Immunomagnetic Cell Separation Research Articles

The Utilization of Optically Induced Dielectrophoresis (ODEP)-Based Cell Manipulation in a Microfluidic System for the Purification and Sorting of Circulating Tumor Cells (CTCs) with Different Sizes.

The analysis of circulating tumor cells (CTCs) at the molecular level holds great promise for several clinical applications. For this goal, the harvest of high-purity, size-sorted CTCs with different subtypes from a blood sample are important. For this purpose, a two-step CTC isolation protocol was proposed, by which the immunomagnetic beads-based cell separation was first utilized to remove the majority of blood cells. After that, an optically induced dielectrophoresis (ODEP) microfluidic system was developed to (1) purify the CTCs from the remaining magnetic microbeads-bound blood cells and to (2) sort and separate the CTCs with different sizes. In this study, the ODEP microfluidic system was designed and fabricated. Moreover, its optimum operation conditions and performance were explored. The results exhibited that the presented technique was able to purify and sort the cancer cells with two different sizes from a tested cell suspension in a high-purity (93.5% and 90.1% for the OECM 1 and HA22T cancer cells, respectively) manner. Overall, this study presented a technique for the purification and sorting of cancer cells with different sizes. Apart from this application, the technique is also useful for other applications in which the high-purity and label-free purification and sorting of cells with different sizes is required.

Investigating the immunomodulatory effects of histotripsy ablation for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary bone cancer in humans and dogs. Canine OS shares many biological similarities with human OS and is a valuable comparative oncology research model. In both species, metastasis remains a major hurdle against improved survival, thus warranting a novel therapeutic approach such as histotripsy, a non-thermal, non-invasive, focused ultrasound ablation modality. Histotripsy can mechanically disintegrate gross tumor and potentially induces an anti-tumor immune response to inhibit metastatic development. The objectiveof this in vitro study was to investigate the immunomodulatory effects of histotripsy ablation of canine OS. We hypothesizedthat histotripsy ablation of OS promotes a pro-inflammatory immune signature in primary monocytes and macrophages. We isolated CD14+ primary monocytes using positive selection immunomagnetic cell separation from peripheral blood mononuclear cells of healthy dogs, and differentiated primary macrophages by culturing primary monocytes with canine GM-CSF for 72 hours. We co-cultured primary monocytes and macrophages with histotripsy-ablated canine D17 OS cells for 24 hours, then characterized their cell surface markers using flow cytometry and performed multiplex supernatant cytokine analysis. We observed upregulation of CD80 and CD62L on primary monocytes and a downregulation of CD206 on macrophages exposed to histotripsy-ablated D17 cells compared to controls, suggesting immune activation. We observed decreased levels of the pro-tumor cytokine, TGF-β, from primary monocytes exposed to histotripsy-ablated D17s compared to controls. Our results indicate that histotripsy ablation of OS can induce a pro-inflammatory immune signature. This work is supported by grants from the American Kennel Club Canine Health Foundation, the Focused Ultrasound Foundation.

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture.

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16(MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of "masked" CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.

Standardised method for cardiomyocyte isolation and purification from individual murine neonatal, infant, and adult hearts

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different age-dependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56–2.2 × 106 cells/heart) and viability (~70–100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell type-specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR < 0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rod-shaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

Abstract 11948: A Standardized Method to Isolate and Purify Cardiomyocytes from Individual Mouse Hearts Irrespective of Postnatal Age

