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hASC characterization (S1); Co-culture of immature adipocytes with mammary epithelial cells increases pro-inflammatory cytokine expression (S2); Immature adipocyte or cytokine exposure increases abundance of sphere and colony forming cells without changing global cell proliferation (S3); Immature adipocyte or cytokine exposure increases matrigel invasion in vitro and tumor formation, vasculogenesis and metastasis in vivo (S4); Src mediates cytokine effects via ES-TF upregulation affecting soft agar colony growth but not cell cycle progression (S5); Cytokine-mediated increase in sphere formation is Sox2-dependent and miR302b upregulation drives further c-MYC and SOX2 gene expression (S6).

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell–like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment. Cancer Res; 76(2); 491–504. ©2016 AACR.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

Objectives Despite successful surgery, 30–50% of patients with resectable non-small cell lung cancer develop tumor recurrence within 5 years of surgery. Materials and methods In this prospective study, we performed CTC enumerations in 40 patients with non-metastatic lung adenocarcinoma (NMLA) using a size-based microfilter. Additionally, cfDNA isolated from plasma was analyzed in 35 out of 40 patients. Results CTCs were identified in 15 out of 40 patients (37.5%) with a range of 1–44 cells, whereas mutated cfDNA was only detected in 3 out of 35 patients (8.6%). Disease-free survival (DFS) was significantly associated with CTC positivity (log-rank p = 0.025), grading (log-rank p = 0.019), tumor stage (log-rank p = 0.025) and lymph node status (log-rank p = 0.029). Multivariate analysis, including tumor stage and grading, showed that CTC positivity (p = 0.006), grading (0.039) and tumor stage (p = 0.022) were independently associated with DFS. Conclusion Our study found that microfilter-based CTC enumeration in NMLA patients is an independent predictor of worse DFS. The used NGS-based cfDNA characterization had limited sensitivity to be clinically informative in our study cohort. CTC assessment before surgery can thus identify NMLA patients at high risk of disease recurrence.

// Theresa Benezeder 1 , Verena Tiran 1 , Alexandra A.N. Treitler 1 , Christoph Suppan 1 , Christopher Rossmann 1 , Herbert Stoeger 1 , Richard J. Cote 2 , Ram H. Datar 2 , Marija Balic 1, 3 and Nadia Dandachi 1, 4 1 Medical University of Graz, Department of Internal Medicine, Division of Oncology, Graz, Austria 2 University of Miami Miller School of Medicine, Department of Pathology, Miami, Florida, U.S.A. 3 Medical University of Graz, Research Unit Circulating Tumor Cells and Cancer Stem Cells, Graz, Austria 4 Medical University of Graz, Research Unit Epigenetic and Genetic Cancer Biomarkers, Division of Oncology, Graz, Austria Correspondence to: Nadia Dandachi, email: nadia.dandachi@medunigraz.at Marija Balic, email: marija.balic@medunigraz.at Keywords: circulating tumor cells, enrichment, metastatic breast cancer, methylation, prognosis Received: April 12, 2017 Accepted: August 27, 2017 Published: September 30, 2017 ABSTRACT Blood-based biomarkers such as circulating tumor cells (CTCs) provide dynamic real-time assessment of molecular tumor characteristics beyond the primary tumor. The aim of this study was to evaluate the feasibility of a size-based microfilter to assess multigene methylation analysis of enriched CTCs in a prospective proof-of principle study. We examined the quantitative methylation status of nine genes ( AKR1B1, BMP6, CST6, HOXB4, HIST1H3C, ITIH5, NEUROD1, RASSF1, SOX17 ) in enriched CTCs from metastatic breast cancer patients. Feasibility and clinical performance testing were assessed in a test set consisting of 37 patients and 25 healthy controls. With established cut-off values from the healthy control group, methylation of enriched CTCs was detected in at least one gene in 18/37 patients (48.6%), while 97.8% of all control samples were unmethylated. Patients with CTCs unmethylated for CST6 , ITIH5 , or RASSF1 showed significantly longer PFS compared to patients with corresponding enriched methylated CTCs. This proof-of-principle study shows the feasibility of a size-based microfilter to enrich and analyze multigene methylation profile of CTCs from metastatic breast cancer patients. For the first time, we report that multigene methylation analysis of enriched CTCs provides prognostic information in metastatic breast cancer patients.

The aim of this study was to assess the prognostic and predictive value of an untargeted assessment of tumor fractions in the plasma of metastatic breast cancer patients and to compare circulating tumor DNA (ctDNA) with circulating tumor cells (CTC) and conventional tumor markers. In metastatic breast cancer patients (n = 29), tumor fractions in plasma were assessed using the untargeted mFAST-SeqS method from 127 serial blood samples. Resulting z-scores for the ctDNA were compared to tumor fractions established with the recently published ichorCNA algorithm and associated with the clinical outcome. We observed a close correlation between mFAST-SeqS z-scores and ichorCNA ctDNA quantifications. Patients with mFAST-SeqS z-scores above three (34.5%) showed significantly worse overall survival (p = 0.014) and progression-free survival (p = 0.018) compared to patients with lower values. Elevated z-score values were clearly associated with radiologically proven progression. The baseline CTC count, carcinoembryonic antigen (CEA), and cancer antigen (CA)15-5 had no prognostic impact on the outcome of patients in the analyzed cohort. This proof of principle study demonstrates the prognostic impact of ctDNA levels detected with mFAST-SeqS as a very fast and cost-effective means to assess the ctDNA fraction without prior knowledge of the genetic landscape of the tumor. Furthermore, mFAST-SeqS-based ctDNA levels provided an early means of measuring treatment response.

