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Melanoma is one of the most aggressive skin cancers due to its potential to metastasize widely in the body. The risk of metastasis is increased with later detection and increased thickness of the primary lesion, thus early identification and surgical removal is critical for higher survival rates for patients. However, even with appropriate treatment, some patients will develop recurrence which may be difficult to identify until advanced or causing symptoms. Recent advances in liquid biopsy have proposed less-invasive alternatives for cancer diagnosis and monitoring using minimal/zero invasion at sample collection, and circulating tumor cells(CTCs) have been considered a promising blood-based surrogate marker of primary tumors. However, previous CTC technologies relying on epithelial-cell adhesion molecules have limited to epithelial cells, thus hampering use of CTCs for non-epithelial cancers such as melanoma. Here, we used the Melanoma-specific OncoBean platform(MelanoBean) conjugated with melanoma specific antibodies(MCAM and MCSP). The device was used in comprehensive studies for diagnosing melanoma and evaluating surgery efficacy based on change in the number and characteristics of CTCs and CTC-clusters pre- and post-surgical treatment. Our study demonstrated that melanoma patients(n=45) at all stages(I-IV) have a noticeable number of MCTCs as well as MCTC-clusters compared to healthy donors(n=9)(

CMOS neural interfaces are aimed at studying the electrical activity of neurons and may help to restore lost functions of the nervous system in the future. The central function of most neural interfaces is the detection of extracellular electrical potentials by means of numerous microelectrodes positioned in close vicinity to the neurons. Modern neural interfaces require compact low-power, low-noise readout circuits, capable of recording from thousands of electrodes simultaneously without excessive area consumption and heat dissipation. In this article, we propose a novel readout technique for neural interfaces. The readout is based on a voltage-controlled oscillator (VCO), the frequency of which is modulated by the input voltage. The novelty of this work lies in the postprocessing of the VCO output, which is based on generating digital timestamps that contain temporal information about the oscillation. This method is potentially advantageous, because it requires mostly digital circuitry, which is more scalable than analog circuitry. Furthermore, most of the digital circuitry required for VCO-timestamping can be shared among several VCOs, rendering the architecture efficient for multi-channel architectures. This article introduces the VCO-timestamping concept, including theoretical derivations and simulations, and presents measurements of a prototype fabricated in 0.18-μm CMOS technology. The measured input-referred noise in the 300 Hz–5 kHz band was 5.7 μ V rms , and the prototype was able to detect pre-recorded extracellular action potentials., IEEE Transactions on Biomedical Circuits and Systems, 17 (3), ISSN:1932-4545, ISSN:1940-9990

Supporting information showing CTC enumeration and gene expression profiling data for CTC clusters from early stage lung cancer.

Early detection of metastasis can be aided by circulating tumor cells (CTC), which also show potential to predict early relapse. Because of the limited CTC numbers in peripheral blood in early stages, we investigated CTCs in pulmonary vein blood accessed during surgical resection of tumors. Pulmonary vein (PV) and peripheral vein (Pe) blood specimens from patients with lung cancer were drawn during the perioperative period and assessed for CTC burden using a microfluidic device. From 108 blood samples analyzed from 36 patients, PV had significantly higher number of CTCs compared with preoperative Pe (P < 0.0001) and intraoperative Pe (P < 0.001) blood. CTC clusters with large number of CTCs were observed in 50% of patients, with PV often revealing larger clusters. Long-term surveillance indicated that presence of clusters in preoperative Pe blood predicted a trend toward poor prognosis. Gene expression analysis by RT-qPCR revealed enrichment of p53 signaling and extracellular matrix involvement in PV and Pe samples. Ki67 expression was detected in 62.5% of PV samples and 59.2% of Pe samples, with the majority (72.7%) of patients positive for Ki67 expression in PV having single CTCs as opposed to clusters. Gene ontology analysis revealed enrichment of cell migration and immune-related pathways in CTC clusters, suggesting survival advantage of clusters in circulation. Clusters display characteristics of therapeutic resistance, indicating the aggressive nature of these cells. Thus, CTCs isolated from early stages of lung cancer are predictive of poor prognosis and can be interrogated to determine biomarkers predictive of recurrence. Cancer Res; 77(18); 5194–206. ©2017 AACR.

