Current Circulating Tumor Cells Detection Research Articles

Utilization of Circulating Tumor Cells in the Management of Solid Tumors.

Circulating tumor cells (CTCs) are tumor cells shed from the primary tumor into circulation, with clusters of CTCs responsible for cancer metastases. CTC detection and isolation from the bloodstream are based on properties distinguishing CTCs from normal blood cells. Current CTC detection techniques can be divided into two main categories: label dependent, which depends upon antibodies that selectively bind cell surface antigens present on CTCs, or label-independent detection, which is detection based on the size, deformability, and biophysical properties of CTCs. CTCs may play significant roles in cancer screening, diagnosis, treatment navigation, including prognostication and precision medicine, and surveillance. In cancer screening, capturing and evaluating CTCs from peripheral blood could be a strategy to detect cancer at its earliest stage. Cancer diagnosis using liquid biopsy could also have tremendous benefits. Full utilization of CTCs in the clinical management of malignancies may be feasible in the near future; however, several challenges still exist. CTC assays currently lack adequate sensitivity, especially in early-stage solid malignancies, due to low numbers of detectable CTCs. As assays improve and more trials evaluate the clinical utility of CTC detection in guiding therapies, we anticipate increased use in cancer management.

Bioinspired nano-plate-coral platform enabled efficient detection of circulating tumor cells via the synergistic capture of multivalent aptamer and tumor cell membrane

Circulating tumor cells (CTCs) offer rich information for early disease diagnosis and therapy evaluation. However, the limited sensitivity, binding affinity, and stability of current monovalent recognition-based CTCs detection techniques remain a challenge for extending their applications. Inspired by the highly efficient predation manner of plate corals, we firstly introduce an efficient and sensitive biomimetic CTCs recognition platform based on the conjugation of multivalent aptamer onto tumor cell membrane-coated magnetic graphene oxide to form a plate coral-like CTCs capture nanoprobe (MNPA-TCMMGO). In this method, the tumor cell membrane was employed to provide a biomimetic homologous fluidic interface for targeting homologous tumor cells. At the same time, multivalent aptamers were used as capture probes, which greatly enhanced the binding affinity and association probability between aptamer and target cells via cooperative multivalent effect. The unique features (robustness, high binding affinity and specificity, and biocompatibility) of MNPA-TCMMGO allow efficient, sensitive, and specific capture of rare tumor cells from biological samples. More importantly, the captured cells could maintain good viability, which is crucial for downstream analysis. Therefore, our developed biomimetic approach offers a new way to address the limitations of current CTCs detection methods and presents considerable potential for clinical cancer diagnostics.

Advances in the Biology, Detection Techniques, and Clinical Applications of Circulating Tumor Cells.

Circulating tumor cells (CTCs) play a crucial role in tumor recurrence and metastasis, and their early detection has shown remarkable benefits in clinical theranostics. However, CTCs are extremely rare, thus detecting them in the blood is very challenging. New CTC detection techniques are continuously being developed, enabling deeper analysis of CTC biology and potential clinical application. This article reviews current CTC detection techniques and their clinical application. CTCs have provided, and will continue to provide, important insights into the process of metastasis, which could lead to development of new therapies for different cancers.

A chimeric virus-based probe unambiguously detects live circulating tumor cells with high specificity and sensitivity

The current methods for detecting circulating tumor cells (CTCs) suffer from several drawbacks. We report a novel method that is based on a chimeric virus probe and can detect CTCs with extremely high specificity and sensitivity. Moreover, it exclusively detects live CTCs, and its detection efficacy is not impacted by the variation of epithelial cell adhesion molecule (EpCAM) expression. The chimeric virus probe is composed of a capsid from human papillomavirus that provides the detection with high specificity and an SV40-based genome that can amplify extensively inside CTCs and, hence, endows the detection with high sensitivity. Furthermore, different marker genes can be incorporated into the probe to provide detection with versatility. These unique capabilities will likely improve the validity and utility of this CTC detection in several clinical applications, which is one of the drawbacks suffered by many of the current CTC detection methods.

