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  • 1
    In: Oncotarget, Impact Journals, LLC, Vol. 5, No. 23 ( 2014-12-15), p. 12383-12397
    Type of Medium: Online Resource
    ISSN: 1949-2553
    URL: Issue
    Language: English
    Publisher: Impact Journals, LLC
    Publication Date: 2014
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  • 2
    In: Advanced Biosystems, Wiley, Vol. 2, No. 12 ( 2018-12)
    Abstract: Although isolation of circulating tumor cells (CTCs) from pancreatic adenocarcinoma patients is feasible, investigating their clinical utility has proven less successful than other cancers due to the limitations of epithelial cellular adhesion molecule (EpCAM)‐only based CTC assays. An integrated technology‐ and biology‐based approach using a microfluidic “Carpet Chip” is presented to study the biological relevance of heterogeneous CTC populations. Both epithelial CTCs (EpCs) and epithelial‐to‐mesenchymal transition (EMT)‐like CTCs (EMTCs) are isolated simultaneously from the whole blood of pancreatic cancer (PaCa) patients ( n = 35) by separately targeting two surface markers: EpCAM and CD133. Recovery of cancer cell lines spiked into whole blood is ≥97% with 〉 76% purity. Thirty‐four patients had ≥5 EpCs mL −1 and 35 patients had ≥15 EMTCs mL −1 . Overall, significantly higher numbers of EMTCs than EpCs are recovered, reflecting the aggressive nature of PaCa. Furthermore, higher numbers of EMTCs are observed in patients with lymph node involvement compared to patients without. Gene expression profiling of CTCs from 17 patients reveals that CXCR1 is significantly upregulated in EpCs, while known stem cell markers POU5F1/Oct‐4 and MYC are upregulated in EMTCs. In conclusion, successful isolation and genomic profiling of heterogeneous CTC populations are demonstrated, revealing genetic signatures relevant to patient outcomes.
    Type of Medium: Online Resource
    ISSN: 2366-7478 , 2366-7478
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2018
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  • 3
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 1595-1595
    Abstract: Background & Aims: A critical factor in the poor outcomes in pancreatic cancer is due to it's silent nature until late in the disease process, so early detection has remained a major issue in this type of cancer. Detection, quantification, and molecular characterization of Epithelial circulating tumor cells (CTCs) as well as Epithelial-to-Mesenchymal Transition (EMT)-like CTCs in peripheral blood have been recognized as a means of “liquid biopsy” technique in the diagnosis of cancer and metastasis. Methods: A modified version of CTC chip, CTC Carpet chip was used to investigate the inextricable relationship between circulating epithelial and EMT-like CTCs in 40 pancreatic cancer patients at both protein and RNA levels. Results: The recovery from whole blood spiked with different cancer cell line was validated, achieving a mean efficiency of ∼100% for both HT-29 and Panc-1 cell lines compared to & lt;80% for PC-3 cell line. Out of 40 patients, 38 patients had ≥5 CK+ CTCs with a mean of 21.84 per mL and all 40 samples had ≥ 15 Vim+ CTCs with mean of 89.77 per mL of whole blood. Up to 67 CK+ epithelial and 257 Vim+ EMT like CTCs were detected in these set of samples. The results indicated the significant differences between the numbers of Epithelial CTCs vs. EMT-like CTCs in both stage I-II and IV of these 40 pancreatic cancer patients’ samples. Conclusion: Although the isolation of CTCs from pancretic cancer is possible nowadays, investigation of their clinical utility has proven less successful than the other cancer types, due to poor sensitivity of many CTC assays for patients with PDAC. Herein, we present an integrated microfluidic technology- and biology-based translational approach using bioengineering tools to identify and study the biological relevance of rare CTCs including both circulating epithelial and EMT-like tumor cells simultaneously from the peripheral blood of pancreatic cancer patients by using multiple markers of interest. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathius Hafner, Shimeng Shao, Ebrahim Azizi, Max S. Wicha, Kyle Cuneo, Diane M. Simeone, Sunitha Nagrath. Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2015-1595
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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  • 4
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 76, No. 14_Supplement ( 2016-07-15), p. 3307-3307
    Abstract: Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis. Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques. Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples. Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics. Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [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 3307.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2016
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  • 5
    In: Nature Nanotechnology, Springer Science and Business Media LLC, Vol. 8, No. 11 ( 2013-11), p. 881-881
    Type of Medium: Online Resource
    ISSN: 1748-3387 , 1748-3395
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2013
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  • 6
    Online Resource
    Online Resource
    American Association for Cancer Research (AACR) ; 2013
    In:  Cancer Research Vol. 73, No. 8_Supplement ( 2013-04-15), p. 5109-5109
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 73, No. 8_Supplement ( 2013-04-15), p. 5109-5109
