Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift.
Ontology highlight
ABSTRACT:
SUBMITTER: Geislinger TM
PROVIDER: S-EPMC3765238 | biostudies-other | 2013
REPOSITORIES: biostudies-other
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Project description:Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.
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Project description:BACKGROUND: In addition to erythrocytes, embryonic blood contains other differentiated cell lineages and potential progenitor or stem cells homed to changing niches as the embryo develops. Using chicken as a model system, we have isolated an enriched pool of circulating non red blood cells (nRBCs) from E4 and E6 embryos; a transition period when definitive hematopoietic lineages are being specified in the peri-aortic region. RESULTS: Transcriptome analysis of both nRBC and RBC enriched populations was performed using chicken Affymetrix gene expression arrays. Comparison of transcript profiles of these two populations, with verification by RT-PCR, reveals in nRBCs an expression signature indicative of hematopoietic stem cells (HSCs) and progenitor cells of myeloid and lymphoid lineages, as well as a number of previously undescribed genes possibly involved in progenitor and stem cell maintenance. CONCLUSION: This data indicates that early circulating embryonic blood contains a full array of hematopoietic progenitors and stem cells. Future studies on their heterogeneity and differentiation potentials may provide a useful alternative to ES cells and perinatal blood.
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Project description:Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.
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Project description:Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.
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Project description:Blood was extracted from embryonic hearts at E4 and E6 and non-red blood was separated by density gradient centrifugation Keywords: Cell type comparison
2007-12-17 | GSE9884 | GEO
Project description:Circulating tumor cells (CTCs), as important liquid biopsy target, can provide valuable information for cancer progress monitoring and individualized treatment. However, current isolation platforms incapable of balancing capture efficiency, specificity, cell viability, and gentle release have restricted the clinical applications of CTCs. Herein, inspired by the structure and functional merits of natural membrane interfaces, we established an antibody-engineered red blood cell (RBC-Ab) affinity interface on microfluidic chip for high-performance isolation and release of CTCs. The lateral fluidity, pliability, and anti-adhesion property of the RBC microfluidic interface enabled efficient CTCs capture (96.5%), high CTCs viability (96.1%), and high CTCs purity (average 4.2-log depletion of leukocytes). More importantly, selective lysis of RBCs by simply changing the salt concentration was utilized to destroy the affinity interface for efficient and gentle release of CTCs without nucleic acid contamination. Using this chip, CTCs were successfully detected in colon cancer samples with 90% sensitivity and 100% specificity (20 patients and 10 healthy individuals). After the release process, KRAS gene mutations of CTCs were identified from all the 5 cancer samples, which was consistent with the results of tissue biopsy. We expect this RBC interface strategy will inspire further biomimetic interface construction for rare cell analysis.
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Project description:Blood was extracted from embryonic hearts at E4 and E6 and non-red blood was separated by density gradient centrifugation Experiment Overall Design: There are a total of 9 samples which have one biological replicate each, except negative control E6 embryonic heart (used to screen heart contamination in our blood samples - which were drawn from the heart) which has only one sample.
2008-11-06 | E-GEOD-9884 | biostudies-arrayexpress
Project description:Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.
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Project description:The systems controlling the number, size, and hemoglobin concentrations of populations of human red blood cells (RBCs), and their dysregulation in anemia, are poorly understood. After release from the bone marrow, RBCs undergo reduction in both volume and total hemoglobin content by an unknown mechanism [Lew VL, et al. (1995) Blood 86:334-341; Waugh RE, et al. (1992) Blood 79:1351-1358]; after ?120 d, responding to an unknown trigger, they are removed. We used theory from statistical physics and data from the hospital clinical laboratory [d'Onofrio G, et al. (1995) Blood 85:818-823] to develop a master equation model for RBC maturation and clearance. The model accurately identifies patients with anemia and distinguishes thalassemia-trait anemia from iron-deficiency anemia. Strikingly, it also identifies many pre-anemic patients several weeks before anemia becomes clinically detectable. More generally we illustrate how clinical laboratory data can be used to develop and to test a dynamic model of human pathophysiology with potential clinical utility.
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Project description:Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.
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