Publications by authors named "Hsian-Rong Tseng"

120 Publications

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Sci Adv 2020 Oct 23;6(43). Epub 2020 Oct 23.

Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging (CIMI), California NanoSystems Institute (CNSI), University of California, Los Angeles, Los Angeles, CA 90095, USA.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.
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http://dx.doi.org/10.1126/sciadv.abb7107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608838PMC
October 2020

Hepatocellular Carcinoma-Circulating Tumor Cells Expressing PD-L1 Are Prognostic and Potentially Associated With Response to Checkpoint Inhibitors.

Hepatol Commun 2020 Oct 4;4(10):1527-1540. Epub 2020 Aug 4.

Department of Surgery University of California Los Angeles Los Angeles CA.

Hepatocellular carcinoma (HCC) is a leading cause of mortality. Checkpoint inhibitors of programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown great efficacy, but lack biomarkers that predict response. Circulating tumor cells (CTCs) have promise as a liquid-biopsy biomarker; however, data on HCC CTCs expressing PD-L1 have not been reported. We sought to detect PD-L1-expressing HCC-CTCs and investigated their role as a prognostic and predictive biomarker. Using an antibody-based platform, CTCs were enumerated/phenotyped from a prospective cohort of 87 patients with HCC (49 early-stage, 22 locally advanced, and 16 metastatic), 7 patients with cirrhosis, and 8 healthy controls. Immunocytochemistry identified total HCC CTCs (4',6-diamidino-2-phenylindole-positive [DAPI+]/cytokeratin-positive [CK+]/clusters of differentiation 45-negative [CD45-]) and a subpopulation expressing PD-L1 (DAPI+/CK+/PD-L1+/CD45-). PD-L1+ CTCs were identified in 4 of 49 (8.2%) early-stage patients, but 12 of 22 (54.5%) locally advanced and 15 of 16 (93.8%) metastatic patients, accurately discriminating early from locally advanced/metastatic HCC (sensitivity = 71.1%, specificity = 91.8%, area under the receiver operating characteristic curve = 0.807;  < 0.001). Compared to patients without PD-L1+ CTCs, patients with PD-L1+ CTCs had significantly inferior overall survival (OS) (median OS = 14.0 months vs. not reached, hazard ratio [HR] = 4.0,  = 0.001). PD-L1+ CTCs remained an independent predictor of OS (HR = 3.22,  = 0.010) even after controlling for Model for End-Stage Liver Disease score (HR = 1.14,  < 0.001), alpha-fetoprotein (HR = 1.55,  < 0.001), and overall stage/tumor burden (beyond University of California, San Francisco, HR = 7.19,  < 0.001). In the subset of 10 patients with HCC receiving PD-1 blockade, all 5 responders demonstrated PD-L1+ CTCs at baseline, compared with only 1 of 5 nonresponders, all of whom progressed within 4 months of starting treatment. We report a CTC assay for the phenotypic profiling of HCC CTCs expressing PD-L1. PD-L1+ CTCs are predominantly found in advanced-stage HCC, and independently prognosticate OS after controlling for Model for End-Stage Liver Disease, alpha-fetoprotein, and tumor stage. In patients with HCC receiving anti-PD-1 therapy, there was a strong association with the presence of PD-L1+ CTCs and favorable treatment response. Prospective validation in a larger cohort will better define the utility of PD-L1+ CTCs as a prognostic and predictive biomarker in HCC.
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http://dx.doi.org/10.1002/hep4.1577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527695PMC
October 2020

Purification of HCC-specific extracellular vesicles on nanosubstrates for early HCC detection by digital scoring.

Nat Commun 2020 09 7;11(1):4489. Epub 2020 Sep 7.

California NanoSystems Institute, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, 90095, USA.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).
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http://dx.doi.org/10.1038/s41467-020-18311-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477161PMC
September 2020

Sexually Dimorphic Crosstalk at the Maternal-Fetal Interface.

J Clin Endocrinol Metab 2020 12;105(12)

Division of Reproductive Endocrinology and Infertility; Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, California.

Context: Crosstalk through receptor ligand interactions at the maternal-fetal interface is impacted by fetal sex. This affects placentation in the first trimester and differences in outcomes. Sexually dimorphic signaling at early stages of placentation are not defined.

Objective: Investigate the impact of fetal sex on maternal-fetal crosstalk.

Design: Receptors/ligands at the maternal-fetal surface were identified from sexually dimorphic genes between fetal sexes in the first trimester placenta and defined in each cell type using single-cell RNA-Sequencing (scRNA-Seq).

Setting: Academic institution.

Samples: Late first trimester (~10-13 weeks) placenta (fetal) and decidua (maternal) from uncomplicated ongoing pregnancies.

