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Circulating tumor cells (CTCs) and tumor-derived extracellular vesicles (tEVs) are two crucial methodologies of liquid biopsy. Given their distinct size differences and release dynamics, CTCs and tEVs potentially offer synergistic capabilities in the non-invasive detection of differentiated thyroid cancer (DTC), a typically indolent tumor. We present the Combined DTC CTC/tEV Assay, integrating dual liquid biopsy processes: i) DTC CTC enrichment by Click Chips, followed by analysis of seven DTC-specific genes, and ii) DTC tEV enrichment by Click Beads, succeeded by mRNA cargo quantification in DTC tEVs. This method utilizes click chemistry, leveraging a pair of biorthogonal and highly reactive functional motifs (tetrazine, Tz, and trans-cyclooctene, TCO), to overcome the challenges encountered in the conventional immunoaffinity-based enrichment of CTCs and tEVs. The Combined DTC CTC/tEV Assay synergistically combines the diagnostic precision of CTCs with the sensitivity of tEVs, demonstrating superior diagnostic accuracy in DTC detection and boasting an AUROC of 0.99. This outperforms the individual diagnostic performance of using either DTC CTC or DTC tEV alone. This integration enables full utilization of a patient's blood sample, and marks a significant evolution in the development of nanomaterial-based liquid biopsy technologies to address challenging unmet clinical needs in cancer care.

Background and aimsThe sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC.Approach and resultsTissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99).ConclusionHCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Exosomes are extracellular vesicles that modulate essential physiological and pathological signals. Communication between cancer cells that express the von Hippel-Lindau (VHL) tumor suppressor gene and those that do not is instrumental to distant metastasis in renal cell carcinoma (RCC). In a novel metastasis model, VHL(-) cancer cells are the metastatic driver, while VHL(+) cells receive metastatic signals from VHL(-) cells and undergo aggressive transformation. This study investigates whether exosomes could be mediating metastatic crosstalk. Exosomes isolated from paired VHL(+) and VHL(-) cancer cell lines were assessed for physical, biochemical, and biological characteristics. Compared to the VHL(+) cells, VHL(-) cells produce significantly more exosomes that augment epithelial-to-mesenchymal transition (EMT) and migration of VHL(+) cells. Using a Cre-loxP exosome reporter system, the fluorescent color conversion and migration were correlated with dose-dependent delivery of VHL(-) exosomes. VHL(-) exosomes even induced a complete cascade of distant metastasis when added to VHL(+) tumor xenografts in a duck chorioallantoic membrane (dCAM) model, while VHL(+) exosomes did not. Therefore, this study supports that exosomes from VHL(-) cells could mediate critical cell-to-cell crosstalk to promote metastasis in RCC.

Serum alpha-fetoprotein and radiologic imaging are the most commonly used tests for early diagnosis and dynamic monitoring of treatment response in hepatocellular carcinoma (HCC). However, the accuracy of these tests is limited, and they may not reflect the underlying biology of the tumor. Thus, developing highly accurate novel HCC biomarkers reflecting tumor biology is a clinically unmet need. Circulating tumor cells (CTCs) have long been proposed as a noninvasive biomarker in clinical oncology. Most CTC assays utilize immunoaffinity-based, size-based, and/or enrichment-free mechanisms followed by immunocytochemical staining to characterize CTCs. The prognostic value of HCC CTC enumeration has been extensively validated. Subsets of CTCs expressing mesenchymal markers are also reported to have clinical significance. In addition, researchers have been devoting their efforts to molecular characterizations of CTCs (e.g. genetics and transcriptomics) as molecular profiling can offer a more accurate readout and provide biological insights. As new molecular profiling techniques, such as digital polymerase chain reaction, are developed to detect minimal amounts of DNA/RNA, several research groups have established HCC CTC digital scoring systems to quantify clinically relevant gene panels. Given the versatility of CTCs to provide intact molecular and functional data that reflects the underlying tumor, CTCs have great potential as a noninvasive biomarker in HCC. Large-scale, prospective studies for HCC CTCs with a standardized protocol are necessary for successful clinical translation.

Well-preserved molecular cargo in circulating extracellular vesicles (EVs) offers an ideal material for detecting oncogenic gene alterations in cancer patients, providing a noninvasive diagnostic solution for detection of disease status and monitoring treatment response. Therefore, technologies that conveniently isolate EVs with sufficient efficiency are desperately needed. Here, a lipid labeling and click chemistry-based EV capture platform ("Click Beads"), which is ideal for EV message ribonucleic acid (mRNA) assays due to its efficient, convenient, and rapid purification of EVs, enabling downstream molecular quantification using reverse transcription digital polymerase chain reaction (RT-dPCR) is described and demonstrated. Ewing sarcoma protein (EWS) gene rearrangements and kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutation status are detected and quantified using EVs isolated by Click Beads and matched with those identified in biopsy specimens from Ewing sarcoma or pancreatic cancer patients. Moreover, the quantification of gene alterations can be used for monitoring treatment responses and disease progression.