In this study, biological cells are sensed and characterized with surface acoustic wave (SAW) devices utilising microcavities. After tumour cells in media are transported to and trapped in microcavities, the proposed platform uses SAW interaction between the substrate and the cells to extract their mechanical stiffness based on the ultrasound velocity. Finite element method (FEM) analysis and experimental results show that output phase information is an indicator of the stiffness modulus of the trapped cells. Small populations of various types of cells such as MCF7, MDA-MB-231, SKBR3, and JJ012 were characterized and characteristic moduli were estimated for each cell population. Results show that high frequency stiffness modulus is a possible biomarker for aggressiveness of the tumour and that microcavity coupled SAW devices are a good candidate for non-invasive interrogation of single cells.

Metastasis is facilitated by cancer-associated fibroblasts (CAF) in the tumor microenvironment through mechanisms yet to be elucidated. In this study, we used a size-based microfilter technology developed by our group to examine whether circulating CAF identified by FAP and α-SMA co-expression (cCAF) could be distinguished in the peripheral blood of patients with metastatic breast cancer. In a pilot study of patients with breast cancer, we detected the presence of cCAFs in 30/34 (88%) patients with metastatic disease (MET group) and in 3/13 (23%) patients with localized breast cancer (LOC group) with long-term disease-free survival. No cCAFs as defined were detected in healthy donors. Further, both cCAF and circulating tumor cells (CTC) were significantly greater in the MET group compared with the LOC group. Thus, the presence of cCAF was associated with clinical metastasis, suggesting that cCAF may complement CTC as a clinically relevant biomarker in metastatic breast cancer. Cancer Res; 75(22); 4681–7. ©2015 AACR.

Nanostructure field-effect transistor (FET) biosensors have shown great promise for ultra sensitive biomolecular detection. Top-down assembly of these sensors increases scalability and device uniformity but faces fabrication challenges in achieving the small dimensions needed for sensitivity. We report top-down fabricated indium oxide (In2O3) nanoribbon FET biosensors using highly scalable radio frequency (RF) sputtering to create uniform channel thicknesses ranging from 50 to 10 nm. We combine this scalable sensing platform with amplification from electronic enzyme-linked immunosorbent assay (ELISA) to achieve high sensitivity to target analytes such as streptavidin and human immunodeficiency virus type 1 (HIV-1) p24 proteins. Our approach circumvents Debye screening in ionic solutions and detects p24 protein at 20 fg/mL (about 250 viruses/mL or about 3 orders of magnitude lower than commercial ELISA) with a 35% conduction change in human serum. The In2O3 nanoribbon biosensors have 100% device yield and use a simple 2 mask photolithography process. The electrical properties of 50 In2O3 nanoribbon FETs showed good uniformity in on-state current, on/off current ratio, mobility, and threshold voltage. In addition, the sensors show excellent pH sensitivity over a broad range (pH 4 to 9) as well as over the physiological-related pH range (pH 6.8 to 8.2). With the demonstrated sensitivity, scalability, and uniformity, the In2O3 nanoribbon sensor platform makes great progress toward clinical testing, such as for early diagnosis of acquired immunodeficiency syndrome (AIDS).

Stimulus responsive release of Circulating Tumor Cells (CTCs), with high recovery rates from their capture platform, is highly desirable for off-chip analyses. Here, we present a temperature responsive polymer coating method to achieve both release as well as culture of viable CTCs captured from patient blood samples.

Microfilters with slot-pore geometry can be used for size-based capture of circulating tumor cells (CTC) from the blood of cancer patients. The slot pore geometry reduces the shear stress that the cells would typically experience during filtration process and allows the cells to remain viable. The microfilter provides a platform capable of high CTC capture efficiency; however, the release of these cells from the filter following capture is nontrivial, possibly due to the strong nonspecific electrostatic adhesion of CTC to the microfilter surface. Techniques such as reverse flow or cell scraping result in recovery of only a small percentage of captured cells. We describe, in detail, a protocol for novel application of thermo-responsive polymer poly(N-iso-propylacrylamide) (PIPAAm) to release viable CTCs from microfilters with slot pores. Following fabrication of the microfilter, a coating of PIPAAm is applied to the surface to exploit its thermoresponsive interfacial properties to release the cells. Typically, cancer patient's blood is filtered at room temperature (below 32 °C) when PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture medium or a buffer maintained at 37 °C, which renders PIPAAm hydrophobic, allowing subsequent release of the electrostatically bound cells with high efficiency. Using this method, viable CTC captured directly from cancer patients' blood can be subjected to downstream off-chip culture, analyses, and characterization.