Main Outcome Measures: Transcriptomic profiling at tissue and single-cell level; immunohistochemistry of select proteins.

Results: We identified 91 sexually dimorphic receptor-ligand pairs across the maternal-fetal interface. We examined fetal sex differences in 5 major cell types (trophoblasts, stromal cells, Hofbauer cells, antigen-presenting cells, and endothelial cells). Ligands from the CC family chemokine ligand (CCL) family were most highly representative in females, with their receptors present on the maternal surface. Sexually dimorphic trophoblast transcripts, Mucin-15 (MUC15) and notum, palmitoleoyl-protein carboxylesterase (NOTUM) were also most highly expressed in syncytiotrophoblasts and extra-villous trophoblasts respectively. Gene Ontology (GO) analysis using sexually dimorphic genes in individual cell types identified cytokine mediated signaling pathways to be most representative in female trophoblasts. Upstream analysis demonstrated TGFB1 and estradiol to affect all cell types, but dihydrotestosterone, produced by the male fetus, was an upstream regulator most significant for the trophoblast population.

Conclusions: Maternal-fetal crosstalk exhibits sexual dimorphism during placentation early in gestation.
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http://dx.doi.org/10.1210/clinem/dgaa503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571453PMC
December 2020

Somatic copy number profiling from hepatocellular carcinoma circulating tumor cells.

NPJ Precis Oncol 2020 2;4:16. Epub 2020 Jul 2.

Department of Surgery, University of California, Los Angeles, Los Angeles, CA USA.

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.
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http://dx.doi.org/10.1038/s41698-020-0123-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331695PMC
July 2020

A ratiometric photoacoustic imaging approach for semi-quantitative determination of aggregation efficiency in vivo.

Nanoscale 2020 Sep 3;12(36):18654-18662. Epub 2020 Jul 3.

Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology (NCNST), No. 11 Beiyitiao, Zhongguancun, Beijing, 100190, China.

In vivo self-assembly not only endows dynamic supramolecules with various biological functions, but also realizes metabolic differences, and improves the level of diagnosis and treatment. However, the method of measuring aggregation efficiency in vivo is still challenging. In this work, we first proposed a ratiometric photoacoustic imaging method to measure the aggregation efficiency of molecules in vivo in real time and semi-quantitatively. Similar to the traditional fluorescence method, the ratiometric photoacoustic signal has a typical exponential relationship with the aggregation efficiency, which is defined as the percentage of aggregation molecules in the total molecules. Then, we proposed a ratiometric photoacoustic (PA) probe, which can be tailored by cathepsin E and self-assembled into nanofibers in situ inside pancreatic cancer cells. The maximum aggregation efficiency of 10 M PA probe was 58% after 2 hours of incubation. After intratumoral administration in xenografted pancreatic tumor mice, the highest aggregation efficiency was found to be 36% 6 hours after the injection. The ratiometric PA probe provides us with a real-time method to detect the aggregation efficiency in vivo, which is helpful to deepen the understanding of the dynamic assembly process and optimize the design of supramolecules.
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http://dx.doi.org/10.1039/d0nr03218bDOI Listing
September 2020

A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

J Mater Chem B 2020 07;8(26):5636-5644

Department of Pathology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, P. R. China.

Determining the status of epidermal growth factor receptor (EGFR) T790M mutation is crucial for guiding further treatment intervention in advanced non-small cell lung cancer (NSCLC) patients who develop acquired resistance to initial EGFR tyrosine kinase inhibitor (TKI) treatment. Circulating tumor cells (CTCs) which contain plentiful copies of well-preserved RNA offer an ideal source for noninvasive detection of T790M mutation in NSCLC. We developed a CTC-based digital assay which synergistically integrates NanoVelcro Chips for enriching NSCLC CTCs and reverse-transcription droplet digital PCR (RT-ddPCR) for quantifying T790M transcripts in the enriched CTCs. We collected 46 peripheral arterial and venous blood samples from 27 advanced NSCLC patients for testing this CTC-based digital assay. The results showed that the T790M mutational status observed by the CTC-based digital assay matched with those observed by tissue-based diagnostic methods. Furthermore, higher copy numbers of T790M transcripts were observed in peripheral arterial blood than those detected in the matched peripheral venous blood. In short, our results demonstrated the potential of the NanoVelcro CTC-digital assay for noninvasive detection of the T790M mutation in NSCLC, and suggested that peripheral arterial blood sampling may offer a more abundant CTC source than peripheral venous blood in advanced NSCLC patients.
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http://dx.doi.org/10.1039/d0tb00589dDOI Listing
July 2020

Dual Supramolecular Nanoparticle Vectors Enable CRISPR/Cas9-Mediated Knockin of Retinoschisin 1 Gene-A Potential Nonviral Therapeutic Solution for X-Linked Juvenile Retinoschisis.