Circulating tumor cell (CTC) clusters are present in cancer patients with severe metastasis, resulting in poor clinical outcomes. However, CTC clusters have not been studied as extensively as single CTCs, and the clinical utility of CTC clusters remains largely unknown. In this study, we aim sought to explore the feasibility of NanoVelcro Chips to simultaneously detect both single CTCs and CTC clusters with negligible perturbation to their intrinsic properties in neuroendocrine tumors (NETs). We discovered frequent CTC clusters in patients with advanced NETs and examined their potential roles, together with single NET CTCs, as novel biomarkers of patient response following peptide receptor radionuclide therapy (PRRT). We observed dynamic changes in both total NET CTCs and NET CTC cluster counts in NET patients undergoing PRRT which correlated with clinical outcome. These preliminary findings suggest that CTC clusters, along with single CTCs, offer a potential non-invasive option to monitor the treatment response in NET patients undergoing PRRT.

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be due to sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing identified miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n = 113) and parturition (n = 47). Sequencing analysis identified 986 expressed mature miRNAs from female and male placentae at first and third trimester (baseMean>10). Of these, 11 sexually dimorphic (FDR 10, with 508 DE miRNAs in common between female-specific and male-specific analysis (269 upregulated in first trimester, 239 upregulated in third trimester). Of those, miR-4483 had the highest fold changes across gestation. There were 62.5% more female exclusive differences with fold change>2 across gestation than male exclusive (52 miRNAs vs 32 miRNAs), indicating miRNA expression across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex-independent and sex-specific placenta miRNA atlas across gestation, which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences.

Early cancer detection by cell-free DNA faces multiple challenges: low fraction of tumor cell-free DNA, molecular heterogeneity of cancer, and sample sizes that are not sufficient to reflect diverse patient populations. Here, we develop a cancer detection approach to address these challenges. It consists of an assay, cfMethyl-Seq, for cost-effective sequencing of the cell-free DNA methylome (with > 12-fold enrichment over whole genome bisulfite sequencing in CpG islands), and a computational method to extract methylation information and diagnose patients. Applying our approach to 408 colon, liver, lung, and stomach cancer patients and controls, at 97.9% specificity we achieve 80.7% and 74.5% sensitivity in detecting all-stage and early-stage cancer, and 89.1% and 85.0% accuracy for locating tissue-of-origin of all-stage and early-stage cancer, respectively. Our approach cost-effectively retains methylome profiles of cancer abnormalities, allowing us to learn new features and expand to other cancer types as training cohorts grow.

Hepatocellular carcinoma (HCC) is among the leading causes of cancer incidence and mortality worldwide. Surveillance of individuals with cirrhosis or other conditions that confer a high risk of HCC development is essential for early detection and improved overall survival. Biannual ultrasonography with or without alpha-fetoprotein is widely recommended as the standard method for HCC surveillance, but it has limited sensitivity in early disease and may be inadequate in certain individuals. This review article will provide a comprehensive overview of the current landscape of HCC surveillance, including the rationale and indications for HCC surveillance, standard methods for HCC surveillance, and their strengths/limitations. Alternative surveillance methods such as the role of cross-sectional imaging, emerging circulating biomarkers, as well as the problem of under-utilization of HCC surveillance and surveillance-related harms will also be discussed in this review.

BackgroundRecent trends of hepatocellular carcinoma (HCC) mortality and outcome remain unknown in the United States. We investigated the recent trends of primary liver cancer (excluding intrahepatic cholangiocarcinoma) mortality and HCC stage, treatment, and overall survival (OS) in the United States.MethodsThe National Center for Health Statistics Database was analyzed to investigate the trend of primary liver cancer mortality. We analyzed the Surveillance, Epidemiology, and End Results 18 Database to assess the temporal trend of tumor size, stage, treatment, and OS of HCC. We investigated the association between HCC diagnosis year and OS using Cox regression analysis. All statistical tests were 2-sided.ResultsDuring 2000-2018, liver cancer mortality rates increased until 2013, plateaued during 2013-2016 (annual percent change = 0.1%/y, 95% confidence interval [CI] = -2.1%/y to 2.4%/y, P = .92), and started to decline during 2016-2018 (annual percent change = -1.5%/y, 95% CI = -3.2%/y to 0.2%/y, P = .08). However, mortality continues to increase in American Indian and Alaska Native, individuals aged 65 years or older, and in 33 states. There was a 0.61% (95% CI = 0.53% to 0.69%, P