Purpose Circulating tumor cell (CTC) enumeration has not been prospectively validated in standard first-line docetaxel treatment for metastatic castration-resistant prostate cancer. We assessed the prognostic value of CTCs for overall survival (OS) and disease response in S0421, a phase III trial of docetaxel plus prednisone with or without atrasentan. Patients and Methods CTCs were enumerated at baseline (day 0) and before cycle two (day 21) using CellSearch. Baseline counts and changes in counts from day 0 to 21 were evaluated for association with OS, prostate-specific antigen (PSA), and RECIST response using Cox regression as well as receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI) analysis, and regression trees. Results Median day-0 CTC count was five cells per 7.5 mL, and CTCs < versus ≥ five per 7.5 mL were significantly associated with baseline PSA, bone pain, liver disease, hemoglobin, alkaline phosphatase, and subsequent PSA and RECIST response. Median OS was 26 months for < five versus 13 months for ≥ five CTCs per 7.5 mL at day 0 (hazard ratio [HR], 2.74 [adjusting for covariates]). ROC curves had higher areas under the curve for day-0 CTCs than for PSA, and IDI analysis showed that adding day-0 CTCs to baseline PSA and other covariates increased predictive accuracy for survival by 8% to 10%. Regression trees yielded new prognostic subgroups, and rising CTC count from day 0 to 21 was associated with shorter OS (HR, 2.55). Conclusion These data validate the prognostic utility of CTC enumeration in a large docetaxel-based prospective cohort. Baseline CTC counts were prognostic, and rising CTCs at 3 weeks heralded significantly worse OS, potentially serving as an early metric to help redirect and optimize therapy in this clinical setting.

Background: Sensitivity of standard urine cytology for detecting urothelial carcinoma of the bladder (UCB) is low, attributable largely to its inability to process entire samples, paucicellularity and presence of background cells. Objective: Evaluate performance and practical applicability of a novel portable microfiltration device for capture, enumeration and characterisation of exfoliated tumour cells in urine, and compare it with standard urine cytology for UCB detection. Methods: A total of 54 urine and bladder wash samples from patients undergoing surveillance for UCB were prospectively evaluated by standard and microfilter-based urine cytology. Head-to-head comparison of quality and performance metrics, and cost effectiveness was conducted for both methodologies. Results: Five samples were paucicellular by standard cytology; no samples processed by microfilter cytology were paucicellular. Standard cytology had 33.3% more samples with background cells that limited evaluation (p < 0.001). Microfilter cytology was more concordant (κ = 50.4%) than standard cytology (κ = 33.5%) with true UCB diagnosis. Sensitivity, specificity and accuracy were higher for microfilter cytology compared to standard cytology (53.3%/100%/79.2% versus 40%/95.8%/69.9%, respectively). Microfilter-captured cells were amenable to downstream on-chip molecular analyses. A 40 ml sample was processed in under 4 min by microfilter cytology compared to 5.5 min by standard cytology. Median microfilter cytology processing and set-up costs were approximately 63% cheaper and 80 times lower than standard cytology, respectively. Conclusions: The microfiltration device represents a novel non-invasive UCB detection system that is economical, rapid, versatile and has potentially better quality and performance metrics than routine urine cytology, the current standard-of-care.

Nanobiosensors have drawn significant research interest in recent years owing to the advantages of label-free, electrical detection. However, nanobiosensors fabricated by bottom-up process are limited in terms of yield and device uniformity due to the challenges in assembly. Nanobiosensors fabricated by top-down process, on the other hand, exhibit better uniformity but require time and costly processes and materials to achieve the critical dimensions required for high sensitivity. In this report, we introduce a top-down nanobiosensor based on polysilicon nanoribbon. The polysilicon nanoribbon devices can be fabricated by conventional photolithography with only materials and equipments used in the standard CMOS process, thus resulting in great time and cost efficiency, as well as scalability. The devices show great response to pH changes with a wide dynamic range and high sensitivity. Biomarker detection is also demonstrated with clinically relevant sensitivity. Such results suggest that polysilicon nanoribbon devices exhibit great potential toward a highly efficient, reliable and sensitive biosensing platform.

Biomarker detection based on nanowire biosensors has attracted a significant amount of research effort in recent years. However, only very limited research work has been directed toward biomarker detection directly from physiological fluids mainly because of challenges caused by the complexity of media. This limitation significantly reduces the practical impact generated by the aforementioned nanobiosensors. In this study, we demonstrate an In(2)O(3) nanowire-based biosensing system that is capable of performing rapid, label-free, electrical detection of cancer biomarkers directly from human whole blood collected by a finger prick. Passivating the nanowire surface successfully blocked the signal induced by nonspecific binding when performing active measurement in whole blood. Passivated devices showed markedly smaller signals induced by nonspecific binding of proteins and other biomaterials in serum and higher sensitivity to target biomarkers than bare devices. The detection limit of passivated sensors for biomarkers in whole blood was similar to the detection limit for the same analyte in purified buffer solutions at the same ionic strength, suggesting minimal decrease in device performance in the complex media. We then demonstrated detection of multiple cancer biomarkers with high reliability at clinically meaningful concentrations from whole blood collected by a finger prick using this sensing system.

Dissemination of tumor cells from primary tumors in the circulatory system is an early event in carcinogenesis. The presence of these single disseminated tumor cells (DTC) in peripheral blood, bone marrow and distant organs is the rationale for adjuvant systemic treatment. Detection of DTC in bone marrow aspirates from breast cancer patients and other solid tumors at the primary diagnosis impacts the prognosis of disease. In peripheral blood these cells are termed as circulating tumor cells (CTC). Due to technical difficulties the clinical significance of CTC detection at early stages is less established. This review focuses on available techniques for detection of DTC and CTC, recent technical advances in development of more sensitive microfluidic methods for capture of DTC and CTC and possibilities for further detection and their potential molecular characterization. Not only the clinical significance of DTC but also the presence of cancer stem cells in dissemination clearly demonstrates the need for development of sensitive technologies allowing for definition of biomarkers and molecular targets on cells in dissemination, thus eventually leading to optimization of systemic therapies.

