus on HNSCC BM-DTCs with high-resolution methods to gain deeper insights into the biology of hematogenous metastasis in this cancer.

Multiparametric Circulating Tumor Cell Analysis to Select Targeted Therapies for Breast Cancer Patients.

BACKGROUND: The analysis of liquid biopsies, e.g., circulating tumor cells (CTCs) is an appealing diagnostic concept for targeted therapy selection. In this proof-of-concept study, we aimed to perform multiparametric analyses of CTCs to select targeted therapies for metastatic breast cancer patients. METHODS: First, CTCs of five metastatic breast cancer patients were analyzed by whole exome sequencing (WES). Based on the results, one patient was selected and monitored by longitudinal and multiparametric liquid biopsy analyses over more than three years, including WES, RNA profiling, and in vitro drug testing of CTCs. RESULTS: Mutations addressable by targeted therapies were detected in all patients, including mutations that were not detected in biopsies of the primary tumor. For the index patient, the clonal evolution of the tumor cells was retraced and resistance mechanisms were identified. The AKT1 E17K mutation was uncovered as the driver of the metastatic process. Drug testing on the patient's CTCs confirmed the efficacy of drugs targeting the AKT1 pathway. During a targeted therapy chosen based on the CTC characterization and including the mTOR inhibitor everolimus, CTC numbers dropped by 97.3% and the disease remained stable as determined by computer tomography/magnetic resonance imaging. CONCLUSION: These results illustrate the strength of a multiparametric CTC analysis to choose and validate targeted therapies to optimize cancer treatment in the future. Furthermore, from a scientific point of view, such studies promote the understanding of the biology of CTCs during different treatment regimens.

GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Magnetic-Based Enrichment of Rare Cells from High Concentrated Blood Samples.

Here, we tested two magnetic-bead based systems for the enrichment and detection of rare tumor cells in concentrated blood products. For that, the defined numbers of cells from three pancreatic cancer cell lines were spiked in 108 peripheral blood mononuclear cells (PBMNCs) concentrated in 1 mL, mimicking diagnostic leukapheresis (DLA) samples, and samples were processed for circulating tumor cells (CTC) enrichment with the IsoFlux or the KingFisher systems, using different types of magnetic beads from the respective technology providers. Beads were conjugated with different anti-EpCAM and MUC-1 antibodies. Recovered cells were enumerated and documented by fluorescent microscopy. For the IsoFlux system, best performance was obtained with IsoFlux CTC enrichment kit, but these beads compromised the subsequent immunofluorescence staining. For the KingFisher system, best recoveries were obtained using Dynabeads Biotin Binder beads. These beads also allowed one to capture CTCs with different antibodies and the subsequent immunofluorescence staining. KingFisher instrument allowed a single and streamlined protocol for the enrichment and staining of CTCs that further prevented cell loss at the enrichment/staining interface. Both IsoFlux and KingFisher systems allowed the enrichment of cell line cells from the mimicked-DLA samples. However, in this particular experimental setting, the recovery rates obtained with the KingFisher system were globally higher, the system was more cost-effective, and it allowed higher throughput.

FGF Signalling in the Self-Renewal of Colon Cancer Organoids.

The progression of colorectal cancer (CRC) is supposedly driven by cancer stem cells (CSC) which are able to self-renew and simultaneously fuel bulk tumour mass with highly proliferative and differentiated tumour cells. However, the CSC-phenotype in CRC is unstable and dependent on environmental cues. Fibroblast growth factor 2 (FGF2) is essential and necessary for the maintenance of self-renewal in adult and embryonic stem cells. Investigating its role in self-renewal in advanced CRC patient-derived organoids, we unveiled that FGF-receptor (FGFR) inhibition prevents organoid formation in very early expanding cells but induces cyst formation when applied to pre-established organoids. Comprehensive transcriptome analyses revealed that the induction of the transcription factor activator-protein-1 (AP-1) together with MAPK activation was most prominent after FGFR-inhibition. These effects resemble mechanisms of an acquired resistance against other described tyrosine kinase inhibitors such as EGF-receptor targeted therapies. Furthermore, we detected elevated expression levels of several self-renewal and stemness-associated genes in organoid cultures with active FGF2 signalling. The combined data assume that CSCs are a heterogeneous population while self-renewal is a common feature regulated by distinct but converging pathways. Finally, we highlight FGF2 signalling as one of numerous components of the complex regulation of stemness in cancer.

Array comparative genomic hybridization of 18 pancreatic ductal adenocarcinomas and their autologous metastases.

BACKGROUND: Mortality rates of pancreatic cancer remain high, which is mainly due to advanced disease and metastasis. We hypothesized that genomic copy number alterations are enriched in metastatic cells compared to autologous primary tumors, which may inform on cancer-related pathways possibly serving as potential targets for specific therapies. We investigated 18 pancreatic ductal adenocarcinomas, including 39 lymph node and 5 distant metastases after surgical resection. Analysis was performed with array-based comparative genomic hybridization (aCGH). RESULTS: Metastases acquire a higher frequency of copy number alterations with the highest in distant metastasis (median = 42, lymph node metastases: median = 23, primary tumors: median = 17). In lymph node metastases, gains were prevalent on chromosome bands 8q11.23-q24.3, 12q14.1, 17p12.1, 21q22.12, and losses on 3p21.31, 4p14, 8p23.3-p11.21,17p12-11.2. Genes on amplified regions are involved in cancer-related pathways such as WNT-signaling, also involved in metastasis. CONCLUSIONS: Pancreatic cancers show a high degree of intratumor heterogeneity, which could lead to resistance of chemotherapy and worse outcome. ACGH analysis reveals regions preferentially gained or lost in synchronous metastases encoding for genes involved in cancer-related pathways, which could lead to novel therapeutic opportunities.

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.

Immune escape and survival mechanisms in circulating tumor cells of colorectal cancer.

The prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer-derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer-derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer-derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694-704. ©2014 AACR.

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.