Rational: Primary cardiomyocytes (CMs) are invaluable for understanding postnatal heart development and elucidating disease mechanisms in genetic and pharmacological models, however, a method to obtain freshly purified CMs at any postnatal age, without the need for different age-dependent isolation procedures and cell culture, is lacking. Objective: To develop a standardized method that allows rapid isolation and purification of CMs in high yield and viability from individual neonatal, infant, and adult mice. Methods and Results: Using a novel in situ aortic cannulation procedure optimized to allow cannulation of even the very small vessel of neonates [postnatal day 0-2 (P0-2)], hearts of C57BL/6J mice were subjected to Langendorff retrograde perfusion and enzymatic digestion. After subsequent tissue disaggregation and filtration, CMs were isolated in high yield (1.55-2.2 x 10 6 CMs/heart) and viability (~78%). The larger size of infant (P10 and P13) and adult (P70), but not neonatal CMs, compared to non-myocytes, allowed their further enrichment by differential centrifugation, before purification at the bench by bead-based immunomagnetic cell separation. This involved depletion of endothelial cells (for infant and adult preparations) or non-myocytes (neonatal preparations). Together, these procedures resulted in highly purified CMs (~95%) from hearts of all ages within 1 hour. Moreover, in situ fixation immediately after tissue digestion via coronary perfusion, preserved the cytoarchitecture of isolated CMs (~94% rod-shaped CMs at all postnatal ages), allowing capture of spindle-shaped neonatal cells undergoing mitosis, as well as enabling accurate quantitation of CM area and nucleation state. RNA-sequencing of CMs purified from one P2 male and female heart per litter (n=4 litters) showed distinct clustering by litters rather than by sexes; a finding consistent with cardiac size and shape being indistinguishable between P2 male (n=6) and female (n=6) hearts as determined by micro-computed tomography (X-ray 3D imaging). Conclusion: The procedures developed here provide a universal protocol for the rapid purification of high-quality CMs from individual hearts at any postnatal age, even those of neonates.

Application and utility of alternative methods in isolation of pure cells from forensic biological mixtures in modern-day: a review

Development of genetic profiles from the biological mixtures has remained challenging, although modern-day technologies may help forensic scientists to attain a reliable genetic profile in the identification of the accused. In the case of rape, vaginal swab exhibits usually contain epithelial cells of victims and sperm cells of accused, such samples are more challenging when there is more than one contributor. In such cases, separation of distinct cells from a mixture that includes blood cells, epithelial cells and sperm cells for their single genetic profile is important. In the last ten decades several new techniques were developed and invented for the separation of single cell from the biological mixture that includes differential lysis, laser micro-dissection, cell sorting (FACS), sieve-based filtration, (vi) micro-fluidic devices or immunomagnetic beads cell separation of fresh samples, and the magnetic activated cell sorting (MACS). Out of them, some techniques have been commonly applied for cell separation in forensic biology. Each technique has its own limitation. Some recent studies showed, magnetic activated cell sorting (MACS), laser capture microdissection (LCM), DEPArray technology and fluorescence activated cell sorting (FACS) has proved to be effective in separation of single cell from cell mixtures. Therefore, in this review we have evaluated these four alternative methods and their potential application in the modern-day over the others for the separation of a single cell from the mixture. In this review we also discuss the advantage of these methods and their modern–day applicability and acceptance in the forensic world.

Future perspectives on in-vitro diagnosis of drug allergy by the lymphocyte transformation test

This article aims to envisage future perspectives of the lymphocyte transformation test (LTT). We describe the select innovative techniques, which can be integrated at different stages of the LTT to potentially improve the sensitivity, specificity, or practicability of the LTT. We first focus upon the cell sorting techniques comprising immunomagnetic cell separation and flow cytometry, which can be implemented prior and after the LTT culturing step to concentrate and quantify specific immune cell types. Further, we elaborate upon three important omics techniques such as transcriptomics, proteomics, and metabolomics, which can be integrated downstream of the LTT to analyze molecular changes in specific immune cells following drug induced activation and proliferation. We also develop visions, how state of the art techniques used in other scientific fields, can be transferred and applied in the context of in-vitro detection of drug allergy.