Placenta accreta spectrum (PAS) is a high-risk obstetrical condition associated with significant morbidity and mortality. Current clinical screening modalities for PAS are not always conclusive. Here, we report a nanostructure-embedded microchip that efficiently enriches both single and clustered circulating trophoblasts (cTBs) from maternal blood for detecting PAS. We discover a uniquely high prevalence of cTB-clusters in PAS and subsequently optimize the device to preserve the intactness of these clusters. Our feasibility study on the enumeration of cTBs and cTB-clusters from 168 pregnant women demonstrates excellent diagnostic performance for distinguishing PAS from non-PAS. A logistic regression model is constructed using a training cohort and then cross-validated and tested using an independent cohort. The combined cTB assay achieves an Area Under ROC Curve of 0.942 (throughout gestation) and 0.924 (early gestation) for distinguishing PAS from non-PAS. Our assay holds the potential to improve current diagnostic modalities for the early detection of PAS.

Aim: To understand miRNA changes across gestation in healthy human placentae. This is essential before miRNAs can be used as biomarkers or prognostic indicators during pregnancy. Materials & methods: Using next-generation sequencing, we characterize the normative human placenta miRNome in first (n = 113) and third trimester (n = 47). Results & conclusion: There are 801 miRNAs expressed in both first and third trimester, including 182 with similar expression across gestation (p ≥ 0.05, fold change ≤2) and 180 significantly different (false discovery rate 2). Of placenta-specific miRNA clusters, chromosome 14 miRNA cluster decreases across gestation and chromosome 19 miRNA cluster is overall highly expressed. Chromosome 13 clusters are upregulated in first trimester. This work provides a rich atlas of healthy pregnancies to direct functional studies investigating the epigenetic differences in first and third trimester placentae.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) is an efficient and precise gene-editing technology that offers a versatile solution for establishing treatments directed at genetic diseases. Currently, CRISPR/Cas9 delivery into cells relies primarily on viral vectors, which suffer from limitations in packaging capacity and safety concerns. These issues with a nonviral delivery strategy are addressed, where Cas9•sgRNA ribonucleoprotein (RNP) complexes can be encapsulated into supramolecular nanoparticles (SMNP) to form RNP⊂SMNPs, which can then be delivered into targeted cells via supramolecular nanosubstrate-mediated delivery. Utilizing the U87 glioblastoma cell line as a model system, a variety of parameters for cellular-uptake of the RNP-laden nanoparticles are examined. Dose- and time-dependent CRISPR/Cas9-mediated gene disruption is further examined in a green fluorescent protein (GFP)-expressing U87 cell line (GFP-U87). The utility of an optimized SMNP formulation in co-delivering Cas9 protein and two sgRNAs that target deletion of exons 45-55 (708 kb) of the dystrophin gene is demonstrated. Mutations in this region lead to Duchenne muscular dystrophy, a severe genetic muscle wasting disease. Efficient delivery of these gene deletion cargoes is observed in a human cardiomyocyte cell line (AC16), induced pluripotent stem cells, and mesenchymal stem cells.

Transcriptomic profiling of tumor tissues introduces a large database, which has led to improvements in the ability of cancer diagnosis, treatment, and prevention. However, performing tumor transcriptomic profiling in the clinical setting is very challenging since the procurement of tumor tissues is inherently limited by invasive sampling procedures. Here, we demonstrated the feasibility of purifying hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) from clinical patient samples with improved molecular integrity using Click Chips in conjunction with a multimarker antibody cocktail. The purified CTCs were then subjected to mRNA profiling by NanoString nCounter platform, targeting 64 HCC-specific genes, which were generated from an integrated data analysis framework with 8 tissue-based prognostic gene signatures from 7 publicly available HCC transcriptomic studies. After bioinformatics analysis and comparison, the HCC CTC-derived gene signatures showed high concordance with HCC tissue-derived gene signatures from TCGA database, suggesting that HCC CTCs purified by Click Chips could enable the translation of HCC tissue molecular profiling into a noninvasive setting.