Background Activated protein C resistance (APCR) due to factor V Leiden (FVL) mutation (R506Q) is a major risk factor in patients with venous thromboembolism (VTE). The present study investigated the clinical manifestations and the risk of venous thromboembolism regarding multiple clinical, laboratory, and demographic properties in FVL patients. Material and methods A retrospective cross-sectional analysis was conducted on a total of 288 FVL patients with VTE according to APCR. In addition, 288 VET control samples, without FVL mutation, were also randomly selected. Demographic information, clinical manifestations, family and treatment history were recorded, and specific tests including t-test, chi-square and uni- and multi-variable regression tests applied. Results APCR was found to be 2.3 times significantly more likely in men (OR: 2.1, p p < 0.05). However, APCR could not be an independent risk factor for arterial thrombosis (AT) and pregnancy complications. Moreover, patients were evaluated for thrombophilia panel tests and showed significantly lower protein C and S than the control group and patients without DVT (p Conclusion FVL mutation and APCR abnormality are noticeable risk factors for VTE. Screening strategies for FVL mutation in patients undergoing surgery, oral contraceptive medication, and pregnancy cannot be recommended, but a phenotypic test for activated protein C resistance should be endorsed in patients with VTE.

Background Myelodysplastic syndrome (MDS), a heterogeneous group of hematopoietic malignancy, has been shown to present different cytogenetic abnormalities, risk factors, and clinico-hematological features in different populations and geographic areas. Herein, we determined the cytogenetic spectrum and clinico-hematological features of Iranian MDS patients for the first time. Methods This prospective cross-sectional study was conducted on 103 patients with MDS in Ahvaz, southwest of Iran, from 2014 to 2018. Clinical presentations, complete blood counts (CBC), and bone marrow (BM) biopsy samples were assessed. Perls' staining was used to evaluate BM iron storage. The cytogenetic evaluation was performed using the conventional G banding method on the BM. Results Patients’ median age was 62.3 (ranged from 50–76), and the majority were male (72.8%). The most common clinical symptom at the time of admission was fatigue (n = 33) followed by pallor (n = 27). The most common subgroup was MDS-Multi Lineage Dysplasia (MDS-MLD) (n = 38, 36.8%), followed by MDS-Single Lineage Dysplasia (MDS-SLD) (n = 28, 18.4%). A normal karyotype was observed in 59 patients (57.3%), while 44 patients (42.7%) had cytogenetic abnormalities. Trisomy 8 (+ 8) was the most common cytogenetic abnormality (n = 14) followed by del 17p (n = 9) and monosomy 7 (− 7) (n = 7). Twelve patients (11.65%) were transformed to AML. Conclusion Our data betokened that among our MDS patients, Trisomy 8 is the predominant cytogenetic abnormality, and MDS-MLD and MDS-SLD are the most common of subtypes. Noteworthy, the male: female ratio was slightly higher in Iran than in previous reports from other parts of the world. Our study is the first report of the clinical, hematological, and cytogenetic spectrum of MDS patients in Iran

Graphical abstract, Tumor heterogeneity is generated through a combination of genetic and epigenetic mechanisms, the latter of which plays an important role in the generation of stem like cells responsible for tumor formation and metastasis. Although the development of single cell transcriptomic technologies holds promise to deconvolute this complexity, a number of these techniques have limitations including drop-out and uneven coverage, which challenge the further delineation of tumor heterogeneity. We adopted deep and full-length single-cell RNA sequencing on Fluidigm’s Polaris platform to reveal the cellular, transcriptomic, and isoform heterogeneity of SUM149, a triple negative breast cancer (TNBC) cell line. We first validate the quality of the TNBC sequencing data with the sequencing data from erythroleukemia K562 cell line as control. We next scrutinized well-defined marker genes for cancer stem-like cell to identify different cell populations. We then profile the isoform expression data to investigate the heterogeneity of alternative splicing patterns. Though classified as triple-negative breast cancer, the SUM149 stem cells show heterogeneous expression of marker receptors (ER, PR, and HER2) across the cells. We identified three cell populations that express patterns of stemness: epithelial-mesenchymal transition (EMT) cancer stem cells (CSCs), mesenchymal-epithelial transition (MET) CSCs and Dual-EMT-MET CSCs. These cells also manifested a high level of heterogeneity in alternative splicing patterns. For example, CSCs have shown different expression patterns of the CD44v6 exon, as well as different levels of truncated EGFR transcripts, which may suggest different potentials for proliferation and invasion among cancer stem cells. Our study identified features of the landscape of previously underestimated cellular, transcriptomic, and isoform heterogeneity of cancer stem cells in triple-negative breast cancers.