A Chimeric Virus-Based Probe Unambiguously Detects Live Circulating Tumor Cells with High Specificity and Sensitivity

The current methods for detecting circulating tumor cells (CTCs) suffer from several drawbacks. We report a novel method that is based on a chimeric virus probe and can detect CTCs with extremely high specificity and sensitivity. Moreover, it exclusively detects live CTCs, and its detection efficacy is not impacted by the variation of EpCAM expression. The chimeric virus probe is composed of a capsid from human papillomavirus that provides the detection with high specificity, and a SV40-based genome that can amplify extensively inside CTCs and, hence, endows the detection n with high sensitivity. Furthermore, different marker genes can be incorporated into the probe to provide detection with versatility. These unique capabilities will likely improve the validity and utility of this CTC detection in several clinical applications, which is one of the drawbacks suffered by many of the current CTC detection methods.

A high-throughput label-free time-stretch acoustofluidic imaging cytometer for single-cell mechanotyping

Current circulating tumor cells (CTC) detection methods have to compromise between sensitivity and throughput. High-throughput imaging cytometer based on serial time-encoded amplified microscopy (STEAM) facilitates CTC detection at single-cell sensitivity from abundant cells. However, this method lacks the information to spot heterogeneity of cells with high morphological similarity. Researches on cell biophysical properties suggest cell mechanotyping can be an indicator of phenotypic heterogeneity to improve classification ability of STEAM cytometer. Here, we present a high-throughput label-free acoustofluidic imaging cytometer for single-cell mechanotyping based on STEAM and acoustofluidic technology. The generated acoustic resonance field translocates cells to different transversal exit positions under continuous flow according to their intrinsic biophysical properties. Such displacements are recorded with images simultaneously using STEAM cytometry at approximately 2000 cells/s. We experimentally verified that our method accounting for both cell images and acoustic displacements can improve mechanotyping accuracy by 12% upon image-based phenotyping method. This new acoustofluidic imaging cytometer facilitates high-accuracy and high-throughput imaging cytometry for single-cell CTC mechanotyping.

Multifunctional Branched Nanostraw-Electroporation Platform for Intracellular Regulation and Monitoring of Circulating Tumor Cells.

Downstream analysis of circulating tumor cells (CTCs) has provided new insights into cancer research. In particular, the detection of CTCs, followed by the regulation and monitoring of their intracellular activities, can provide valuable information for comprehensively understanding cancer pathogenesis and progression. However, current CTC detection techniques are rarely capable of in situ regulation and monitoring of the intracellular microenvironments of cancer cells over time. Here, we developed a multifunctional branched nanostraw (BNS)-electroporation platform that could effectively capture CTCs and allow for downstream regulation and monitoring of their intracellular activities in a real-time and in situ manner. The BNSs possessed numerous nanobranches on the outer sidewall of hollow nanotubes, which could be conjugated with specific antibodies to facilitate the effective capture of CTCs. Nanoelectroporation could be applied through the BNSs to nondestructively porate the membranes of the captured cells at a low voltage, allowing the delivery of exogenous biomolecules into the cytosol and the extraction of cytosolic contents through the BNSs without affecting cell viability. The efficient delivery of biomolecules (e.g., small molecule dyes and DNA plasmids) into cancer cells with spatial and temporal control and, conversely, the repeated extraction of intracellular enzymes (e.g., caspase-3) for real-time monitoring were both demonstrated. This technology can provide new opportunities for the comprehensive understanding of cancer cell functions that will facilitate cancer diagnosis and treatment.

Prognostic significance of circulating tumor cells in non-small cell lung cancer patients undergoing chemotherapy.

The utility of circulating tumor cells (CTCs) as prognostic biomarkers in non-small cell lung cancer (NSCLC) is inconclusive due to the limitations of current CTC detection methods. Using a novel high-efficiency detection method, we determined the ability of CTCs to predict survival and chemotherapeutic responses in NSCLC. In 127 patients with advanced NSCLC, CTCs were counted and analyzed at baseline and during follow-up. Median overall survival (OS) and progression-free survival (PFS) were longer in patients with baseline CTC counts <8 CTCs/3.2 mL (20.0 vs. 10.4 months [P = 0.009] and 7.2 vs. 5.5 months [P < 0.001], respectively). Patients with post-treatment increases in the CTC count had poorer OS and PFS than those without increases (12.0 vs. 13.3 months [P = 0.028] and 5.2 vs. 6.4 months [P = 0.022], respectively). There was no association between the baseline CTC count and chemotherapeutic response (P = 0.734). However, the rate of progressive disease in patients with and without post-treatment increases in the CTC count were 15.6% and 2.4% (P = 0.042), respectively. The baseline CTC count and the change in the CTC count during treatment were both valuable prognostic indicators for NSCLC.