    Abstract: Introduction and Objective: Circulating tumor cells (CTCs) have been identified in the art in peripheral blood from cancer patients and are likely the origin of metastatic disease. Isolation and capture of CTCs represent a potential alternative to invasive biopsies during diagnosis of disease. In the recent past, vVarious microfluidic- based CTC isolation technologieschips have been developed to capture, indentify, sort, and enumerate CTCs. but, they need to be more sensitive, reliable, and specific for CTC detection in early stage cancer patients. Here we present a microfluidic CTC-chip with functionalized nanomaterials to capture and culture CTCs. Methods: Micro-sized structuresposts functionalized with anti-epithelial-adhesion-molecule antibodies (Anti-EpCAM) are is the common approachstructures to capture CTCs. Here we present a microfluidic device with functionalized planar structure to capture CTCs. The CTC-chipmicrofluidic device has a PDMS chamber and patterned gold postatterns . functionalized with graphene oxides as nanomaterial and anti-epithelial-cell-adhesion-molecule antibodies (Anti-EpCAM). To investigate the capture efficiency before running cancer patients’ samples, a low number of cells were spiked into 1 mL of whole blood. We stained the captured cells with cytokeratin, CD45, and DAPI to identify CTCs. For clinical studies, we searched for CTCs in 10 pancreatic cancer patients, 6 breast cancer patients, 4 early lung cancer patients, and 5 healthy donors. Results: It is much easier to scan, count and grow captured cells in comparison with previous micro-sized post-based structure. CTCs were isolated by assembling Graphene oxide on the planar structures for enhanced sensitivity. is easy to functionalize the surface and sensitive to detect biomaterial. The functionalized graphene oxide has been used to selectively capture MCF-7 breast cancer cells with a capture rate higher than 8790 %. This new CTC-chip identified CTCs in the peripheral blood of patients with pancreatic, breast, and lung cancer in 20 of 20 samples (100%). Conclusion: We present a sensitive CTC-chip using graphene oxides as nanomaterial to isolate, capture, identify, and characterize extremely rare CTCs. This CTC-chipIt is much easier to scan, count, and grow captured cells in comparison with previous micro-sized post-based structure. We believe this CTC-chip offers great potential for clinical research on cancer. Citation Format: Hyeun Joong Yoon, Tae Hyun Kim, Zhuo Zhang, Nithya Ramnath, Max S. Wicha, Daniel F. Hayes, Diane M. Simeone, Sunitha Nagrath. Integrated micro nanotechnology for the sensitive isolation of circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2013-5109
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2013
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  • 7
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3069-3069
    Abstract: Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process accounting for the majority of cancer-related deaths. They offer a non-invasive biopsy technique to study tumors and are shown to be useful prognostic indicators. Because of their rarity in the bloodstream, microfluidic isolation techniques are complex and time-consuming, and provide yields of CTCs insufficient or non-viable for studies other than enumeration. Additionally, due to the low processing speeds, the volumes of blood processed remain limited and can be a hindrance to obtaining higher yields of CTCs and their potential use as biomarkers of early diagnosis. Here we report a novel high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device and enables efficient cell capture by affinity at high flow rates. The cell recovery from whole blood was validated with cancer cell lines H1650 and MCF7, and the OncoBean Chip achieved an efficiency & gt;80% at a throughput of 10 mL/hr, a flow rate yet achieved only in physical size based separation techniques. A cell viability of 92.91% shows that the cells recovered at high throughput could still be used for downstream analysis. Clinical competence was demonstrated in blood specimens from breast, pancreatic and lung cancer patients. The OncoBean Chip thus holds potential applications in the diagnosis of early stage cancers, where the low numbers of CTCs could be enriched using this novel device. Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Mina Zeinali, Svetlana Grabauskiene, Mostafa Ghannad-Rezaie, Max S. Wicha, Diane M. Simeone, Nithya Ramnath, Sunitha Nagrath. Radial flow microfluidic device for high-throughput affinity-based isolation of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2014-3069
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2014
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  • 8
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 1593-1593
    Abstract: Introduction and Objective: A necessary step in distant metastasis is the hematogenous dissemination of cancer cells from the primary tumor site to remote sites. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival in many solid malignancies. Immune-affinity based capture is the most commonly used method for the isolation of CTCs which utilizes antibodies to capture tumor cells expressing specific proteins. However, immune-affinity based approaches offer low throughput (∼1mL/hr) and considerable cell loss (∼20-40%) resulting from heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing physical properties of CTCs have been developed to overcome the limitations of immune-affinity based isolation techniques, including micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs that yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. Methods: The PDMS-made microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. The device was optimized with MCF-7 and Panc-1 cell lines spiked into PBS buffer and also diluted blood. It was then tested on 10 mL blood samples from patients with metastatic breast cancer. The separated cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, CD45 negative cells. Results: Samples were processed through the inertial microfluidic device that enriches CTCs in the second outlet based on size difference between CTCs and blood cells. The device was optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (92% for both spiked samples of MCF-7 and PANC-1 cell lines) and high white blood cells (WBCs) removal (91%). To determine the efficiency of capture of rare cell populations, healthy donor blood samples were spiked with MCF-7/GFP at 100 cells and 95% recovery was obtained. In patient samples, we identified CTCs in 24 of 27 (89%) breast patients with metastatic disease (4.3±4.8 CTCs/mL) with low WBCs contamination (465±473 WBCs/mL). Conclusion: The study of CTCs could have a direct impact upon patient care by presenting a novel CTC isolation method. This technology may be applicable to early detection, and also for monitoring response to treatment. Our approach is superior to current strategies because it is independent of cell surface markers, which may be varied in tumor cells, and may exclude cancer stem cells. Citation Format: Eric Lin, Lianette Rivera, Hyeun Joong Yoon, Shamileh Fouladdel, Jacob Wieger, Stephanie Guthrie, Yadwinder S. Deol, Shawn G. Clouthier, Tahra K. Luther, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2015-1593
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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  • 9
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 75, No. 15_Supplement ( 2015-08-01), p. 370-370
    Abstract: Establishing the role of circulating tumor cells (CTCs) in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of cells for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. One approach to overcome this limitation is to expand CTCs, i.e. increase their numbers as a result of cell division. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully identified CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs expressed tumor specific markers and formed spheroids in culture. CTCs derived xenograft demonstrated tumorigenic capability of these cells. Furthermore, CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis. Citation Format: Zhuo Zhang, Hiroe Shiratsuchi, Jules Lin, Guoan Chen, Rishindra M. Reddy, Ebrahim Azizi, Shamileh Fouladdel, Andrew C. Chang, Lin Lin, Hui Jiang, Meghna Waghray, Diane M. Simeone, Max S. Wicha, David G. Beer, Gary Luker, Nithya Ramnath, Sunitha Nagrath. Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 370. doi:10.1158/1538-7445.AM2015-370
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2015
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    detail.hit.zdb_id: 1432-1
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  • 10
    In: Cancer Research, American Association for Cancer Research (AACR), Vol. 78, No. 13_Supplement ( 2018-07-01), p. 5579-5579
    Abstract: Objective: Although circulating tumor cells' (CTCs) isolation from pancreatic adenocarcinoma (PaCa) patients is feasible, investigation of their clinical utility has proven less successful than other cancer types, due to poor technology sensitivity and EpCAM-only based CTC assays. Design: We present an integrated technology and biology-based approach using a microfluidic “Carpet chip” to study the biologic relevance of rare CTCs. We isolate both circulating epithelial CTCs (EpCs) and EMT-like CTCs (EMTCs) simultaneously from whole blood of PaCa patients (n=35), targeting two different surface markers: anti-EpCAM and anti-CD133. Gene expression profiling of CTCs was performed with 96 genes (n=17). Results: Recovery of cancer cell lines spiked into whole blood is ≥97% with & gt;76% purity. Of the 35 PaCa patients analyzed, 34 had ≥5 EpCs/mL and 35 had ≥15 EMTCs/mL. Higher numbers of EMTCs were observed in patients with lymph node involvement (N1) compared to patients without. The ratio of EpCs to total number of CTCs marginally decreased with advanced stage, while the ratio of EMTCs increased. Gene expression profiling showed CXCR1 was significantly upregulated in EpCs whereas POU5F1/Oct-4 and MYC were upregulated in EMTCs, demonstrating inherent phenotypic differences. Early-stage patients with significantly better overall survival and/or progression-free survival showed significant higher expression of genes including ERCC1 and TTF1. Conclusions: We demonstrated successful isolation of heterogeneous population of CTCs (EpCs and EMTCs). We recovered significantly higher number of EMTCs in PaCa patients compared to EpCs, reflecting aggressive nature of PaCa. Profiling of CTCs revealed genetic signatures relevant to predicting patients' outcomes. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathias Hafner, Shimeng Shao, Lili Zhao, Heather Cameron, Armand Iii Bankhead, Jiaqi Shi, Kyle C. Cuneo, Vaibhav Sahai, Ebrahim Azizi, Max S. Wicha, Diane M. Simeone, Sunitha Nagrath. Increased number of EMT-like CTCs relative to epithelial CTCs reveals signatures of poor outcomes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5579.
    Type of Medium: Online Resource
    ISSN: 0008-5472 , 1538-7445
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    Language: English
    Publisher: American Association for Cancer Research (AACR)
    Publication Date: 2018
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