Adv Sci (Weinh) 2020 May 16;7(10):1903432. Epub 2020 Apr 16.

Division of Basic Research Department of Medical Research and Department of Ophthalmology Taipei Veterans General Hospital Taipei 112 Taiwan.

The homology-independent targeted integration (HITI) strategy enables effective CRISPR/Cas9-mediated knockin of therapeutic genes in nondividing cells in vivo, promising general therapeutic solutions for treating genetic diseases like X-linked juvenile retinoschisis. Herein, supramolecular nanoparticle (SMNP) vectors are used for codelivery of two DNA plasmids-CRISPR-Cas9 genome-editing system and a therapeutic gene, Retinoschisin 1 (RS1)-enabling clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) knockin of the RS1 gene with HITI. Through small-scale combinatorial screenings, two SMNP vectors, with Cas9 and single guide RNA (sgRNA)-plasmid in one and Donor-RS1 and green fluorescent protein (GFP)-plasmid in the other, with optimal delivery performances are identified. These SMNP vectors are then employed for CRISPR/Cas9 knockin of RS1/GFP genes into the mouse Rosa26 safe-harbor site in vitro and in vivo. The in vivo study involves intravitreally injecting the two SMNP vectors into the mouse eyes, followed by repeated ocular imaging by fundus camera and optical coherence tomography, and pathological and molecular analyses of the harvested retina tissues. Mice ocular organs retain their anatomical integrity, a single-copy 3.0-kb RS1/GFP gene is precisely integrated into the Rosa26 site in the retinas, and the integrated RS1/GFP gene is expressed in the retinas, demonstrating CRISPR/Cas9 knockin of RS1/GFP gene.
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http://dx.doi.org/10.1002/advs.201903432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237855PMC
May 2020

Detection of Circulating Tumor Cells and Their Implications as a Biomarker for Diagnosis, Prognostication, and Therapeutic Monitoring in Hepatocellular Carcinoma.

Hepatology 2021 Jan 18;73(1):422-436. Epub 2021 Jan 18.

Division of Digestive and Liver Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA.

Hepatocellular carcinoma (HCC) is among the leading causes of worldwide cancer-related morbidity and mortality. Poor prognosis of HCC is attributed primarily to tumor presentation at an advanced stage when there is no effective treatment to achieve the long term survival of patients. Currently available tests such as alpha-fetoprotein have limited accuracy as a diagnostic or prognostic biomarker for HCC. Liver biopsy provides tissue that can reveal tumor biology but it is not used routinely due to its invasiveness and risk of tumor seeding, especially in early-stage patients. Liver biopsy is also limited in revealing comprehensive tumor biology due to intratumoral heterogeneity. There is a clear need for new biomarkers to improve HCC detection, prognostication, prediction of treatment response, and disease monitoring with treatment. Liquid biopsy could be an effective method of early detection and management of HCC. Circulating tumor cells (CTCs) are cancer cells in circulation derived from the original tumor or metastatic foci, and their measurement by liquid biopsy represents a great potential in facilitating the implementation of precision medicine in patients with HCC. CTCs can be detected by a simple peripheral blood draw and potentially show global features of tumor characteristics. Various CTC detection platforms using immunoaffinity and biophysical properties have been developed to identify and capture CTCs with high efficiency. Quantitative abundance of CTCs, as well as biological characteristics and genomic heterogeneity among the CTCs, can predict disease prognosis and response to therapy in patients with HCC. This review article will discuss the currently available technologies for CTC detection and isolation, their utility in the clinical management of HCC patients, their limitations, and future directions of research.
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http://dx.doi.org/10.1002/hep.31165DOI Listing
January 2021

Nanostructured Substrates for Detection and Characterization of Circulating Rare Cells: From Materials Research to Clinical Applications.

Adv Mater 2020 Jan 30;32(1):e1903663. Epub 2019 Sep 30.

California NanoSystems Institute, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, 90095, USA.

Circulating rare cells in the blood are of great significance for both materials research and clinical applications. For example, circulating tumor cells (CTCs) have been demonstrated as useful biomarkers for "liquid biopsy" of the tumor. Circulating fetal nucleated cells (CFNCs) have shown potential in noninvasive prenatal diagnostics. However, it is technically challenging to detect and isolate circulating rare cells due to their extremely low abundance compared to hematologic cells. Nanostructured substrates offer a unique solution to address these challenges by providing local topographic interactions to strengthen cell adhesion and large surface areas for grafting capture agents, resulting in improved cell capture efficiency, purity, sensitivity, and reproducibility. In addition, rare-cell retrieval strategies, including stimulus-responsiveness and additive reagent-triggered release on different nanostructured substrates, allow for on-demand retrieval of the captured CTCs/CFNCs with high cell viability and molecular integrity. Several nanostructured substrate-enabled CTC/CFNC assays are observed maturing from enumeration and subclassification to molecular analyses. These can one day become powerful tools in disease diagnosis, prognostic prediction, and dynamic monitoring of therapeutic response-paving the way for personalized medical care.
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http://dx.doi.org/10.1002/adma.201903663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946854PMC
January 2020

Noninvasive Prenatal Diagnostics: Recent Developments Using Circulating Fetal Nucleated Cells.