Automated Large-Scale T Cell Isolation in a New Closed Cell Separation System

Abstract Large-scale T cell isolation is commonly performed in a wide range of laboratory settings, including for cell therapy and drug discovery research, as well as in core facilities as part of cell manufacturing and banking. However, current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day just for sample processing and cell isolation. Furthermore, many applications have stringent requirements for purity, sterility, and standardization of the cell isolation procedure. To address these needs, we have developed RoboSep™-C, an instrument for efficient and automated cell isolation in a closed system. RoboSep™-C automates the established EasySep™ technology for immunomagnetic cell separation to enable isolation of untouched T cells, CD4+ T cells, or CD8+ T cells from leukapheresis samples. To set up the system, the user follows the on-screen prompts to install a sterile single-use tubing set and load the recommended medium, cell isolation reagents, and starting leukopak. The instrument then performs all necessary cell processing steps including sample washing, cell labeling, magnetic separation, and cell concentration in a 50-minute protocol. Starting with samples ranging from 2.5 to 20 billion nucleated cells, we obtained purities of 95.4 ± 4.1%, 95.7 ± 2.5%, and 89.3 ± 3.5% for T cell (n=16), CD4+ T cell (n=14), and CD8+ T cell (n=12) isolation, respectively (mean ± SD). The recoveries of the isolated T, CD4+ T, and CD8+ T cells were 64.2 ± 12.4%, 67.0 ± 8.2%, and 54.3 ± 13.5%, respectively. Automated isolation of high-purity T cells with RoboSep™-C enables researchers to scale up their operations and can be easily integrated upstream of existing T cell expansion, genome editing, and cryopreservation protocols.

Microspheres made of polyvinyl alcohol crosslinked with glutaraldehyde for immunomagnetic cell separation

Microspheres made of polyvinyl alcohol crosslinked with glutaraldehyde for immunomagnetic cell separation

A Two-Step Immunomagnetic Microbead-Based Method for the Isolation of Human Primary Skin Telocytes/CD34+ Stromal Cells.

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31−/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα−/vimentin+) and fibroblasts (CD31−/CD34−/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.

Immunomagnetic cell separation is a suitable method for T cell electrophysiology and ion channel pharmacology

Abstract Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation. PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration. The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells. Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.

185 - Linear relationship of cells in vs. cells out with the clinimacs plus

There is a substantial body of experience using the CliniMacs Plus immunomagnetic cell separator to produce modified grafts for hematopoietic progenitor transplantation. In general, graft preparation has been performed on starting products of varying cellularity, and the total recovered product from CliniMacs manipulation has been administered in its entirety. This, in turn, leaves cell dosing as a confounding variable when evaluating clinical outcomes resulting from treatment with CliniMacs processed grafts. We hypothesize that the relationship between starting numbers of targeted cell types and final numbers may be sufficiently linear that final cell numbers might be pre-determined by controlling the number of targeted cells in the pre-processing products. We have reviewed the data of pre-processing cell numbers and the resultant targeted cell recoveries from our CD34 selection and CD45RA depletion single step processes and from our NK cell selection double step process. Using a Pearson's linear regression model, we analyzed each identified cell type, comparing the number of those cells input into the CliniMacs process with the number of the same cell type recovered at the end of processing. From these analyses, we obtained for each cell type a regression equation, y=ax+b, wherein a is the slope of the regression line and also the average recovery of the targeted cell, and b is the y axis intercept of the best fit line. Additionally, we obtained an R2 value, which identifies the fractional certainty that the input cell value predicts the output value. This analysis has revealed that for the identified cell types, there is a linear relationship, suggesting a predictability of final product counts from starting product counts with 65–94 percent certainty (R2). The resultant predictive model incorporates all processing steps, including washes, antibody incubations and the actual CliniMacs separation processing. It should be noted in particular that a linear relationship is present even in our NK cell enrichment processes which comprise a CD3 depletion followed serially by a CD56 selection. We conclude that maintaining minimal variation in the conduct of processing steps, combined with a non-varying CliniMacs process, produces a sufficiently predictable final product to allow for increased precision in cell dosing. Table ICliniMacs plus processing linearity. Manipulation Cell Type Targeted N Regression Line Slope R-square CD34 Selection CD34 positive 309 0.60 0.94 CD45RA depletion CD34 positive 64 0.56 0.88 CD45RA depletion CD3 positive/CD45RA negative 64 0.39 0.65 CD3 depletion followed by CD56 selection CD56 positive 151 0.53 0.80 CD3 depletion followed by CD56 selection CD56 positive/CD3 negative 151 0.53 0.80 Open table in a new tab