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.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and one of the leading causes of cancer-related death worldwide. Despite the improvements in surveillance and treatment, the prognosis of HCC remains poor. Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed particles circulating in the bloodstream and mediating intercellular communication. Emerging studies have shown that EVs play a crucial role in regulating the proliferation, immune escape, and metastasis of HCC. In addition, because EVs are present in the circulation at relatively early stages of disease, they are getting attention as an attractive biomarker for HCC detection. Over the past decade, dedicated efforts have been made to isolate EVs more efficiently and make them useful tools in different clinical settings. In this review article, we provide an overview of the EVs isolation methods and highlight the role of EVs as mediators in the pathogenesis and progression of HCC. Lastly, we summarize the potential applications of EVs in early-stage HCC detection.

Tumor-derived extracellular vesicles (EVs) play essential roles in intercellular communication during tumor growth and metastatic evolution. Currently, little is known about the possible roles of tumor-derived EVs in sarcoma because the lack of specific surface markers makes it technically challenging to purify sarcoma-derived EVs. In this study, a specific purification system is developed for Ewing sarcoma (ES)-derived EVs by coupling covalent chemistry-mediated EV capture/ release within a nanostructure-embedded microchip. The purification platform-ES-EV Click Chip-takes advantage of specific anti-LINGO-1 recognition and sensitive click chemistry-mediated EV capture, followed by disulfide cleavage-driven EV release. Since the device is capable of specific and efficient purification of intact ES EVs with high purity, ES-EV Click Chip is ideal for conducting downstream functional studies of ES EVs. Absolute quantification of the molecular hallmark of ES (i.e., EWS rearrangements) using reverse transcription Droplet Digital PCR enables specific quantification of ES EVs. The purified ES EVs can be internalized by recipient cells and transfer their mRNA cargoes, exhibiting their biological intactness and potential role as biological shuttles in intercellular communication.

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.

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; P

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%).

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.

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.

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.

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.

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.

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.

Rationale: 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. Methods: 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. Results: 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). Conclusions: 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.

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.

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

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.

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.

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.

Tattooing has been utilized by the medical community for precisely demarcating anatomic landmarks. This practice is especially important for identifying biopsy sites of nonmelanoma skin cancer (NMSC) due to the long interval (i.e., up to 3 months) between the initial diagnostic biopsy and surgical treatment. Commercially available tattoo pigments possess several issues, which include causing poor cosmesis, being mistaken for a melanocytic lesion, requiring additional removal procedures when no longer desired, and potentially inducing inflammatory responses. The ideal tattoo pigment for labeling of skin biopsy sites for NMSC requires (i) invisibility under ambient light, (ii) fluorescence under a selective light source, (iii) a finite intradermal retention time (ca. 3 months), and (iv) biocompatibility. Herein, we introduce cross-linked fluorescent supramolecular nanoparticles (c-FSNPs) as a "finite tattoo" pigment, with optimized photophysical properties and intradermal retention time to achieve successful in vivo finite tattooing. Fluorescent supramolecular nanoparticles encapsulate a fluorescent conjugated polymer, poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV), into a core via a supramolecular synthetic approach. FSNPs which possess fluorescent properties superior to those of the free MPS-PPV are obtained through a combinatorial screening process. Covalent cross-linking of FSNPs results in micrometer-sized c-FSNPs, which exhibit a size-dependent intradermal retention. The 1456 nm sized c-FSNPs display an ideal intradermal retention time (ca. 3 months) for NMSC lesion labeling, as observed in an in vivo tattoo study. In addition, the c-FSNPs induce undetectable inflammatory responses after tattooing. We believe that the c-FSNPs can serve as a "finite tattoo" pigment to label potential malignant NMSC lesions.

Recent cancer research has demonstrated the existence of circulating tumor cells (CTCs) in cancer patient's blood. Once identified, CTC biomarkers will be invaluable tools for clinical diagnosis, prognosis and treatment. In this review, we propose ex vivo culture as a rational strategy for large scale amplification of the limited numbers of CTCs from a patient sample, to derive enough CTCs for accurate and reproducible characterization of the biophysical, biochemical, gene expressional and behavioral properties of the harvested cells. Because of tumor cell heterogeneity, it is important to amplify all the CTCs in a blood sample for a comprehensive understanding of their role in cancer metastasis. By analyzing critical steps and technical issues in ex vivo CTC culture, we developed a cost-effective and reproducible protocol directly culturing whole peripheral blood mononuclear cells, relying on an assumed survival advantage in CTCs and CTC-like cells over the normal cells to amplify this specified cluster of cancer cells.

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs.