Detection of circulating tumor cells has emerged as a promising minimally invasive diagnostic and prognostic tool for patients with metastatic cancers. We report a novel three dimensional microfilter device that can enrich viable circulating tumor cells from blood. This device consists of two layers of parylene membrane with pores and gap precisely defined with photolithography. The positions of the pores are shifted between the top and bottom membranes. The bottom membrane supports captured cells and minimize the stress concentration on cell membrane and sustain cell viability during filtration. Viable cell capture on device was investigated with scanning electron microscopy, confocal microscopy, and immunofluorescent staining using model systems of cultured tumor cells spiked in blood or saline. The paper presents and validates this new 3D microfiltration concept for circulation tumor cell enrichment application. The device provides a highly valuable tool for assessing and characterizing viable enriched circulating tumor cells in both research and clinical settings.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell–like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment. Cancer Res; 76(2); 491–504. ©2016 AACR.

Circulating Tumor Cells (CTCs) are tumor cells found in cancer patients’ peripheral blood. Enumeration of CTCs can provide prognosis information for cancer management (Cristofanilli et al., N Engl J Med 351(8):781–791, 2004; Cohen et al., J Clin Oncol 26(19):3213–3221, 2008; de Bono et al., Clin Cancer Res, 14(19):6302–6309, 2008; Poveda et al., Gynecol Oncol, 122(3):567–572, 2011). However, the technical hurdle for studying CTCs is their rare presence in blood, thus, isolating them is a non-trivial task. Two major categories of technologies have been developed in the past to isolate CTCs based on their biological expression of antigens (affinity-based capture) or based on their physical properties (non-affinity based capture). This chapter dedicates itself to the non-affinity based method for CTC capture. CTCs, as tumor cells, are inherently distinct from normal blood components. The chapter touches on the how these differences are reflected in their gene expression profiles, as well as their physical properties. We discuss how researchers utilized the unique biomechanical and electrical properties of CTCs to isolate them from enormous numbers of erythrocytes and leukocytes present in peripheral blood. We begin the chapter with technologies utilizing biomechanical properties (cell density, size, deformability) to isolate CTCs and then move on to discuss the development of dielectrophoresis (DEP) based CTC isolation, based on their distinct electrical properties.

Study of CTC in cancer has always been hampered by its rare existence in blood. In this chapter, we discuss one of first principles employed to capture CTC from cancer patients’ peripheral blood—the affinity-based enrichment of CTC. We briefly discuss the different technologies utilizing antibodies to capture CTC based on specific antigen expression. Then we address the downstream molecular and functional characterization of CTC by utilizing these technologies. We also discuss the limitations of affinity-based CTC enrichment.

Circulating tumor cells (CTCs) that detach and migrate from primary tumors are implicated in the metastatic spread of cancer. The identification of CTCs in peripheral blood samples has been associated with poor survival outcomes in various cancer types. As a readily accessible source of tumor tissue there is a vast potential to develop CTCs as a biomarker to advance cancer diagnosis, prognosis and the development of novel and targeted therapies. The fact that CTCs occur as extremely rare events in whole blood presents a technical challenge for characterization, requiring enrichment techniques that are both highly sensitive and sufficiently specific. The culture and expansion of CTCs is desirable as a means of yielding a population suitable for comprehensive functional characterization and drug testing. Reports of successful in vitro culture of CTCs are rare, but various approaches have been attempted and significant progress has been made. The development of protocols for reliable and efficient culture of viable CTCs will advance our biological understanding of cancer metastasis and facilitate the development of personalized therapies.

Circulating Tumor Cells (CTC) are tumor cells released into blood. They are considered the pivotal component of the metastatic cascade and are being extensively studied only in the last decade or so. Understanding the biological and clinical impact of CTC is likely to reveal important information of the metastatic process and contribute to better management of cancer. We briefly discuss here the current clinical utility of CTC and their emerging clinical applications.

Purpose The p53 gene status (mutation) and protein alterations (nuclear accumulation detectable by immunohistochemistry; p53 protein status) are associated with bladder cancer progression. Substantial discordance is documented between the p53 protein and gene status, yet no studies have examined the relationship between the gene-protein status and clinical outcome. This study evaluated the clinical relationship of the p53 gene and protein statuses. Materials and Methods The complete coding region of the p53 gene was queried using DNA from paraffin-embedded tissues and employing a p53 gene–sequencing chip. We compared p53 gene status, mutation site, and protein status with time to recurrence. Results The p53 gene and protein statuses show significant concordance, yet 35% of cases showed discordance. Exon 5 mutations demonstrated a wild-type protein status in 18 of 22 samples. Both the p53 gene and protein statuses were significantly associated with stage and clinical outcome. Specific mutation sites were associated with clinical outcome; tumors with exon 5 mutations showed the same outcome as those with the wild-type gene. Combining the p53 gene and protein statuses stratifies patients into three distinct groups, based on recurrence-free intervals: patients showing the best outcome (wild-type gene and unaltered protein), an intermediate outcome (either a mutated gene or an altered protein) and the worst outcome (a mutated gene and an altered protein). Conclusion We show that evaluation of both the p53 gene and protein statuses provides information in assessing the clinical recurrence risk in bladder cancer and that the specific mutation site may be important in assessing recurrence risk. These findings may substantially impact the assessment of p53 alterations and the management of bladder cancer.