An integrated microfluidic chip for one-step isolation of circulating tumor cells

The presence of circulating tumor cells (CTCs) in blood of cancer patients is thought to be an important indicator of disease progression and survival. Due to the rarity of CTCs in the bloodstream, CTCs must be enriched for molecular and cellular analyses for clinical use. Current CTC detection methods mostly rely on epithelial cell adhesion molecule (EpCAM); however, these methods may underestimate CTC number and potentially miss a critical subpopulation because of large heterogeneity among CTCs. We describe an integrated microfluidic chip, termed the μ-MixMACS chip, that can negatively exclude background cells from a CTC population. It comprises a micromixer that generates multivortexing flows to enhance mixing, which increasing binding between white blood cells and magnetic nanoparticles coated CD45 antibody, and a magnetic sorter to remove magnet-coated cells and minimize interference from white blood cells during subsequent CTC analysis. As all processes were carried out continuous manner, any CTC loss during isolation was minimized. Blood samples from 10 patients with adjuvant breast cancer were analyzed. CTCs were sorted as unfixed cells in solution. CTCs were isolated from 5 of 10 blood samples, 1–3 CTCs were found in each of these 5 samples. The proposed μ-MixMACS chip is expected to be useful for isolating CTCs from a variety of tumor types without any changes.

Abstract 1607: Identification of low number circulating tumor cells (CTCs) for cancer treatment monitoring

Abstract For most solid tumors, the clinical use of CTCs is limited by current instrument restrictions on detection of infrequent CTCs. Based on the FDA-approved criteria used in current CTC detection instruments, we developed a microfluidic device capable of capturing, enumerating and releasing low numbers of CTCs in the peripheral blood of patients with solid tumors. The device is the size of a microscope slide, consisting of an inlet, an outlet, and eight parallel channels connecting them. Micromixers inside the channels enhance interactions between CTCs and capture agents on the channel surfaces. Epithelial cell-adhesion molecule (EpCAM) antibodies are immobilized onto the surfaces via avidin-biotin chemistry. A blood sample from a cancer patient is introduced through the inlet. Cells captured by anti-EpCAM studded channels are verified as CTCs using triple-color IHC definitions (EpCAM+, cytokeratin+, CD45-, and DAPI+). Using pancreatic cancer as a model for a low frequency CTC condition, device capture efficiency and cell purity was tested under various flow rates (shear stresses) using L3.6pl cells spiked into blood. Capture efficiency was defined as the% of target cells isolated relative to target cells present in a sample, representing device sensitivity. Cell purity was defined as the% of target cells isolated relative to all cells captured (both target and non-target cells), representing device specificity. Capture efficiency (90%) and cell purity (84%) were further optimized by changing the flow rate. A higher flow rate resulted in higher cell purity as non-target bound cells are dislodged by shear forces. This capability of parameter adjustment and performance control is enabled by the microfluidics platform used as opposed to CTC isolation using other commercially approved technologies. The device was then clinically tested in detecting CTCs and monitoring the response to anti-cancer drug treatment in patients with advanced pancreatic cancer. We identified CTCs in 113 out of 115 clinical blood samples (98%). CTC enumeration (CTC number/mL of blood) both correlated with and predated changes in the radiographic tumor size over the treatment period, ranging from 10 to 35 cycles (2 weeks per cycle) of therapy. Microfluidic devices possess the capacity to enumerate infrequent CTCs which also appear to correlate with treatment response. Note: This abstract was not presented at the meeting. Citation Format: Thomas J. George, Weian Sheng, Jose I. Varillas, Chen Liu, Z. Hugh Fan. Identification of low number circulating tumor cells (CTCs) for cancer treatment monitoring. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1607. doi:10.1158/1538-7445.AM2015-1607