Background: This study investigated the possible role of Genistein as a combination with Imatinib in controlling leukemia cell line proliferation. Methods: Three cell lines, K562, Kcl22, and CCRF, were cultured and analyzed for MTT, LDH, apoptosis, and cycle cell gene expression in the presence of different dosages of Imatinib and Genistein in combination or separately. Results: Data has shown a decrease in proliferation and an increase in apoptosis activity during combination treatment. LDH assay has shown no additional toxicity due to Genistein consumption in combination therapy. Analysis of the expression of responsible genes for cell cycle demonstrated both G1 (p53, p21 upregulation) and G2 (cdc25c downregulation) inhibitory effect in combination treatment. Conclusion: Altogether, this study suggests thatthe combination treatment of Imatinib and Genistein for leukemia cells resistant to Imatinib can increase treatment efficiency.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

Preclinical studies demonstrated that radiation up-regulates PD-L1 expression in tumor cells, providing a rationale for combining PD-1/PD-L1 inhibitors with radiation. However this has not been validated in patients with non-small cell lung cancer due to the difficulty to obtain serial biopsies. Measuring PD-L1 expression in circulating tumor cells (CTCs), may allow real-time monitoring of immune activation in tumor. In this study, whole blood from non-metastatic NSCLC patients was collected before, during, and after radiation or chemoradiation using a microfluidic chip. PD-L1 expression in CTCs was assessed by immunofluorescence and qPCR and monitored through the course of treatment. Overall, PD-L1(+) CTCs were detected in 25 out of 38 samples (69.4%) with an average of 4.5 cells/ml. After initiation of radiation therapy, the proportion of PD-L1(+) CTCs increased significantly (median 0.7% vs. 24.7%, P

Background: Activated protein C resistance (APCR) due to factor V R506Q (Leiden) mutation is a major risk factor in patients with venous thromboembolism. The present study investigated the symptoms patterns and the risk for venous thromboembolism regarding multiple clinical, laboratory, and demographic properties in APCR patients.Material and Methods: A retrospective cross-sectional analysis was conducted on a total of 288 APCR patients with age interval ranging between 1 to 80 years. In addition, 288 control samples, reported healthy after confirmatory tests, were also randomly selected. Demographic information, clinical manifestations, family and treatment history were recorded, and specific tests applied.Results: APCR was found to be 2.3 times significantly more likely in men (OR: 2.1, p < 0.05) than women. The risk of DVT and PE in APCR patients was 4.5 and 3.2 times more than the normal group, respectively (p < 0.05). However, APCR could not be an independent risk factor for arterial thrombosis and pregnancy complications. Moreover, patients were evaluated for thrombophilia panel tests and showed significantly lower protein C and S than the control group and patients without DVT (pConclusion: Factor V Leiden mutation and APCR abnormality are noticeable independent risk factors for venous thromboembolism. Screening strategies for factor V Leiden mutation in patients undergoing surgery, oral contraceptive medication, and pregnancy cannot be recommended, but a phenotypic test for activated protein C resistance should be endorsed in patients with venous thromboembolism.

Reactive Oxygen Species Modulator 1 (ROMO1) plays a pivotal role in the regulation of mitochondrial structure integrity, and the production of reactive oxygen species (ROS). Increased ROMO1 expression was reported in various cancer cell lines; however, the possible association between ROMO1 expression and bladder cancer was not well studied. The present study aimed to investigate the rate of ROMO1 expression and the correlation of oxidative stress with the development of bladder cancer. In this study, a total of 35 cancerous and healthy adjacent tissues were examined using quantitative real-time polymerase chain reaction (qRT-PCR) to analyze the gene expression of ROMO1. Also, we evaluated the serum level of ROMO1 and Total Antioxidant Capacity (TAC), as well as Total Oxidant Status (TOS) in patients with bladder cancer along with age- and sex-matched healthy individuals. The ROMO1 gene was significantly higher in cancerous tissues than that of adjacent healthy tissues. Also, the serum levels of ROMO1, TAC, TOS, and Oxidative Stress Index (OSI) were increased in patients with bladder cancer compared with healthy subjects. It can be concluded that the overexpression of the ROMO1 gene is associated with advanced grades of bladder cancer as well as an increase in oxidative stress conditions. Our findings also suggest that the serum level of ROMO1 might be a promising tumor marker for bladder cancer.