Curr Obstet Gynecol Rep 2019 Mar 21;8(1):1-8. Epub 2019 Jan 21.

Department of Molecular and Medical Pharmacology, California NanoSystems Institute, Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, CA, USA.

Purpose Of Review: The purpose of this review is to highlight recent research advances in noninvasive prenatal diagnostic methods.

Recent Findings: Recent studies developing noninvasive prenatal diagnostic (NIPD) methods have been focused on either fetal nucleated red blood cells (fNRBCs) or circulating trophoblasts (cTBs). Enriched cTBs were successfully utilized for whole genome profiling and short tandem repeat (STR) identification to confirm feto-maternal relationship. However, further analysis of isolated fNRBCs remains confined to examining fetal cytogenetics.

Summary: Invasive prenatal diagnostic procedures, amniocentesis and chorionic villus sampling, are the gold standard for the diagnosis of fetal chromosomal abnormalities and genetic disorders. Meanwhile, noninvasive techniques of analyzing circulating cell-free fetal DNA (cffDNA) have been limited to screening tools and are highly fragmented and confounded by maternal DNA. By detecting circulating fetal nucleated cells (CFNCs) we are able to noninvasively confirm fetal chromosomal abnormalities, truly realizing the concept of "noninvasive prenatal diagnostics". The primary technical challenge is the enrichment of the low abundance of CFNCs in maternal peripheral blood. For any cell-based NIPD method, both fetal whole genome profiling and confirmation of the feto-parental relationship are essential. This has been successfully performed using enriched and isolated cTBs, making cTB a better candidate for NIPD. cTB enumeration also correlates with abnormal fetal or placental development. On the other hand, downstream analysis of fNRBCs remains limited to examining fetal sex and aneuploidies. Furthermore, trophoblast-based NIPD via an endocervical sample is also promising because of reduced dilution from hematologic cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764767PMC
March 2019

Covalent chemistry on nanostructured substrates enables noninvasive quantification of gene rearrangements in circulating tumor cells.

Sci Adv 2019 07 31;5(7):eaav9186. Epub 2019 Jul 31.

California NanoSystems Institute, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.
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http://dx.doi.org/10.1126/sciadv.aav9186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669017PMC
July 2019

A Circulating Tumor Cell-RNA Assay for Assessment of Androgen Receptor Signaling Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer.

Theranostics 2019 13;9(10):2812-2826. Epub 2019 Apr 13.

Urologic Oncology Program and Uro-Oncology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, California, USA.

: Our objective was to develop a circulating tumor cell (CTC)-RNA assay for characterizing clinically relevant RNA signatures for the assessment of androgen receptor signaling inhibitor (ARSI) sensitivity in metastatic castration-resistant prostate cancer (mCRPC) patients. : We developed the NanoVelcro CTC-RNA assay by combining the Thermoresponsive (TR)-NanoVelcro CTC purification system with the NanoString nCounter platform for cellular purification and RNA analysis. Based on the well-validated, tissue-based Prostate Cancer Classification System (PCS), we focus on the most aggressive and ARSI-resistant PCS subtype, i.e., PCS1, for CTC analysis. We applied a rigorous bioinformatic process to develop the CTC-PCS1 panel that consists of prostate cancer (PCa) CTC-specific RNA signature with minimal expression in background white blood cells (WBCs). We validated the NanoVelcro CTC-RNA assay and the CTC-PCS1 panel with well-characterized PCa cell lines to demonstrate the sensitivity and dynamic range of the assay, as well as the specificity of the PCS1 Z score (the likelihood estimate of the PCS1 subtype) for identifying PCS1 subtype and ARSI resistance. We then selected 31 blood samples from 23 PCa patients receiving ARSIs to test in our assay. The PCS1 Z scores of each sample were computed and compared with ARSI treatment sensitivity. : The validation studies using PCa cell line samples showed that the NanoVelcro CTC-RNA assay can detect the RNA transcripts in the CTC-PCS1 panel with high sensitivity and linearity in the dynamic range of 5-100 cells. We also showed that the genes in CTC-PCS1 panel are highly expressed in PCa cell lines and lowly expressed in background WBCs. Using the artificial CTC samples simulating the blood sample conditions, we further demonstrated that the CTC-PCS1 panel is highly specific in identifying PCS1-like samples, and the high PCS1 Z score is associated with ARSI resistance samples. In patient bloods, ARSI-resistant samples (ARSI-R, n=14) had significantly higher PCS1 Z scores as compared with ARSI-sensitive samples (ARSI-S, n=17) (Rank-sum test, P=0.003). In the analysis of 8 patients who were initially sensitive to ARSI (ARSI-S) and later developed resistance (ARSI-R), we found that the PCS1 Z score increased from the time of ARSI-S to the time of ARSI-R (Pairwise T-test, P=0.016). : Using our new methodology, we developed a first-in-class CTC-RNA assay and demonstrated the feasibility of transforming clinically-relevant tissue-based RNA profiling such as PCS into CTC tests. This approach allows for detecting RNA expression relevant to clinical drug resistance in a non-invasive fashion, which can facilitate patient-specific treatment selection and early detection of drug resistance, a goal in precision oncology.
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http://dx.doi.org/10.7150/thno.34485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568173PMC
May 2020