An immunomagnetic cell separation system based on a retroviral vector containing a chimeric, recombinant human-murine CD4 gene

An immunomagnetic cell separation system based on a retroviral vector containing a chimeric, recombinant human-murine CD4 gene

Homogeneity evaluation of mesenchymal stem cells based on electrotaxis analysis

Stem cell therapy that can restore function to damaged tissue, avoid host rejection and reduce inflammation throughout body without use of immunosuppressive drugs. The established methods were used to identify and to isolate specific stem cell markers by FACS or by immunomagnetic cell separation. The procedures for distinguishing population of stem cells took a time and needed many preparations. Here we suggest an electrotaxis analysis as a new method to evaluate the homogeneity of mesenchymal stem cells which can observe the stem cell population in culture condition and wide use to various types of stem cells. Human mesenchymal stem cell, adipose derived stem cell, tonsil derived stem cell and osteogenic differentiated cells migrated toward anode but the migration speed of differentiated cells was significantly decreased versus that of stem cells. In mixture of stem cells and differentiated cells condition, we identified that the ratio of stem cell versus differentiated cell was matched with the homogeneity evaluation data of stem cells based on electrotaxis analysis. As a result, our evaluation tool has the possibility of the wide use to stem cell homogeneity evaluation and might be used as the stem cell quality control during stem cell culture without any additional antibodies.

One-Step Facile Synthesis of Highly Magnetic and Surface Functionalized Iron Oxide Nanorods for Biomarker-Targeted Applications.

We report a one-step method for facile and sustainable synthesis of magnetic iron oxide nanorods (or IONRs) with mean lengths ranging from 25 to 50 nm and mean diameters ranging from 5 to 8 nm. The prepared IONRs are highly stable in aqueous media and can be surface functionalized for biomarker-targeted applications. This synthetic strategy involves the reaction of iron(III) acetylacetonate with polyethyleneimine in the presence of oleylamine and phenyl ether, followed by thermal decomposition. Importantly, the length and diameter as well as the aspect ratio of the prepared IONRs can be controlled by modulating the reaction parameters. We show that the resultant IONRs exhibit stronger magnetic properties compared to those of the widely used spherical iron oxide nanoparticles (IONPs) at the same iron content. The increased magnetic properties are dependent on the aspect ratio, with the magnetic saturation gradually increasing from 10 to 75 emu g-1 when increasing length of the IONRs, 5 nm in diameter, from 25 to 50 nm. The magnetic resonance imaging (MRI) contrast-enhancing effect, as measured in terms of the transverse relaxivity, r2, increased from 670.6 to 905.5 mM-1 s-1, when increasing the length from 25 to 50 nm. When applied to the immunomagnetic cell separation of the transferrin receptor (TfR)-overexpressed medulloblastoma cells using transferrin (Tf) as the targeting ligand, Tf-conjugated IONRs can capture 92 ± 3% of the targeted cells under a given condition (2.0 × 104 cells/mL, 0.2 mg Fe/mL concentration of magnetic materials, and 2.5 min of incubation time) compared to only 37 ± 2% when using the spherical IONPs, and 14 ± 2% when using commercially available magnetic beads, significantly improving the efficiency of separating the targeted cells.