Abstract 1586: Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples

Abstract Introduction: Current CTC detection platforms require the processing of patient blood within 48 hrs after collection to preserve CTC integrity and biomarker expression. Here we demonstrate the recovery of CTCs from banked patient blood samples that have been cryopreserved for up to 7 years. Our results show that cryopreserved CTCs have equivalent morphology and protein expression patterns as CTCs in freshly processed blood and are suitable for genomic analyses. This process enables retrospective studies of banked patient blood samples inclusive of CTC enumeration, biomarker quantification, FISH and genomic analysis via the Epic Platform. Methods: 30 cryopreserved mCRPC patient blood samples were sent to Epic Sciences alongside 10 matched fresh blood draws (&amp;lt;48 h after draw). All nucleated cells from mCRPC patient samples or cell line-spiked healthy donor (HD) controls were plated onto glass slides and subjected to IF staining, CTC identification by fluorescent scanners and algorithmic analysis. Traditional CTCs (CK+, CD45-, intact DAPI nuclei and morphologically distinct) and CK- CTCs (CK-, CD45-, intact and distinct) were identified. Androgen receptor (AR) expression was assessed by IF staining and PTEN status determined by FISH. Relative CTC enumeration, morphology, protein expression and genomic status were compared between freshly processed and cryopreserved samples. Results: Initial testing in VCaP (AR+) and PC3 (AR-) cell line-spiked HD blood demonstrated preservation of cell morphology and biomarker expression. In VCaP, CK signal decreased 20% and AR decreased 6% after freezing, but CK and AR expression were 90- and 8-fold higher than negative controls, respectively. In patients, CTCs were detected in 7/10 freshly processed samples and in equal relative abundance in 6/10 matched frozen samples. Cryopreserved CTCs retain CK and AR expression as compared to freshly processed samples: there were no significant differences in mean CK or AR expression among all matched samples compared, and% AR+ CTCs correlated strongly between fresh and frozen samples (r2 = 0.96). Furthermore, PTEN FISH on cryopreserved CTCs resulted in a near-equal percentage of PTEN variant cells as compared to matched fresh draws. After concordance studies, an additional 20 frozen mCRPC patient samples were processed: CTCs were detected in 16/20 samples, with 11/20 harboring AR+ CTCs. Conclusion: Recovery of CTCs in cryopreserved patient blood samples is feasible using the Epic Platform. These data demonstrate concordance of CTC recovery, protein characterization and genomic analysis in an initial cohort of cryopreserved mCRPC samples. Further studies characterizing cryopreserved CTCs in mCRPC and in cancers of other tissues are warranted, with the goal of enabling retrospective protein and genomic characterization of banked patient samples. Citation Format: David Lu, Melissa Harvey, Ravi Madan, Christopher Heery, Jennifer Marte, Sharon Beasley, Mark Landers, Rachel Krupa, Jessica Louw, Justin Wahl, Natalee Bales, Dena Marrinucci, Jeffrey Schlom, James Gulley, Ryan Dittamore. Recovery and characterization of circulating tumor cells (CTCs) in cryopreserved metastatic castrate-resistant prostate cancer (mCRPC) patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2015-1586

63. Rapid and Sensitive Detection of Circulating Tumor Cells with Nuclease-Activated Oligonucleotide Probes

Metastatic breast cancer is the second leading cause of female cancer deaths in the United States. Despite substantial progress in its treatment, metastatic breast cancer remains incurable. Early identification of breast cancer patients at greatest risk of developing metastatic disease is thus an important goal that would enable oncologists to aggressively treat these patients while the cancer is still vulnerable. In addition, this would spare patients who do not need or would not benefit from further treatments from having to endure the harmful side-effects of chemotherapeutic drug regimens. Circulating tumor cells (CTCs) are rare cancer cells found in the blood circulation of cancer patients that provide a non-invasively accessible cancer cell specimen (liquid biopsy) from patients. The number of circulating tumor cells (CTCs) in cancer patients has recently been shown to be a valuable diagnostic indicator of the state of metastatic breast cancer. In particular, patients with few or no CTCs were found to have a better overall prognosis compared to patients with high numbers of CTCs.Despite the implications of CTCs as diagnostics for advanced breast cancer treatment, a critical challenge for adopting CTC-based diagnostic tests has been the development of methods with sufficient sensitivity to reliably detect the small number of CTCs that are present in the circulation. Furthermore, current tests for CTC detection are expensive, have high false positives and negatives, have high background noise, are time consuming and require a significant level of expertise to conduct. To overcome the limitations of current CTC detection assays and develop more sensitive, rapid and cost effective CTC detection methods, we explored the potential of detecting CTCs by measuring their nuclease activity with nuclease-activated probes (1). We present data towards the development of a rapid and highly-sensitive CTC detection assay based on nuclease-activated oligonucleotide probes that are selectively digested (activated) by target nucleases expressed in breast cancer cells. We confirm that these probes are not activated by serum nucleases or nucleases from a lymphoblastic cell line (e.g. K-562). Furthermore, we present extensive data towards the optimization of activity and sensitivity of these probes in cell lysates from various breast cancer cell lines and in blood from breast cancer patients. Future studies will focus on developing similar probes for the detection of CTCs in patients with pancreatic and ovarian cancer where early detection would greatly improve survival outcomes. In conclusion, this work describes a robust assay for detection of breast cancer CTCs that will be straightforward to implement in most clinical diagnostic labs.

