Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

Circulating tumor cells detected by lab-on-adisc: Role in early diagnosis of gastric cancer

[Background] The use of circulating tumor cells (CTCs) as an early diagnostic biomarker and prognostic indicator after surgery or chemotherapy has been suggested for various cancers. This study aimed to evaluate CTCs in patients who underwent gastrectomy for gastric cancer and to explore their clinical usefulness in the early diagnosis of gastric cancer. [Methods] A total of 116 patients with gastric cancer who underwent gastrectomy and 31 healthy volunteers were prospectively included between 2014 and 2015. Peripheral blood samples were collected before gastrectomy, and CTCs were examined using a centrifugal microfluidic system with a new fluid-assisted separation technique. [Results] After creating a receiver operating characteristic curve to identify the discriminative CTC value needed differentiate patients with gastric cancer from healthy volunteers, sensitivity and specificity were nearly optimized at a CTC threshold of 2 per 7.5 mL of blood. Of the 102 persons with a CTC level >= 2 per 7.5 mL of blood, 99 (97.1%) had gastric cancer, and of the 45 persons with a CTC level <2 per 7.5 mL of blood, 28 (62.2%) were healthy controls. Accordingly, the sensitivity and specificity for the differentiation of patients with gastric cancer from healthy controls were 85.3% and 90.3%, respectively. However, the presence of CTCs was not associated with any clinicopathologic features such as staging, histologic type, or mucin phenotype. [Conclusion] Although we could not prove the clinical feasibility of CTCs for gastric cancer staging, our results suggest a potential role of CTCs as an early diagnostic biomarker of gastric cancer

Circulating melanoma cells and survival in metastatic melanoma.

A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146+ cells were immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with DAPI, anti-molecular weight melanoma-associated antigen (HMW-MAA), anti-CD45 and CD34 and Ki67. CMCs were identified as CD146+, HMW-MAA+, CD45-, CD34-, Ki67-/+ cells. Eighty-eight percent of spiked SK-MEL28 cells in 7.5 ml blood were recovered. In all 55 healthy donors ≤1 CMCs were detected in 7.5 ml of blood. A retrospective analysis was conducted comparing CMC counts and overall survival in 79 blood samples from 44 melanoma patients. CMCs ranged from 0 to 8,042 per 7.5 ml. Two or more CMCs were detected in 18 (23%) of the patients and 30-100% (mean 84%) of the CMCs expressed the proliferation marker Ki67. Patients with ≥2 CMCs per 7.5 ml of whole blood, as compared with the group with <2 CMCs, had a shorter overall survival (2.0 months vs. 12.1 months, P=0.001)

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.

BACKGROUND: Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) . OBJECTIVES: To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques. METHODS: The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS). RESULTS: Each technique gave a different size distribution and a different concentration for the same vesicle sample. CONCLUSION: Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research

Fiber-Optic Array Scanning Technology (FAST) for Detection and Molecular Characterization of Circulating Tumor Cells.

Circulating tumor cell (CTC) as an important component in "liquid biopsy" holds crucial clinical relevance in cancer prognosis, treatment efficiency evaluation, prediction and potentially early detection. Here, we present a Fiber-optic Array Scanning Technology (FAST) that enables antigen-agnostic, size-agnostic detection of CTC. By immunofluorescence staining detection of a combination of a panel of markers, FAST technology can be applied to detect rare CTC in non-small cell lung cancer (NSCLC) setting with high sensitivity and specificity. In combination with Automated Digital Microscopy (ADM) platform, companion markers on CTC such as Vimentin and Programmed death-ligand 1 (PD-L1) can also be analyzed to further characterize these CTCs. FAST data output is also compatible with downstream single cell picking platforms. Single cell can be isolated post ADM confirmation and used for "actionable" genetic mutations analysis