Summary During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/− or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH−CD44+CD24− human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24− by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity., Highlights • Isolation and RNA-seq of ALDH+ and CD44+CD24− breast cells • Unlike in cancer, there is substantial overlap in ALDH+ and CD44+CD24− populations • Single-cell analysis of ALDH+ cells identifies unexpected subpopulation structure • Hybrid epithelial/mesenchymal ALDH+ cells have a cancer-like expression signature, In this article, Colacino and colleagues use flow-cytometry-sorted populations and single-cell analyses to investigate human mammary stem cells. They discover unexpected phenotypic and functional heterogeneity at the single-cell level, including a subpopulation of ALDH+ stem cells with a hybrid epithelial/mesenchymal phenotype and triple-negative breast cancer-like gene expression pattern.

Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells (CSCs), a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photo-mechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube-polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.

This paper aimed to design a sustainable agile retail supply chain using multi-objective optimization methods. To this end, a mathematical model was presented for the sustainable agile supply chain with five objectives, including "minimizing costs", "minimizing unanswered demand", "maximizing the quality of goods purchased from suppliers," "maximizing social responsibility or social benefits", and "minimizing environmental impacts". The NSGA-II, PESA and SPEA-II algorithms were used to solve the proposed model, which were run in MATLAB software. After collecting data from the SAIPA Company’s supply chain, the model was solved using the three algorithms. The results indicate that the SPEA-II algorithm produces more high quality responses, compared to the other two algorithms. Furthermore, the SPEA-II algorithm was found to be among the Pareto Front responses. A decrease of environmental impacts had no effect on the problem responses due to the lack of a specific structure in the current system.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α

Background Chronic Myeloid Leukemia (CML) is characterized by the overproduction of BCR-ABL, a tyrosine kinase with constitutive activity, in which the majority of CML patients have e13a2 or e14a2 transcripts. Reckoned the possible associations between the hematologic and molecular features of the disease, a profound understanding of different aspects of this neoplasm would be provided. Method The authors implemented a systematic literature search, utilizing the terms published articles or internationally accepted abstracts from PubMed, Embase, Medline, Cochrane library before January 2019. Weighted mean proportion and 95 % confidence intervals (CIs) of CML prevalence calculated using a fixed-effects and a random-effects model. Statistical heterogeneity was evaluated using the I2 statistic. Results 34 studies for a total of 54,034 Patients were selected and included in the review. Results revealed that compared to e13a2 group, the overall estimated prevalence is much higher in the e14a2 (39 % and 54 %, respectively). Besides, the overall estimated prevalence ratio of male to female was higher in the e13a2 group in comparison to e14a2 (1.08 and 0.856 respectively). The overall estimated prevalence of dual transcription of e13a2/e14a2 was 1.11 %, and male/female overall estimated prevalence ratio was 1.18. Conclusion This meta-analysis of CML patients demonstrated the e14a2 as the more common transcript type. Usually, the e14a2 transcript is prevalent in females, whereas e13a2 and dual transcription of e13a2/e14a2 are more common in men. These data explicate that the differences in proportion are not by chance. This is crucial, as the transcript type is a variable suspected to be of prognostic importance for the treatment-related response, the outcome of treatment, and the rate of treatment-free remission.

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis.