This paper presents development of a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells (CTCs) in human blood, and the potential of this device to allow genomic analysis. The presence and number of CTCs in blood has recently been demonstrated to provide significant prognostic information for patients with metastatic breast cancer. While finding as few as five CTCs in about 7.5mL of blood (i.e., 10(10) blood cells in) is clinically significant, detection of CTCs is currently difficult and time consuming. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. Work presented here provides a micro-electro-mechanical system (MEMS)-based option to make this process simpler, faster, better and cheaper. We exploited the size difference between CTCs and human blood cells to achieve the CTC capture on filter with approximately 90% recovery within 10 min, which is superior to current approaches. Following capture, we facilitated polymerase chain reaction (PCR)-based genomic analysis by performing on-membrane electrolysis with embedded electrodes reaching each of the individual 16,000 filtering pores. The biggest advantage for this on-membrane in situ cell lysis is the high efficiency since cells are immobilized, allowing their direct contact with electrodes. As a proof-of-principle, we show beta actin gene PCR, the same technology can be easily extended to real time PCR for CTC-specific transcript to allow molecular identification of CTC and their further characterization.

It is a challenge to eradicate tumor cells while sparing normal cells. We used magnetoelectric nanoparticles (MENs) to control drug delivery and release. The physics is due to electric-field interactions (i) between MENs and a drug and (ii) between drug-loaded MENs and cells. MENs distinguish cancer cells from normal cells through the membrane’s electric properties; cancer cells have a significantly smaller threshold field to induce electroporation. In vitro and in vivo studies (nude mice with SKOV-3 xenografts) showed that (i) drug (paclitaxel (PTX)) could be attached to MENs (30-nm CoFe2O4@BaTiO3 nanostructures) through surface functionalization to avoid its premature release, (ii) drug-loaded MENs could be delivered into cancer cells via application of a d.c. field (~100 Oe), and (iii) the drug could be released off MENs on demand via application of an a.c. field (~50 Oe, 100 Hz). The cell lysate content was measured with scanning probe microscopy and spectrophotometry. MENs and control ferromagnetic and polymer nanoparticles conjugated with HER2-neu antibodies, all loaded with PTX were weekly administrated intravenously. Only the mice treated with PTX-loaded MENs (15/200 μg) in a field for three months were completely cured, as confirmed through infrared imaging and post-euthanasia histology studies via energy-dispersive spectroscopy and immunohistochemistry.

Analysis of isolated cancer cells in circulation is proven to help determine the success of the cancer treatment and understand the genetic signature of cancer disease. Scarcity of these cells in blood circulation (1–10 CTC in 1ml blood) however, makes the isolation process extremely challenging. Ever improving CTC isolation methods fall into two main categories: 1.Immunomagnetic separation based on antibody binding to tumor specific biomarkers expressed on the cell 2. Physical separation based on the size of the CTCs. Efficiency in cell isolation is still low in these techniques due to the variation in expression level of tumor specific antigens and tumor cell size. Therefore, tumor cell isolation strategies using new CTC biomarkers must be explored. In this study, we investigated the feasibility of using mechanical stiffness difference in order to detect and isolate the circulating tumor cells from the blood cells. AFM nanindentation experiments revealed that cancer cells are significantly softer than the surrounding white blood cells and therefore, stiffness can be used as a biomarker for CTC isolation. In addition, finite element analysis simulations have shown that CTC isolation can be performed at high efficiency using stiffness-based isolation. Therefore, stiffness based isolation has a potential to achieve fast, label-free isolation of CTCs at high efficiency for clinical applications.

Papillary and invasive cancers of the urinary bladder appear to evolve and progress through distinct molecular pathways. Invasion in bladder cancer forebodes a graver prognosis, and these tumors are generally characterized by alterations in the p53 and retinoblastoma (RB) pathways that normally regulate the cell cycle by interacting with the Ras–mitogen activated protein kinase signal transduction pathway. Tumor angiogenesis further contributes to the neoplastic growth by providing a constant supply of oxygen and nutrients. Distinct epigenetic and genetic events characterize the interplay between the molecules involved in these pathways, thus affording their use as indicators of prognosis. Efforts are now underway to construct molecular panels comprising multiple markers that can serve as more robust predictors of outcome. While clinical trials for targeted chemotherapy for bladder cancer have commenced, novel genetic and pharmacologic agents that can target pathway-specific molecules are currently under development. The next generation of clinical management for urothelial carcinoma will witness the use of multimarker panels for prognostic prediction and combination therapy directed at novel molecular targets for treatment.