Bio-Inspired NanoVilli Chips for Enhanced Capture of Tumor-Derived Extracellular Vesicles: Toward Non-Invasive Detection of Gene Alterations in Non-Small Cell Lung Cancer.

ACS Appl Mater Interfaces 2019 Apr 2;11(15):13973-13983. Epub 2019 Apr 2.

Department of Pathology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine , Guangdong Provincial Academy of Chinese Medical Sciences , Guangzhou 510120 , P. R. China.

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.
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http://dx.doi.org/10.1021/acsami.9b01406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545291PMC
April 2019

Emerin Deregulation Links Nuclear Shape Instability to Metastatic Potential.

Cancer Res 2018 11 28;78(21):6086-6097. Epub 2018 Aug 28.

Division of Cancer Biology and Therapeutics, Department of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, California.

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane-associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis. This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer. http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0608DOI Listing
November 2018

Cross-Linked Fluorescent Supramolecular Nanoparticles for Intradermal Controlled Release of Antifungal Drug-A Therapeutic Approach for Onychomycosis.

ACS Nano 2018 07 27;12(7):6851-6859. Epub 2018 Jun 27.

Department of Molecular and Medical Pharmacology, Crump Institute for Molecular Imaging (CIMI), California NanoSystems Institute (CNSI) , University of California, Los Angeles , Los Angeles , California 90095-1770 , United States.

The existing approaches to onychomycosis demonstrate limited success since the commonly used oral administration and topical cream only achieve temporary effective drug concentration at the fungal infection sites. An ideal therapeutic approach for onychomycosis should have (i) the ability to introduce antifungal drugs directly to the infected sites; (ii) finite intradermal sustainable release to maintain effective drug levels over prolonged time; (iii) a reporter system for monitoring maintenance of drug level; and (iv) minimum level of inflammatory responses at or around the fungal infection sites. To meet these expectations, we introduced ketoconazole-encapsulated cross-linked fluorescent supramolecular nanoparticles (KTZ⊂c-FSMNPs) as an intradermal controlled release solution for treating onychomycosis. A two-step synthetic approach was adopted to prepare a variety of KTZ⊂c-FSMNPs. Initial characterization revealed that 4800 nm KTZ⊂c-FSMNPs exhibited high KTZ encapsulation efficiency/capacity, optimal fluorescent property, and sustained KTZ release profile. Subsequently, 4800 nm KTZ⊂c-FSMNPs were chosen for in vivo studies using a mouse model, wherein the KTZ⊂c-FSMNPs were deposited intradermally via tattoo. The results obtained from (i) in vivo fluorescence imaging, (ii) high-performance liquid chromatography quantification of residual KTZ, (iii) matrix-assisted laser desorption/ionization mass spectrometry imaging mapping of KTZ distribution in intradermal regions around the tattoo site, and (iv) histology for assessment of local inflammatory responses and biocompatibility, suggest that 4800 nm KTZ⊂c-FSMNPs can serve as an effective treatment for onychomycosis.
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http://dx.doi.org/10.1021/acsnano.8b02099DOI Listing
July 2018

Structure and function analysis in circulating tumor cells: using nanotechnology to study nuclear size in prostate cancer.

Am J Clin Exp Urol 2018 1;6(2):43-54. Epub 2018 Apr 1.

Urologic Oncology Program & Uro-Oncology Laboratories, Samuel Oschin Comprehensive Cancer InstituteLos Angeles, CA 90048, USA.