A pretargeted oncologic positron emission tomography (PET) imaging that leverages the power of supramolecular nanoparticles with in vivo bioorthogonal chemistry was demonstrated for the clinically relevant problem of tumor imaging. The advantages of this approach are that (i) the pharmacokinetics (PKs) of tumor-targeting and imaging agents can be independently altered via chemical alteration to achieve the desired in vivo performance and (ii) the interplay between the two PKs and other controllable variables confers a second layer of control toward improved PET imaging. In brief, we utilized supramolecular chemistry to synthesize tumor-targeting nanoparticles containing transcyclooctene (TCO, a bioorthogonal reactive motif), called TCO⊂SNPs. After the intravenous injection and subsequent concentration of the TCO⊂SNPs in the tumors of living mice, a small molecule containing both the complementary bioorthogonal motif (tetrazine, Tz) and a positron-emitting radioisotope ((64)Cu) was injected to react selectively and irreversibly to TCO. High-contrast PET imaging of the tumor mass was accomplished after the rapid clearance of the unreacted (64)Cu-Tz probe. Our nanoparticle approach encompasses a wider gamut of tumor types due to the use of EPR effects, which is a universal phenomenon for most solid tumors.

Simultaneous delivery of multiple genes and proteins (e.g., transcription factors; TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications.

Liquid biopsy of tumor through isolation of circulating tumor cells (CTCs) allows non-invasive, repetitive, and systemic sampling of disease. Although detecting and enumerating CTCs is of prognostic significance in metastatic cancer, it is conceivable that performing molecular and functional characterization on CTCs will reveal unprecedented insight into the pathogenic mechanisms driving lethal disease. Nanomaterial-embedded cancer diagnostic platforms, i.e., NanoVelcro CTC Assays represent a unique rare-cell sorting method that enables detection isolation, and characterization of CTCs in peripheral blood, providing an opportunity to noninvasively monitor disease progression in individual cancer patients. Over the past decade, a series of NanoVelcro CTC Assays has been demonstrated for exploring the full potential of CTCs as a clinical biomarker, including CTC enumeration, phenotyping, genotyping and expression profiling. In this review article, the authors will briefly introduce the development of three generations of NanoVelcro CTC Assays, and highlight the clinical applications of each generation for various types of solid cancers, including prostate cancer, pancreatic cancer, lung cancer, and melanoma.

Previous studies have demonstrated focal but limited molecular similarities between circulating tumor cells (CTCs) and biopsies using isolated genetic assays. We hypothesized that molecular similarity between CTCs and tissue exists at the single cell level when characterized by whole genome sequencing (WGS). By combining the NanoVelcro CTC Chip with laser capture microdissection (LCM), we developed a platform for single-CTC WGS. We performed this procedure on CTCs and tissue samples from a patient with advanced prostate cancer who had serial biopsies over the course of his clinical history. We achieved 30X depth and ≥ 95% coverage. Twenty-nine percent of the somatic single nucleotide variations (SSNVs) identified were founder mutations that were also identified in CTCs. In addition, 86% of the clonal mutations identified in CTCs could be traced back to either the primary or metastatic tumors. In this patient, we identified structural variations (SVs) including an intrachromosomal rearrangement in chr3 and an interchromosomal rearrangement between chr13 and chr15. These rearrangements were shared between tumor tissues and CTCs. At the same time, highly heterogeneous short structural variants were discovered in PTEN, RB1, and BRCA2 in all tumor and CTC samples. Using high-quality WGS on single-CTCs, we identified the shared genomic alterations between CTCs and tumor tissues. This approach yielded insight into the heterogeneity of the mutational landscape of SSNVs and SVs. It may be possible to use this approach to study heterogeneity and characterize the biological evolution of a cancer during the course of its natural history.

Conformal bioelectronics enable wearable, noninvasive, and health-monitoring platforms. We demonstrate a simple and straightforward method for producing thin, sensitive In2O3-based conformal biosensors based on field-effect transistors using facile solution-based processing. One-step coating via aqueous In2O3 solution resulted in ultrathin (3.5 nm), high-density, uniform films over large areas. Conformal In2O3-based biosensors on ultrathin polyimide films displayed good device performance, low mechanical stress, and highly conformal contact determined using polydimethylsiloxane artificial skin having complex curvilinear surfaces or an artificial eye. Immobilized In2O3 field-effect transistors with self-assembled monolayers of NH2-terminated silanes functioned as pH sensors. Functionalization with glucose oxidase enabled d-glucose detection at physiologically relevant levels. The conformal ultrathin field-effect transistor biosensors developed here offer new opportunities for future wearable human technologies.

Supramolecular nanosubstrate-mediated delivery (SNSMD) leverages the power of molecular self-assembly and a nanostructured substrate platform for the low toxicity, highly efficient co-delivery of biological factors encapsulated in a nanovector. Human fibroblasts are successfully reprogrammed into induced pluripotent stems and transdifferentiated into induced neuronal-like cells.