Loss of heterozygosity, mutations or deletions of the RB1 gene usually result in loss of pRb expression, which has been regarded as an indicator of loss of pRb function in human tumours. It has previously been shown that in addition to loss of pRb expression, aberrantly high (pRb2+) pRb expression also indicates loss of pRb function in bladder tumours compared with moderate (normal, pRb1+) pRb expression. The aim of this study was to elucidate the mechanism by which pRb is functionally inactivated in bladder tumours expressing aberrantly high levels of pRb. Constitutive phosphorylation was therefore investigated as a mechanism of pRb inactivation in bladder tumours. Of 28 bladder tumours examined, western blotting demonstrated pRb hyperphosphorylation in 5/7 (71%) pRb2+ bladder tumours compared with only 4/11 (36%) pRb1+ tumours (p = 0.002). All cases with undetectable pRb showed moderate to high p16 expression and none showed cyclin D1 expression by immunohistochemistry. All pRb1+ tumours with underphosphorylated pRb showed p16 but not cyclin D1 expression. All pRb2+ tumours with hyperphosphorylated pRb showed loss of p16 expression and/or cyclin D1 overexpression. Thus, elevated pRb expression was associated with pRb hyperphosphorylation, which, in turn, was associated with loss of p16 expression and/or increased cyclin D1 expression. In order to analyse this association in vitro, T24 cells, which express high levels of pRb, were transfected with p16 cDNA. Transfection with p16 cDNA resulted in a marked decrease in pRb phosphorylation, decreased cell proliferation, and a change in expression of pRb from high to moderate phenotype as assessed by immunohistochemistry. This paper gives the biological basis for constitutive alteration of pRb function in human tumours in the presence of an intact, expressed pRb protein; the mechanism of pRb inactivation is through hyperphosphorylation, which results from loss of p16 expression and/or cyclin D1 overexpression. Immunohistochemical expression of pRb appears to be a reliable indicator of pRb function.

Purpose To determine the combined effects of p53, p21, and pRb alterations in predicting the progression of bladder transitional cell carcinoma. Patients and Methods p53, p21, and pRb expression was examined immunohistochemically on archival radical cystectomy samples from 164 patients with invasive or high-grade recurrent superficial transitional cell carcinoma (TCC; lymph node–negative, 117 patients; lymph node–positive, 47 patients). Median follow-up was 8.6 years. Based on percentage of nuclear reactivity, p53 was considered as wild-type (0% to 10%) or altered (> 10%); p21 was scored as wild-type (>10%) or altered (< 10%); and pRb status was considered wild-type (1% to 50%) or altered (0% or > 50%). Results As individual determinants, the p53, p21, and pRb status were independent predictors of time to recurrence (P < .001, P < .001, and P < .001, respectively), and overall survival (P < .001, P = .002, and P = .001, respectively). By examining these determinants in combination, patients were categorized as group I (no alteration in any determinant, 47 patients), group II (any one determinant altered, 51 patients), group III (any two determinants altered, 42 patients), and group IV (all three determinants altered, 24 patients). The 5-year recurrence rates in these groups were 23%, 32%, 57%, and 93%, respectively (log-rank P < .001), and the 5-year survival rates were 70%, 58%, 33%, and 8%, respectively (log-rank P < .001). After stratifying by stage, the number of altered proteins remained significantly associated with time to recurrence and overall survival. Conclusion This study suggests that alterations in p53, p21, and pRb act in cooperative or synergistic ways to promote bladder cancer progression. Examining these determinants in combination provides additional information above the use of a single determinant alone.

To measure and compare placental mRNA expression in the maternal circulation among women with intrauterine and ectopic pregnancies.Plasma was collected from patients in early pregnancy at risk of ectopic pregnancy. Clinical information was prospectively collected and entered into a dedicated database. mRNA was isolated from maternal plasma and quantitative RT-PCR was performed to measure mRNA for human gonadotropin (hCG) and human placental lactogen (hPL). GAPDH mRNA expression was used as an internal control.Twelve women with ectopic pregnancy and 13 women with intrauterine pregnancy were enrolled. Patients with ectopic pregnancy were 6 times more likely to have undetectable levels of hPL mRNA (relative risk [RR] 6.36; 95% confidence interval [CI], 1.70-23.20; P0.01). They were also 8 times more likely to have undetectable levels of hCG mRNA (RR 8.64, 95% CI, 1.30-57.10; P0.01). mRNA copy numbers for hPL and hCG (normalized by GAPDH) were significantly lower in the ectopic group than in the intrauterine group.Placental mRNA is present in the maternal circulation in significantly lower copies in cases of ectopic pregnancy compared with cases of intrauterine pregnancy. Measurement of placental mRNA in the maternal circulation may help to distinguish between intrauterine and ectopic pregnancies.

CORRIGENDUM: Separable Bilayer Microfiltration Device for Viable Label-free Enrichment of Circulating Tumour Cells

The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 µg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein‐protein binding, DNA hybridization, and DNA‐protein interactions, as well as drug discovery. It is becoming increasingly evident that high-throughput identification and quantitation of a large number of biological molecules is important for generating a molecular profile that is critical in diagnosis, monitoring, and prognostic evaluation of complex diseases such as cancer

Characterization of single-nucleotide polymorphisms is a major focus of current genomics research. We demonstrate the discrimination of DNA mismatches using an elegantly simple microcantilever-based optical deflection assay, without the need for external labeling. Gold-coated silicon AFM cantilevers were functionalized with thiolated 20- or 25-mer probe DNA oligonucleotides and exposed to target oligonucleotides of varying sequence in static and flow conditions. Hybridization of 10-mer complementary target oligonucleotides resulted in net positive deflection, while hybridization with targets containing one or two internal mismatches resulted in net negative deflection. Mismatched targets produced a stable and measurable signal when only a four-base pair stretch was complementary to the probe sequence. This technique is readily adaptable to a high-throughput array format and provides a distinct positive/negative signal for easy interpretation of oligonucleotide hybridization.