Professor Donald Coffey and his laboratory pioneered studies showing the relationships between nuclear shape and cellular function. In doing so, he and his students established the field of nuclear morphometry in prostate cancer. By using perioperative tissues via biopsies and surgical sampling, Dr. Coffey's team discovered that nuclear shape and other pathologic features correlated with clinical outcome measures. Cancer cells also exist outside of solid tumor masses as they can be shed from both primary and metastatic lesions into the circulatory system. The pool of these circulating tumor cells (CTCs) is heterogeneous. While some of these CTCs are passively shed into the circulation, others are active metastasizers with invasive potential. Advances in nanotechnology now make it possible to study morphologic features such as nuclear shape of CTCs in the bloodstream via liquid biopsy. Compared to traditional tissue sampling, liquid biopsy allows for minimally invasive, repetitive, and systemic disease sampling, which overcomes disease misrepresentation issues due to tumor temporospatial heterogeneity. Our team developed a novel liquid biopsy approach, the NanoVelcro assay, which allows us to identify morphologic heterogeneity in the CTC compartment. By applying classical methods of nuclear morphometry, we identified very small nuclear CTCs (vsnCTCs) in prostate cancer patients. Our initial studies showed that vsnCTCs strongly correlated with unfavorable clinical behaviors including the disposition to visceral metastases. These approaches may continue to yield additional insights into dynamic clinical behaviors, which creates an opportunity for more comprehensive and accurate cancer profiling. Ultimately, these advancements will allow physicians to employ more accurate and personalized treatments, helping the field reach the goal of true precision medicine.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902722PMC
April 2018

CTHRC1 induces non-small cell lung cancer (NSCLC) invasion through upregulating MMP-7/MMP-9.

BMC Cancer 2018 04 10;18(1):400. Epub 2018 Apr 10.

Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, No. 58, ZhongShan Second Road, Guangdong, 510080, China.

Background: The strong invasive and metastatic nature of non-small cell lung cancer (NSCLC) leads to poor prognosis. Collagen triple helix repeat containing 1 (CTHRC1) is involved in cell migration, motility and invasion. The object of this study is to investigate the involvement of CTHRC1 in NSCLC invasion and metastasis.

Methods: A proteomic analysis was performed to identify the different expression proteins between NSCLC and normal tissues. Cell lines stably express CTHRC1, MMP7, MMP9 were established. Invasion and migration were determined by scratch and transwell assays respectively. Clinical correlations of CTHRC1 in a cohort of 230 NSCLC patients were analysed.

Results: CTHRC1 is overexpressed in NSCLC as measured by proteomic analysis. Additionally, CTHRC1 increases tumour cell migration and invasion in vitro. Furthermore, CTHRC1 expression is significantly correlated with matrix metalloproteinase (MMP)7 and MMP9 expression in sera and tumour tissues from NSCLC. The invasion ability mediated by CTHRC1 were mainly MMP7- and MMP9-dependent. MMP7 or MMP9 depletion significantly eradicated the pro-invasive effects mediated by CTHRC1 on NSCLC cells. Clinically, patients with high CTHRC1 expression had poor survival.

Conclusions: CTHRC1 serves as a pro-metastatic gene that contributes to NSCLC invasion and metastasis, which are mediated by upregulated MMP7 and MMP9 expression. Targeting CTHRC1 may be beneficial for inhibiting NSCLC metastasis.
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http://dx.doi.org/10.1186/s12885-018-4317-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891957PMC
April 2018

A novel multimarker assay for the phenotypic profiling of circulating tumor cells in hepatocellular carcinoma.

Liver Transpl 2018 07;24(7):946-960

Departments of Surgery, University of California, Los Angeles, Los Angeles, CA.

Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel "liquid-biopsy" assay capable of detecting HCC circulating tumor cells (CTCs) and characterizing phenotypic subpopulations with prognostic significance. Using HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), glypican-3, and epithelial cell adhesion molecule was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (P < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive CTCs accurately discriminated early-stage, LT eligible patients (median, 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median, 6 CTCs; AUROC = 0.89; P = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [HR], 2.21; P = 0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early-stage HCC (HR, 3.14; P = 0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC CTCs with high efficiency and accuracy. A phenotypic subpopulation of VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.
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http://dx.doi.org/10.1002/lt.25062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097911PMC
July 2018

NanoVelcro rare-cell assays for detection and characterization of circulating tumor cells.

Adv Drug Deliv Rev 2018 02 15;125:78-93. Epub 2018 Mar 15.

Department of Molecular and Medical Pharmacology, California NanoSystems Institute, Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, CA, USA. Electronic address:

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.
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http://dx.doi.org/10.1016/j.addr.2018.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5993593PMC
February 2018

Precision-Guided Nanospears for Targeted and High-Throughput Intracellular Gene Delivery.

ACS Nano 2018 05 14;12(5):4503-4511. Epub 2018 Mar 14.