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.

Abstract B51: ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers.

Abstract Background: Detection of circulating tumor cells (CTCs) is an indicator of poor prognosis in patients with metastatic breast cancer and not in primary breast cancer (PBC). The classical phenotypic definition of a CTC is a nucleated cell that is cytokeratin (CK) positive but CD45 negative. Several reports have shown that EpCAM based capture methods detect only a fraction of CTCs and not the heterogeneous subpopulations of CTCs. Moreover, subsets of CTCs may acquire a more aggressive phenotype with features of invasiveness and motility by undergoing an epithelial to mesenchymal transition (EMT) and down regulate the epithelial cell adhesion molecule, EpCAM. EMT is a hallmark of cellular invasion and metastasis and CTCs undergoing EMT may express the putative cancer stem cell like phenotype, CD24lowCD44+. CTCs undergoing EMT (CTC-EMT) are not readily detected by current CTC detection technologies. Thus, it is desirable to isolate CTCs using capture methods independent of EpCAM to recover a heterogeneous CTC population for more extensive characterization. Here we use ApoStream™, a novel antibody-free CTC isolation device that does not rely on EpCAM to capture circulating rare cells, to evaluate the molecular heterogeneity of CTCs. Methods: Baseline blood samples from 14 newly diagnosed PBC patients were collected and processed using ApoStream™. Isolated cells were stained with anti-CK and anti-CD45, and DAPI. In addition, a multiplexed immunofluorescence assay and laser scanning cytometry analysis were applied to identify multiple combinations of CK+CD45− cells for the expression and distribution of EpCAM, vimentin, CD44, CD24, β-catenin and E-cadherin. Results: ApoStream™ recovered both EpCAM+ and EpCAM− cells. CK+CD45− cells were detected in 9 out of 14 PBC patients. The expression of EpCAM− vimentin+ in the CK+CD45− population was heterogeneous across the patient population. E-cadherin and β-catenin were detected in 0-94% (Mean 52 %) and 0-37% (Mean 8 %), respectively of the CK+CD45− population. All patients with CK+CD45− cells had a subset of cells with the putative phenotype of CD44+CD24low cells. Conclusions: Heterogeneous CTC phenotypes with CD44+CD24lowin both EpCAM+ and EpCAM− cells were observed in patients with PBC. Our aim is to correlate ApoStream™ EMT-CTC counts in patients with PBC with the pathological clinical response (pCR). This study will continue to enroll PBC patients and test the hypothesis that low EMT-CTC count patients have higher pCR rates compared to high EMT-CTC count patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B51. Citation Format: Kenna L. Anderes, Insiya Jafferji, Vladislava O. Melnikova, Jackson A. Summer, Darren W. Davis, James M. Reuben, Naoto T. Ueno. ApoStream™ isolated circulating tumor cells from primary breast cancer patients reveals heterogeneous phenotypes related to epithelial-mesenchymal transition and stem cell markers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B51.

Development of Coagulation Factor Probes for the Identification of Procoagulant Circulating Tumor Cells

Metastatic cancer is associated with a hypercoagulable state, and pathological venous thromboembolic disease is a significant source of morbidity and the second leading cause of death in patients with cancer. Here we aimed to develop a novel labeling strategy to detect and quantify procoagulant circulating tumor cells (CTCs) from patients with metastatic cancer. We hypothesize that the enumeration of procoagulant CTCs may be prognostic for the development of venous thrombosis in patients with cancer. Our approach is based on the observation that cancer cells are capable of initiating and facilitating cell-mediated coagulation in vitro, whereby activated coagulation factor complexes assemble upon cancer cell membrane surfaces. Binding of fluorescently labeled, active site-inhibited coagulation factors VIIa, Xa, and IIa to the metastatic breast cancer cell line, MDA-MB-231, non-metastatic colorectal cell line, SW480, or metastatic colorectal cell line, SW620, was characterized in a purified system, in anticoagulated blood and plasma, and in plasma under conditions of coagulation. We conclude that a CTC labeling strategy that utilizes coagulation factor-based fluorescent probes may provide a functional assessment of the procoagulant potential of CTCs, and that this strategy is amenable to current CTC detection platforms.

A Novel Platform for Detection of CK+ and CK− CTCs

Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.