A serum-free medium for differentiation of monocytes to macrophages

Abstract Macrophages play an important role in defense against pathogens and in tissue homeostasis. They are classified into two main groups depending on how they are activated: 1) M1 (activated by IFN-γ and LPS) and 2) M2, which include M2a (activated by IL-4 or IL-13), M2b (immune complexes with IL-1β or LPS) and M2c (IL-10) subsets. It is difficult to control macrophage activation and polarization in cultures containing serum, in which variable amounts of M1- and M2-specific cytokines and other factors may be present. Here we describe a serum-free culture system that selectively supports the differentiation of monocytes into either M1 or M2a macrophages. Monocytes were isolated using EasySep immunomagnetic cell separation and cultured for 4 or 6 days in serum-free ImmunoCult medium with 50 ng/mL M-CSF. The cells were then stimulated by adding 10 ng/mL LPS plus 50 ng/mL IFN-γ for M1 or 10 ng/mL IL-4 for M2a macrophage polarization and cultured for 2 more days. The yields of M1 and M2a macrophage were 51±18% and 58±14% (mean±SD), respectively. M1 macrophages expressed high levels of CD80 and CCR7 (96±3% and 62±15%, n=57), but very low levels of CD206 and CD209. In contrast, M2a macrophages expressed CD206 and CD209 (90±10% and 92±13%, n=59), but were negative or low for CD80 and CCR7. M1 macrophages produced TNF-α and IL-12 (mean±SEM: 2821±396 and 656±86, pg/mL, n=24). M2a macrophages produced small amounts of IL-10 (29±6 pg/mL, n=21). Both M1 and M2a macrophages were functional as demonstrated in a phagocytosis assay using fluorescently-labeled E.coli. By selecting appropriate stimuli this culture method can be easily adapted to generate other macrophage subsets as well and should prove useful for the study of macrophage biology.

Relevance of Chimerism Analysis After Allogeneic Stem Cell Transplantation

Hematopoietic stem cell transplantation is a potentially curative therapy for a range of malignant and non-malignant hematological diseases. Analysis of chimerism following allogeneic stem cell transplantation has been a routine method for the assessment of engraftment and early detection of graft failure. Lineage-specific chimerism monitoring is progressively used to specifically detect chimerism in one or more cell subsets, which may be undetected in assessment of the whole leukocyte population. The chimerism study in different leukocyte subpopulations increases sensitivity and specificity in the monitoring after transplantation, especially the analysis of T lymphocytes. All peripheral blood samples were separated into mononuclear cells and granulocytes by Ficoll density gradient centrifugation and T, B, and CD34+ was separated by immunomagnetic automatic cell separator. After DNA extraction, chimerism monitoring was performed using short tandem repeat by multiplex polymerase chain reaction followed by capillary electrophoresis. Quantification of chimerism was performed by determining the ratio of peak areas from donor and recipient informative short tandem repeat. Donor-recipient chimerism analysis in patients after allogeneic stem cell transplantation is a practical, feasible, and useful tool that predicts clinical outcomes and provides a guide for suitable therapeutic interventions.

The Importance of Positive Immunomagnetic Cell Separation Prior to Autologous Hematopoetic Stem Cell Transplantation for Advanced Stage Lymphomas

Abstract We present the method of immunomagnetic stem cell separation with the ISOLEX 300i device (Isolex® 300i Magnetic Cell Selection System, Nextell Therapeutics Inc. Irvine California 21618 USA) and the results obtained using this method in patients admitted to the Hematology and Bone Marrow Transplantation Clinic of Tîrgu Mureş, Romania. Cell selection has a great importance in separating stem cells from tumor cells, therefore contributing to the success of autologous stem cell transplantation.

Circulating tumor stem like cells in oral squamous cell carcinoma: An unresolved paradox