While cancer cell populations are known to be highly heterogeneous within a tumor, the current gold standard of tumor profiling is through a tumor biopsy. These biopsies are invasive and prone to missing these clones due to spatial heterogeneity, and this bulk analysis approach can miss information from rare subpopulations. To noninvasively investigate tumor cell heterogeneity, a streamlined workflow is developed to scrutinize rare cells, such as circulating tumor cells (CTCs), for simultaneous analysis of mutations and gene expression profiles at the single cell level. This powerful workflow overcomes low-input limitations of single cell analysis techniques. The utility of this multiplexed workflow to unravel inter- and intra-patient heterogeneity is demonstrated using non-small-cell lung cancer (NSCLC) CTCs (n = 58) from six epidermal growth factor receptor (EGFR) mutant positive NSCLC patients. CTCs are isolated using a high-throughput microfluidic technology, the Labyrinth, and their EGFR mutation status and gene expression profiles are characterized.

The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) who possess activating epidermal growth factor receptor (EGFR) mutations that responded to tyrosine kinase inhibitors (TKIs). Current standard of care is to screen metastatic patients for the presence of EGFR mutations to determine if they qualify for TKI treatment. Due to the invasiveness of a tumor biopsy, this characterization is typically only done at the time of diagnosis. This single time-point determines the entire treatment plan. Despite strong initial response to TKI therapy, most patients develop resistance within months, most commonly by acquiring a secondary EGFR mutation. Identifying resistance through current monitoring techniques, such as measuring tumor volume, can lead to a delay in detection. Alternatively, circulating tumor cells (CTCs) are cancer cells present in the blood that provide access to tumor cells without the requirement of a biopsy. Shed from the tumor into the vasculature, CTCs circulate throughout the body before a fraction extravasate, leading to metastatic sites. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Due to their ease of access, they can be used to serially track patients’ conditions to detect the early emergence of new tumor clones and, potentially, therapeutic resistance. One of the major limitations of CTCs’ clinical utility is their low abundance in the blood. Our group previously developed the Labyrinth, a high-throughput, label-free microfluidic technology, which enables rapid and efficient CTC enrichment. These CTCs are used for enumeration or single-cell transcriptomic analysis. Initial single-cell analysis was performed through highly multiplexed RT-qPCR that compared 96-gene expression profiles to unravel inter- and intrapatient CTC heterogeneity. We recently developed a complimentary single-cell analysis approach using digital PCR (dPCR) to detect the presence of sensitizing EGFR mutations, exon 19 deletion and L858R, and resistance mutation, T790M. dPCR is an ultrasensitive approach with single-molecule resolution, which enables detection and quantification at a single-cell level by partitioning the sample into individual PCR reaction droplets. This resolution can distinguish if a cell is homo- or heterozygous for a mutation and each allele’s relative expression level. We validated this system for single-cell analysis using lung cancer cell lines and demonstrated its agreement with qPCR results. We present initial findings of EGFR mutation screening in CTCs from a small cohort of NSCLC patients with known EGFR status. We highlight the single-cell heterogeneity of CTCs within a patient at both the gene expression and EGFR mutation level. By tracking these patients’ CTCs over time, we may be able to identify the emergence of resistant clones sooner than can be done in the clinic. This may allow for more rapid treatment modification and lead to improved patient outcomes. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Mina Zeinali, Evan Keller, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Identifying single-cell gene expression and EGFR mutation profile heterogeneity in NSCLC patients’ CTCs [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B36.

Rates of progression and treatment response in advanced prostate cancer are highly variable, necessitating non‐invasive methods to assess the molecular characteristics of these tumors in real time. The unique potential of circulating tumor cells (CTCs) to serve as a clinically useful liquid biomarker is due to their ability to inform via both enumeration and RNA expression. A microfluidic graphene oxide‐based device (GO Chip) is used to isolate CTCs and CTC clusters from the whole blood of 41 men with metastatic castration‐resistant prostate cancer. Additionally, the expression of 96 genes of interest is determined by RT‐qPCR. Multivariate analyses are conducted to determine the genes most closely associated with overall survival, PSA progression, and radioclinical progression. A preliminary signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis in this setting.

The concept of the stem cell of origin for cancers was first proposed more than 100 years ago. In this theoretical model, certain cells with self-renewal capacity would form tumors from “embryonic rests.” More evidence has accumulated since that time as a result of extensive research that strongly supports the cancer stem cell (CSC) hypothesis, suggesting the existence of self-renewing cells that generate heterogenous populations of cells within the tumor mass. The preponderance of evidence now suggests that the majority of cancers are hierarchically organized and sustained by a population of cells that display stem cell properties—CSCs. As is the case for normal tissue stem cells, CSCs are able to self-renew and differentiate, generating cells that comprise the tumor bulk. Furthermore, preclinical and clinical studies demonstrate that CSCs mediate tumor metastasis and contribute to resistance to chemotherapy and radiation therapy. The CSC hypothesis has fundamental biological and clinical implications that are discussed in detail in this chapter.