Generation of nanomechanical cantilever motion from biomolecular interactions can have wide applications, ranging from high-throughput biomolecular detection to bioactuation. Although it has been suggested that such motion is caused by changes in surface stress of a cantilever beam, the origin of the surface-stress change has so far not been elucidated. By using DNA hybridization experiments, we show that the origin of motion lies in the interplay between changes in configurational entropy and intermolecular energetics induced by specific biomolecular interactions. By controlling entropy change during DNA hybridization, the direction of cantilever motion can be manipulated. These thermodynamic principles were also used to explain the origin of motion generated from protein–ligand binding.

BACKGROUND: The purpose of the current study was to determine telomerase activity as a sensitive biomarker for the detection of malignant cells in fine-needle aspiration (FNA) specimens. METHODS: FNA specimens with parallel samples of fresh tumor tissue were obtained from surgical specimens after surgical excision. Using a polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) assay, telomerase activity was determined systematically in FNA specimens (n = 21) and corresponding available tissue biopsy specimens (n = 16) containing malignant cells. In addition to a case of myelolipoma, normal counterparts for 3 of 16 cancer cases, including both biopsy and FNA specimens, also were available for the determination of telomerase activity. RESULTS: Telomerase activity was observed in 14 of 16 of the FNA specimens (88%) and 15 of 16 of the corresponding biopsy specimens (94%). Telomerase activity was detected in both the biopsy specimen and the corresponding FNA specimen, with one exception (a case of mucinous adenocarcinoma of the cecum). In contrast, specimens from three normal tissue biopsies and FNA specimens of normal tissue adjacent to the malignant lesions, as well as the myelolipoma, exhibited no telomerase activity. It is interesting to note that both tissue biopsy specimens and FNA specimens from a patient with high grade sarcoma were negative for telomerase activity. The examination of hematoxylin and eosin-stained adjacent tissue biopsy sections or FNA smears revealed similar low populations of lymphocytes, including those cases that were negative for telomerase activity. There was agreement in the detection of telomerase activity between tissue biopsies and their corresponding FNA specimens in 15 of the 16 patients, indicating a 94% concordance rate (95% confidence interval, 70%, 98%). CONCLUSIONS: The results of the current study clearly suggest that the telomerase activity in FNA specimens was comparable to that of their corresponding biopsy specimens, and that this activity was associated with the presence of malignant cells. The TRAP assay has potential for use in the detection of malignant cells in FNA specimens, particularly cases in which the cytology is not characteristically malignant and/or is present in insufficient numbers. Cancer (Cancer Cytopathol)

BACKGROUND. Telomeres that protect chromosomes at both ends are shortened with each somatic cell division through replication-dependent sequence loss at DNA termini. The chromosomes with shortened telomeres tend to become unstable, leading to cell death. Due largely to reactivation/upregulation of telomerase, a ribonucleoprotein that adds nucleotide sequences onto chromosome ends, cancer cells become immortal and neoplastically transformed. METHODS. The purpose of the present study was to study three newly established human prostate cancer cell lines and three prostate-derived fibroblastic cell cultures at different passages for telomeric DNA signal intensity, telomeric restriction fragment length (TRFL), telomerase activity, and spontaneous apoptotic index. RESULTS. Compared with the three fibroblastic cell cultures, the three new prostate cancer cell lines showed: 1) telomerase activity, 2) stronger telomeric signals, 3) relatively longer TRFLs, and 4) much lower apoptotic indices. On the other hand, three fibroblastic cell cultures showed: 1) no telomerase activity, 2) weaker telomeric signals, 3) shorter TRFLs (fibroblasts derived from surrounding tissue of prostate tumor showed intermediate TRFLs), and 4) comparatively higher apoptotic indices. CONCLUSIONS. Based on these results, we conclude that telomeric DNA signal intensity, TRFL, and telomerase activity can be used to distinguish prostate cancer cells from adjacent fibroblasts.

Significant changes in the global levels and regional patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancers. 1 Alterations in DNA methylation have a direct impact on both the mutational and epigenetic components of neoplastic transformation.ImportanteffectsofDNAmethylationonthe genome include mutational burden of 5-methylcytosine, epigenetic effects of promoter methylation on gene transcription and potential gene activation, and induction of chromosomal instability by DNA hypomethylation. 2,3 Hypermethylationofcytosine-guaninedinucleotide(CpG)islands is associated with transcriptional repression, whereas hypomethylation may lead to increased potential for gene activity or chromosomal instability. CpG islands located in tumor suppressor gene promoters are normally unmethylated. Abnormal methylation of these regions may lead to progressive reduction in gene expression, thus transcriptionally silencing suppressor genes by a variety of mechanisms, including remodeling of local chromatin structure or inhibition of transcription factor binding, ultimately altering normal cellular growth properties. Application of novel molecular biologic techniques has increased our appreciation of the widespread changes in methylation patterns that occur during urinary bladder carcinogenesis. Epigenetic events, such as methylation, occur throughout all stages of tumorigenesis, including the early phases, and are increasingly recognized as major mechanisms involved in silencing tumor suppressor genes. A large number of genes have been reported to be hypermethylated in bladder cancer. The frequency of such events suggests that widespread alterations in the patterns of DNA methylation are common in bladder carcinogenesis. Some examples include the p16 gene (at CDKN2A INK4a locus on chromosome 9p21), E-cadherin gene (CDH1, encoding a transmembrane glycoprotein that modulates calcium-dependent intercellular adhesion), and RASSF1A gene (ras association domain family gene 1, isoform A). 4-6 Diminished CDH1 expression may lead to increase in betacateninactivityandproliferationinurothelialcarcinomas. 7 Aside from deletion and mutation, the p16 gene has been shown to be inactivated by promoter methylation. 8-11 Mutationordeletionofonep16alleleandhypermethylationof the remaining allele may be sufficient for loss of functional activity. Progressive inhibition of p16 expression by DNA methylation may result in loss of adequate growth regulatory function. Methylation of p16 has been observed in 27% to 60% of primary urothelial carcinomas. 12,13 Both