ObjectiveCirculating tumor cells (CTCs) are increasingly gaining importance due to their immense potential in enhancing diagnosis, prognosis and response to therapy in solid malignancies. Therefore, we aimed to comprehend the molecular diversity and critical role of this disseminated tumor population in OSCC. MethodologyCD44+ subpopulation was isolated using immuno-magnetic cell separation and their purity was validated using flow cytometry. Characterisation of self renewal potential and resistance to chemotherapy was assessed using tumor sphere forming and cytotoxicity assay. Gene expression profile of pertinent CSC (CD44s, CD44v3, CD44v6) and stemness markers (Bmi1 and Nanog) was carried out in CD44+ cells using Real Time PCR. Predominantly expressed markers and their association with clinico-pathological conditions were substantiated in 30 OSCC patients. ResultFlow cytometry analysis depicted a predominant population of CD44+CD24−CD45− cells suggesting that circulating tumor cells had a subpopulation of CSC like cells in the circulation. These cells demonstrated increased sphere forming capability and intrinsic chemo-resistance compared to non-CSC, thus indicating the CSC features of self-renewal and chemo-resistance. Additionally, CD44+ cells showed significantly increased expression levels of CD44v6 and Nanog compared to CD44− cells. Clinically, expression pattern of CD44v6 and Nanog correlated with different anatomical subsites, loco-regional aggressiveness of the disease and recurrence, thus opening newer avenues that can be explored for better prognostic and therapeutic implications. ConclusionThis study explored the inevitable role of CD44v6 and Nanog as circulating stem like cell markers in assessment of loco-regional aggressiveness, detection of relapse and therapeutic response and resistance.

Abstract 3357: Role of miRNA dynamics in governing cancer stem cell properties via CD44v6/Nanog/ PTEN axis in head and neck cancer: Modulating the master regulators

Abstract Background: Head and Neck Squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality worldwide. Late diagnosis, metastasis and therapeutic resistance are major confounders for the poor 5 year survival rate and are attributed to the existence of Cancer Stem Cells (CSCs). Several evidences have demonstrated the key role of nicotine in Tobacco related Cancers; however, the plausibility of nicotine modulating CSC markers still needs to be elucidated. Propelling evidences strongly suggest that HNSCC is a genetic disease, regulated by epigenetic mechanisms. Thus, understanding the role of miRNAs in regulation of CSCs and its contributions to improvise the prognostic and therapeutic implications is of great importance. Methodology: Orosphere was generated from primary habituated and non-habituated HNSCC tumors using CD44+ immunomagnetic cell separation and ex-vivo expansion. Characterization of these in-vitro models to evaluate spheroid formation ability and expression profile of CSC markers (CD44v3, CD44v6, BMI1 and Nanog) was carried out using quantitative Real Time PCR. Further, miRNA profiling of the upstream regulators (miR5423p, miR34a and miR21) was conducted in order to understand the miRNA mediated pathway that regulate these CSC features in tumor development, metastasis and response to chemotherapy. Correlation analysis of these miRNA profile along with the expression levels of its CSC target gene, were analyzed in specimens from patients with HNSCC. Result: Orosphere assay facilitated the propagation of CD44+ cells that retained CSC phenotype and self-renewal property. In-vitro model of habituated tumor showed an increased spheroid forming efficiency as compared to non-habituated models. In addition, these models showed higher expressions of CSC markers (CD44v3, CD44v6, Nanog &amp; BMI1) and a significantly reduced expression of PTEN and ATM as compared to the healthy individual; however the habituated and non-habituated were found to have a trivial difference in their behavioral patterns. Furthermore, a significantly increased expression of upstream miRNA was observed which eventually leads to the modulation CD44 interaction with Nanog/PTEN, thus forming an unexplored axis which can have prognostic and therapeutic implications. Clinically, altered miRNA expression not only positively correlated with CSC expression patterns but also associated with increased progression of the disease. Conclusion: Collectively, this study indicates that miR5423p and miR34a targets the HA-induced CD44v6 -Nanog-PTEN axis playing a vital role in regulating the CSC properties comprising of self renewal, clonal proliferation and resistance to chemotherapy in Head and Neck Cancers. Furthermore, our results depict an undisputable role of these miRNA mediated pathways in the development of tobacco related carcinogenesis Citation Format: Shanaya Patel, Dr. Rakesh Rawal. Role of miRNA dynamics in governing cancer stem cell properties via CD44v6/Nanog/ PTEN axis in head and neck cancer: Modulating the master regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3357.