Early detection of metastasis can be aided by circulating tumor cells (CTC), which also show potential to predict early relapse. Because of the limited CTC numbers in peripheral blood in early stages, we investigated CTCs in pulmonary vein blood accessed during surgical resection of tumors. Pulmonary vein (PV) and peripheral vein (Pe) blood specimens from patients with lung cancer were drawn during the perioperative period and assessed for CTC burden using a microfluidic device. From 108 blood samples analyzed from 36 patients, PV had significantly higher number of CTCs compared with preoperative Pe (P < 0.0001) and intraoperative Pe (P < 0.001) blood. CTC clusters with large number of CTCs were observed in 50% of patients, with PV often revealing larger clusters. Long-term surveillance indicated that presence of clusters in preoperative Pe blood predicted a trend toward poor prognosis. Gene expression analysis by RT-qPCR revealed enrichment of p53 signaling and extracellular matrix involvement in PV and Pe samples. Ki67 expression was detected in 62.5% of PV samples and 59.2% of Pe samples, with the majority (72.7%) of patients positive for Ki67 expression in PV having single CTCs as opposed to clusters. Gene ontology analysis revealed enrichment of cell migration and immune-related pathways in CTC clusters, suggesting survival advantage of clusters in circulation. Clusters display characteristics of therapeutic resistance, indicating the aggressive nature of these cells. Thus, CTCs isolated from early stages of lung cancer are predictive of poor prognosis and can be interrogated to determine biomarkers predictive of recurrence. Cancer Res; 77(18); 5194–206. ©2017 AACR.

The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

During development and pregnancy, the human mammary gland undergoes extensive remodeling in processes driven by populations of stem and progenitor cells. We recently reported that breast cancers are also hierarchically organized and driven by distinct populations of cancer stem cells characterized as CD44+CD24low/−or by expression of Aldehyde dehydrogenase (ALDH). These sets of markers identify largely non-overlapping mesenchymal and epithelial populations, each of which is capable of tumor initiation when transplanted into immunosuppressed mice. Less is known about these two populations, individually or their overlap, in the normal human mammary gland. The goal of this study was to understand the biology of the ALDH+and CD44+CD24−populations in the normal human breast, using flow cytometry based sorting paired with functionalex vivoanalyses, RNA-sequencing, and single cell RNA expression profiling. ALDH+cells and ALDH−CD44+CD24−cells, generally, have epithelial-like and mesenchymal-like characteristics, respectively. Despite this, there are substantial similarities in the biological pathways activated in both populations when compared to differentiated cells. Additionally, we found a substantial proportion of cells that simultaneously express ALDH+and CD44+CD24−whose abundance varies between individuals. At the single cell level, these cells have the greatest mammosphere forming capacity and express high levels of stemness and EMT-associated genes includingID1, SOX2, TWIST1, and ZEB2.Through unbiased analysis of individual ALDH+ cells, we find cells with either epithelial or mesenchymal expression phenotypes. We also identify a subpopulation of cells with a hybrid epithelial/mesenchymal expression phenotype that overexpress genes associated with aggressive triple negative breast cancers. These results highlight the utility of single cell analyses to characterize tissue heterogeneity, even in marker enriched cell populations, and further identifies the genes and pathways that define this heterogeneity.

Purpose: Fibroblastic growth factor-10 (FGF-10) has an important role in type I epithelial mesenchymal transition (EMT) during the embryonic period of life (gastrulation). Since EMT has a critical role during cancer cells invasion and metastasis (type III) this study sought to investigate the possible role of FGF-10 in type III EMT by monitoring breast cancer cell lines’ behavior by FGF-10 regulation. Methods: MCF-7 and MDA-MB-231 cell lines with different levels of FGF10 expression were treated with FGF-10 recombinant protein and FGF-10 siRNA, respectively. Results: The cell viability, migration, colony formation and wound healing have a direct relationship with FGF-10 expression, while FGF-10 expression decreased apoptosis. All mesenchymal factors (such as vimentin, N cadherin, snail, slug, TGF-β) increased due to FGF-10 expression with contrary expression of epithelial markers (such as E-cadherin). Moreover, GSK3β phosphorylation (inactivation) increased with FGF-10 expression. Conclusion: The important role of FGF-10 in type III EMT on cancer cells and initiation of metastasis via various kinds of signaling pathways has been suggested.