California NanoSystems Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States.

An efficient nonviral platform for high-throughput and subcellular precision targeted intracellular delivery of nucleic acids in cell culture based on magnetic nanospears is reported. These magnetic nanospears are made of Au/Ni/Si (∼5 μm in length with tip diameters <50 nm) and fabricated by nanosphere lithography and metal deposition. A magnet is used to direct the mechanical motion of a single nanospear, enabling precise control of position and three-dimensional rotation. These nanospears were further functionalized with enhanced green fluorescent protein (eGFP)-expression plasmids via a layer-by-layer approach before release from the underlying silicon substrate. Plasmid functionalized nanospears are guided magnetically to approach target adherent U87 glioblastoma cells, penetrating the cell membrane to enable intracellular delivery of the plasmid cargo. After 24 h, the target cell expresses green fluorescence indicating successful transfection. This nanospear-mediated transfection is readily scalable for the simultaneous manipulation of multiple cells using a rotating magnet. Cell viability >90% and transfection rates >80% were achieved, which exceed conventional nonviral intracellular methods. This approach is compatible with good manufacturing practices, circumventing barriers to the translation and clinical deployment of emerging cellular therapies.
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http://dx.doi.org/10.1021/acsnano.8b00763DOI Listing
May 2018

Reduction of Circulating Cancer Cells and Metastases in Breast-Cancer Models by a Potent EphA2-Agonistic Peptide-Drug Conjugate.

J Med Chem 2018 03 27;61(5):2052-2061. Epub 2018 Feb 27.

Division of Biomedical Sciences, School of Medicine , University of California, Riverside , 900 University Avenue , Riverside , California 92521 , United States.

EphA2 overexpression has been associated with metastasis in multiple cancer types, including melanomas and ovarian, prostate, lung, and breast cancers. We have recently proposed the development of peptide-drug conjugates (PDCs) using agonistic EphA2-targeting agents, such as the YSA peptide or its optimized version, 123B9. Although our studies indicated that YSA- and 123B9-drug conjugates can selectively deliver cytotoxic drugs to cancer cells in vivo, the relatively low cellular agonistic activities (i.e., the high micromolar concentrations required) of the agents toward the EphA2 receptor remained a limiting factor to the further development of these PDCs in the clinic. Here, we report that a dimeric version of 123B9 can induce receptor activation at nanomolar concentrations. Furthermore, we demonstrated that the conjugation of dimeric 123B9 with paclitaxel is very effective at targeting circulating tumor cells and inhibiting lung metastasis in breast-cancer models. These studies represent an important step toward the development of effective EphA2-targeting PDCs.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5907794PMC
March 2018

Circulating Tumor Cells Predict Occult Metastatic Disease and Prognosis in Pancreatic Cancer.

Ann Surg Oncol 2018 Apr 13;25(4):1000-1008. Epub 2018 Feb 13.

Department of Surgery, University of California Los Angeles, Los Angeles, CA, USA.

Background: Occult metastatic tumors, below imaging thresholds, are a limitation of staging systems that rely on cross-sectional imaging alone and are a cause of the routine understaging of pancreatic ductal adenocarcinomas (PDACs). We investigated circulating tumor cells (CTCs) as a preoperative predictor of occult metastatic disease and as a prognostic biomarker for PDAC patients.

Experimental Design: A total of 126 patients (100 with cancer, 26 with benign disease) were enrolled in our study and CTCs were identified and enumerated from 4 mL of venous blood using the microfluidic NanoVelcro assay. CTC enumeration was correlated with clinicopathologic variables and outcomes following both surgical and systemic therapies.

Results: CTCs were identified in 78% of PDAC patients and CTC counts correlated with increasing stage (ρ = 0.42, p < 0.001). Of the 53 patients taken for potentially curative surgery, 13 (24.5%) had occult metastatic disease intraoperatively. Patients with occult disease had significantly more CTCs than patients with local disease only (median 7 vs. 1 CTC, p < 0.0001). At a cut-off of three or more CTCs/4 mL, CTCs correctly identified patients with occult metastatic disease preoperatively (area under the receiver operating characteristic curve 0.82, 95% confidence interval (CI) 0.76-0.98, p < 0.0001). CTCs were a univariate predictor of recurrence-free survival following surgery [hazard ratio (HR) 2.36, 95% CI 1.17-4.78, p = 0.017], as well as an independent predictor of overall survival on multivariate analysis (HR 1.38, 95% CI 1.01-1.88, p = 0.040).