Objectives This study aimed to measure the effects of peroxisome proliferator-activated receptor-δ (PPARδ) agonist GW501516 (GW) and zinc sulfate (ZS) on ovariectomized rats' vaginal histomorphology and collagen expression. Methods Two weeks after ovariectomy, rats received daily treatment with vaginal suppositories containing placebo, ZS, GW, ZS with GW, or estradiol for 2 weeks. Macroscopic measurements were taken and the midsection of the vagina was used for histology. Immunofluorescence was performed with antibodies against collagen I, III, and anti-actin or collagen I and V and anti-actin. Gene expression analysis of 3 collagen genes was performed by qRT-PCR. Results Macroscopic measurements revealed that the genital hiatus was narrower in the ZS and ZS with GW groups, and the vagina was significantly longer in the animals treated with GW, ZS with GW, and estradiol compared to the placebo group. Microscopic measurements of the vaginal layers showed that the lamina propria and the vaginal muscularis were significantly thicker in the ZS and ZS with GW group compared to the placebo.The ratio of vaginal Col1a1/Col3a1 mRNA expression was significantly up-regulated by ZS with GW compared to placebo, whereas the ratio of vaginal Col1a1/Col5a1 expression was significantly up-regulated by ZS, GW, and ZS with GW. The ratio of vaginal collagen I/III protein expression was significantly up-regulated by ZS and ZS with GW, whereas the ratio of vaginal collagen I/V expression was significantly up-regulated by estradiol, ZS, and ZS with GW compared to control. Conclusions Vaginal suppositories containing zinc and PPARδ agonist significantly altered the vagina of ovariectomized rats.

The most important determinant of prognosis and management of cancer is the presence or absence of metastasis [1]. The road to metastasis involves tumor cells to become detached from the primary tumor and travel in the blood to distant sites, causing secondary tumors. These tumor cells traveling in blood are termed Circulating tumor cells (CTC). Capture of CTC from whole blood has been a challenging feat. The fact that these CTC are few in number, to effectively and efficiently isolate them from whole blood can be thought of as looking for a needle in a haystack. Our microfilter technology exploits the use of size based capture of the larger CTC from the smaller white blood cells and components of whole blood. The effective capture potential of the microfilter platform has driven the area of CTC analysis into a new age of research in the field of cancer. The ability to finally analyze CTC at a molecular level, leads to a deeper understanding of metastatic process, while providing an opportunity to evaluate, monitor and manage treatment options as well as the adherent possibility of having an “on-chip” drug sensitivity assay for focused treatment options. We have demonstrated through clinical trials the ability to effectively identify, enumerate and characterize CTC based on immunfluorescence and FISH assays and provide a companion endpoint for monitoring and evaluating treatment management. Our work on viable CTC capture has resulted in successfully capturing and culturing CTC from blood in mouse models that have been inoculated with breast cancer cell lines to form primary and secondary metastatic cancer sites. The future potential within the microfilter technology to capture viable CTC for culture, will catapult therapeutic interventions to a new level of personalized medicine in cancer management.

Circulating tumor cells (CTCs) represent a surrogate biomarker of hematogenous metastases. In recent years, their detection has gained increasing interest. There is ample evidence regarding the ability to detect CTCs and their prognostic relevance, but their demonstrated predictive value in therapeutic response monitoring is clinically even more meaningful. Many clinical trials in the early and metastatic cancer setting now include CTCs as a monitoring parameter, and numerous translational studies attempting their molecular characterization are under way. There has been great progress in defining the clinical importance of CTCs, and it now seems likely that we may expect wider implementation of CTCs as a diagnostic oncology tool to monitor therapeutic response in real time. Novel technologies may further facilitate molecular characterization of CTCs and development of novel therapeutic targets, possibly leading to more powerful treatment strategies for cancer patients. As the detection and evaluation of CTCs are becoming an increasingly important diagnostic and prognostic tool, the goal of this review is to communicate the knowledge obtained through analysis of primary tumors and CTCs to oncologists and medical specialists in managing patients with cancer.

The hematogenous dissemination of cancer and development of distant metastases is the cause of nearly all cancer deaths. Detection of circulating tumor cells (CTCs) as a surrogate biomarker of metastases has gained increasing interest. There is accumulating evidence on development of novel technologies for CTC detection, their prognostic relevance and their use in therapeutic response monitoring. Many clinical trials in the early and metastatic cancer setting, particularly in breast cancer, are including CTCs in their translational research programs and as secondary end points. We summarize the progress of detection methods in the context of their clinical importance and speculate on the possibilities of wider implementation of CTCs as a diagnostic oncology tool, the likelihood that CTCs will be used as a useful biomarker, especially to monitor therapeutic response, and what may be expected from the future improvements in technologies.