Background: Human endometrial-derived stem cells (hEnSCs) as multipotent accessible source of cells are known as useful cell candidates in the field of bone tissue engineering. However, the effect of bone morphogenic protein-2 (BMP-2) as an osteoinductive growth factor has not been clearly ascertained. Objective: To evaluate the effect of the remarkable osteoinductive growth factor BMP-2, on promotion of osteogenic differentiation in hEnSCs. Methods: Endometrial biopsies were obtained from healthy women referred to the hospital for infertility treatment. After tissue digestion in collagenase, the isolated endometrial cells were expanded in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS. The propagated cells were characterized based on the expression of endometrial (CD90, CD105), endothelial (CD31), and hematopoietic (CD34, CD133) stem cell markers. Cells were differentiated in osteogenic medium containing DMEM supplemented with 10% FBS, 10 nM dexamethasone, 50 μg/ml Ascorbic acid, and 10 mM β-glycerophosphate in the presence or absence of BMP-2 for 21 days. Alizarin red staining was performed to verify the matrix mineralization. Immunocytochemical staining was conducted to detect the expression of OCT-4, CD133, and osteopontin as well as osteocalcin. The expression of osteoblast transcripts, including osteopontin, osteonectin, and alkaline phosphatase (ALP) were analyzed by semi quantitative PCR. Results: The expanded EnSCs were spindle shaped. They were positive for the expression of Oct-4, CD90, and CD105, while they were negative for endothelial and hematopoietic markers. The matrix mineralization was confirmed by Alizarin red in both groups at day 21. Although the expression of osteopontin and osteocalcin was detected in both groups by immunological staining, the expression of osteocalcin was more intense in the presence of BMP-2. ALP, Osteonectin and osteopontin transcripts were expressed in all groups; however, the expression of ALP and osteopontin was upregulated in the presence of BMP-2. Conclusion: BMP-2 as an osteoinductive growth factor, could promote the osteogenic differentiation of EnSCs in vitro.

Summary We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations.

In the era of personalized medicine, many targeted therapies have been developed against cancer-specific antigens, which have greatly improved patient outcome. The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors (TKIs). To determine if a patient qualifies for targeted therapy, the patient must undergo a tumor biopsy. Usually done at the time of diagnosis, this single time-point examination dictates the treatment plan. Despite strong initial response to TKI therapy, nearly all patients develop resistance, most commonly by acquiring a secondary EGFR mutation. At the time of halted response, an additional biopsy may be necessary to screen for the presence of these secondary, resistance-causing mutations. Circulating tumor cells (CTCs) are cancer cells present in the blood that provide an alternative to an invasive tumor biopsy. Shed from the primary tumor, CTCs circulate throughout the vasculature, and extravasate leading to metastasis. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Easily accessed through a blood draw, they can be used to serially track patients throughout treatment to detect the early emergence of new tumor clones. One of the biggest hurdles with CTCs being clinical prognostic markers is their low abundance in the blood. We have previously developed a high-throughput, label-free microfluidic technology, the Labyrinth, to isolate CTCs from the blood of breast, lung, and pancreatic cancer patients. This technology allows for quick and efficient isolation of CTCs from the millions of surrounding blood cells. After isolation, the CTCs are used for enumeration or single cell analysis. The single cell gene expression analysis performs a highly multiplexed RT-qPCR of a pre-designed 96 gene panel. Here we report a complementary single cell analysis approach to detect the presence of EGFR point mutations, L858R and T790M, implicated in TKI response using digital PCR (dPCR). dPCR is a highly sensitive approach which enables detection and quantification a single-cell level. dPCR relies on small sample loading and partitions the sample into individual PCR reaction droplets. This single molecule resolution provides the ability to distinguish if a cell is homo- or heterozygous for a mutation as well as their relative expression levels. We have validated this system for single cell analysis using lung cancer cell lines with known L858R and T790M mutation status. We present initial findings of EGFR mutation screening of lung CTCs from patients with known EGFR status based on tissue profiling. By tracking patients over time, comparing the CTC profiles, and correlating it to patient outcomes, we may be able to better identify early treatment resistant clones and adjust their treatment to improve patient outcome. Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Gene expression and mutation profiles of single lung circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 407.