Conclusions: CTCs show promise as a prognostic biomarker for PDAC patients at all stages of disease being treated both medically and surgically. Furthermore, CTCs demonstrate potential as a preoperative biomarker for identifying patients at high risk of occult metastatic disease.
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http://dx.doi.org/10.1245/s10434-017-6290-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896564PMC
April 2018

Glycan Stimulation Enables Purification of Prostate Cancer Circulating Tumor Cells on PEDOT NanoVelcro Chips for RNA Biomarker Detection.

Adv Healthc Mater 2018 02 11;7(3). Epub 2017 Sep 11.

Smart Organic Material Laboratory, Institute of Chemistry, Academia Sinica, No. 128, Sec. 2, Academia Rd., Nankang, Taipei, 11529, Taiwan.

A glycan-stimulated and poly(3,4-ethylene-dioxythiophene)s (PEDOT)-based nanomaterial platform is fabricated to purify circulating tumor cells (CTCs) from blood samples of prostate cancer (PCa) patients. This new platform, phenylboronic acid (PBA)-grafted PEDOT NanoVelcro, combines the 3D PEDOT nanosubstrate, which greatly enhances CTC capturing efficiency, with a poly(EDOT-PBA-co-EDOT-EG3) interfacial layer, which not only provides high specificity for CTC capture upon antibody conjugation but also enables competitive binding of sorbitol to gently release the captured cells. CTCs purified by this PEDOT NanoVelcro chip provide well-preserved RNA transcripts for the analysis of the expression level of several PCa-specific RNA biomarkers, which may provide clinical insights into the disease.
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http://dx.doi.org/10.1002/adhm.201700701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803304PMC
February 2018

Imprinted NanoVelcro Microchips for Isolation and Characterization of Circulating Fetal Trophoblasts: Toward Noninvasive Prenatal Diagnostics.

ACS Nano 2017 08 19;11(8):8167-8177. Epub 2017 Jul 19.

California NanoSystems Institute, Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, University of California, Los Angeles , Los Angeles, California 90095-1770, United States.

Circulating fetal nucleated cells (CFNCs) in maternal blood offer an ideal source of fetal genomic DNA for noninvasive prenatal diagnostics (NIPD). We developed a class of nanoVelcro microchips to effectively enrich a subcategory of CFNCs, i.e., circulating trophoblasts (cTBs) from maternal blood, which can then be isolated with single-cell resolution by a laser capture microdissection (LCM) technique for downstream genetic testing. We first established a nanoimprinting fabrication process to prepare the LCM-compatible nanoVelcro substrates. Using an optimized cTB-capture condition and an immunocytochemistry protocol, we were able to identify and isolate single cTBs (Hoechst+/CK7+/HLA-G+/CD45-, 20 μm > sizes > 12 μm) on the imprinted nanoVelcro microchips. Three cTBs were polled to ensure reproducible whole genome amplification on the cTB-derived DNA, paving the way for cTB-based array comparative genomic hybridization (aCGH) and short tandem repeats analysis. Using maternal blood samples collected from expectant mothers carrying a single fetus, the cTB-derived aCGH data were able to detect fetal genders and chromosomal aberrations, which had been confirmed by standard clinical practice. Our results support the use of nanoVelcro microchips for cTB-based noninvasive prenatal genetic testing, which holds potential for further development toward future NIPD solution.
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http://dx.doi.org/10.1021/acsnano.7b03073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614709PMC
August 2017

Precision oncology using a limited number of cells: optimization of whole genome amplification products for sequencing applications.

BMC Cancer 2017 Jul 1;17(1):457. Epub 2017 Jul 1.

Department of Surgery, University of California Los Angeles, 10833 Le Conte Ave, California, Los Angeles, 90095, USA.

Background: Sequencing analysis of circulating tumor cells (CTCs) enables "liquid biopsy" to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies.

Methods: Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score.

Results: Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA.

Conclusion: The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5-10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.
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http://dx.doi.org/10.1186/s12885-017-3447-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493892PMC
July 2017

Circulating tumor cells in prostate cancer: beyond enumeration.

Clin Adv Hematol Oncol 2017 Jan;15(1):63-73

Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, California.

Circulating tumor cells (CTCs) are a population of rare cancer cells that have detached from the primary tumor and/or metastatic lesions and entered the peripheral circulation. Enumeration of CTCs has demonstrated value as a prognostic biomarker, and newer studies have pointed to information beyond enumeration that is of critical importance in prostate cancer. Technologic advances that permit examination of the morphology, function, and molecular content of CTCs have made it possible to measure these factors as part of liquid biopsy. These advances provide a way to study tumor evolution and the development of resistance to therapy. Recent breakthroughs have created new applications for CTCs that will affect the care of patients with prostate cancer.
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January 2017

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PLoS One 2017 26;12(1):e0170897. Epub 2017 Jan 26.

Department of Surgery, University of California Los Angeles, Los Angeles, California, United States of America.

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268428PMC
August 2017