Transdermal drug delivery of proteins is challenging because the skin acts as a natural and protective barrier. Several techniques including using the cell-penetrating peptides have been studied to increase the penetration of therapeutic proteins into and through the skin. Cell-penetrating peptides facilitate and improve the transduction of large and hydrophilic cargo molecules through plasma membrane. We have recently reported an efficient skin delivery of elastin protein in complex with a cell-penetrating peptide called Pep-1. As the biophysical characteristics of cell-penetrating peptide/protein complexes have been linked with their biological responses, in this study, we investigated biophysical properties of Pep-1/elastin complexes (ratio 10:1) stored in three temperatures (-20 °C, 4 °C and 25 °C) by photon correlation spectroscopy, circular dichroism and isothermal denaturation. We also evaluated the ability of transduction of this complex into cells and skin tissue using both fluorescence microscopy and Kodak In-Vivo FX Pro Imaging System.

Recent studies showed that organophosphorus pesticides can increase incidence of metabolic disorders and diabetes. Considering the importance of pancreas in regulating blood glucose, the effect of malathion on essential elements of glucose-stimulated insulin secretion (GSIS) in in vitro and in vivo conditions were evaluated. After dividing of rats into control and treatment groups, oral glucose tolerance test (OGTT) was examined and then kinetic of glucose as well as pancreatic response to raise blood glucose were evaluated. After isolation of islets from pancreas, its function as well as oxidative stress markers and essential elements of GSIS were examined. Malathion at dose of 400 mg/kg impaired GTT and increased AUC0–180 min (P = 0.047) and T1/2β of glucose (P = 0.0016), and reduced insulin response (P = 0.005) 30 min after oral administering of glucose. In addition to impaired glucose tolerance, there were significant increases in lipid peroxidation (P Conclusion Dysfunction of glucose metabolism and impairment of insulin secretion are associated with a depletion of energy and induction of oxidative stress following acute exposure to malathion.

Disorder of glucose homeostasis is one of the most important complications following exposure to organophosphorous (OPs) pesticides. Regarding the importance of adipose tissue in regulating blood glucose and the role of oxidative stress in toxicity of OPs and in the continue of our previous works, in the present study we focused on tumor necrosis factor alpha (TNFα), glucose transporter type 4 (GLUT4), and nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-κB) in a sublethal model of toxicity by diazinon as a common OPs. Following time-course study of various doses of diazinon in impairing blood glucose, dose of 70mg/kg/day was found the optimum. Animals were treated for 4 weeks and after gavage of glucose (2g/kg), the glucose change was evaluated at time-points of 0, 30, 60, 120 and 180min to identify oral glucose tolerance test (GTT). In addition, serum insulin was measured in fasting condition. In adipose tissue, oxidative stress markers including reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and TNFα were evaluated. The mRNA expression of GLUT4, Nf-κB and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were also determined by real time reverse transcription polymerase chain reaction (RT-PCR). Diazinon at dose of 70mg/kg/day impaired GTT and diminished insulin level while augmented ROS, NADPH oxidase, and TNFα. The GLUT4 mRNA expression was amplified by diazinon while unlikely, the expression of Nf-κB gene did not change. On the basis of biochemical and molecular findings, it is concluded that diazinon impairs glucose homeostasis through oxidative stress and related proinflammatory markers in a way to result in a reduced function of insulin inside adipose tissue. Although, diazinon interfered with pancreatic influence on the adipose tissue most probably via stimulation of muscarinic receptors, current data are not sufficient to introduce adipose tissue as a target organ to OPs toxicity. Considering the potential of OPs to accumulate in adipose tissue, it seems a good candidate organ for future studies. Although, hyperglycemia was not induced by diazinon but increased AUC0-180min leads us to the point that diazinon induces kind of instability in glucose homostasis and diabetes.

Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.