Dynamic Metal-Phenolic Coordination Complexes for Versatile Surface Nanopatterning.

We report a general nanopatterning strategy that takes advantage of the dynamic coordination bonds between polyphenols and metal ions (e.g., Fe3+ and Cu2+) to create structures on surfaces with a range of properties. With this methodology, under acidic conditions, 29 metal-phenolic complex-based precursors composed of different polyphenols and metal ions are patterned using scanning probe and large-area cantilever free nanolithography techniques, resulting in a library of deposited metal-phenolic nanopatterns. Significantly, post-treatment of the patterns under basic conditions (i.e., ammonia vapor) triggers a change in coordination state and results in the in situ generation of more stable networks firmly attached to the underlying substrates. The methodology provides control over feature size, shape, and composition, almost regardless of substrate (e.g., Si, Au, and silicon nitride). Under reducing conditions (i.e., H2) at elevated temperatures (180-600 °C), the patterned features have been used as nanoreactors to synthesize individual metal nanoparticles. At room temperature, the ammonia-treated features can reduce Ag+ to form metal nanostructures and be modified with peptides, proteins, and thiolated DNA via Michael addition and/or Schiff base reaction. The generality of this technique should make it useful for a wide variety of researchers interested in modifying surfaces for catalytic, chemical and biological sensing, and template-directed assembly purposes.

Clinical significance of circulating tumor cells in predicting disease progression and chemotherapy resistance in patients with gestational choriocarcinoma.

Gestational choriocarcinoma (GC) is a highly aggressive tumor. In our study, we systematically investigated EpCAM/CD147 expression characteristics in patients with GC and assessed the role of circulating tumor cells (CTCs) in predicting chemotherapy response and disease progression. GC tissues were positive for either epithelial cellular adhesion molecule (EpCAM) or CD147, and all samples exhibited strong human chorionic gonadotropin (HCG) expression. Among all the recruited patients (n = 115), 103 had at least 1 CTC in a 7.5-mL peripheral blood sample, and the percentage of patients with ≥4 CTCs in a particular FIGO stage group increased with a higher FIGO stage (p < 0.001). Furthermore, the pretreatment CTC count was related to tumor size (r = 0.225, p = 0.015) and the number of metastases (r = 0.603, p < 0.001). A progression analysis showed that among the 115 included patients who qualified for further examination, 52 of the 64 patients defined as progressive had ≥4 pretreatment CTCs, while only 7 of the 51 non-progressive patients had ≥4 pretreatment CTCs (p < 0.001). In multivariate analysis, CTCs (≥4) remained the strongest predictor of PFS when other prognostic markers, FIGO score and FIGO stage were included. Moreover, based on the chemotherapy response, patients with ≥4 CTCs were more likely to be resistant to chemotherapy than those with <4 CTCs (P < 0.001). These findings demonstrates the feasibility of CTC detection in cases of GC by adopting EpCAM/CD147 antibodies together as capturing antibodies. The CTC count is a promising indicator in the evaluation of biological activities and the chemotherapy response in GC patients.

Nanostructure embedded microchips for detection, isolation, and characterization of circulating tumor cells.

Circulating tumor cells (CTCs) are cancer cells that break away from either a primary tumor or a metastatic site and circulate in the peripheral blood as the cellular origin of metastasis. With their role as a "tumor liquid biopsy", CTCs provide convenient access to all disease sites, including that of the primary tumor and the site of fatal metastases. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status without the flaws and limitations encountered in performing conventional tumor biopsies. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies. Inspired by the nanoscale interactions observed in the tissue microenvironment, 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 high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of Velcro: when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding. Through continuous evolution, three generations (gens) of NanoVelcro CTC chips have been established to achieve different clinical utilities. The first-gen NanoVelcro chip, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Side-by-side analytical validation studies using clinical blood samples suggested that the sensitivity of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch. In conjunction with the use of the laser microdissection (LMD) technique, second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation. The individually isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger sequencing and next-generation sequencing, NGS) to verify the CTC's role as tumor liquid biopsy. Created by grafting of thermoresponsive polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive NanoVelcro) have demonstrated the capture and release of CTCs at 37 and 4 °C, respectively. The temperature-dependent conformational changes of polymer brushes can effectively alter the accessibility of the capture agent on SiNS, allowing for rapid CTC purification with desired viability and molecular integrity. This Account summarizes the continuous evolution of NanoVelcro CTC assays from the emergence of the original idea all the way to their applications in cancer research. We envision that NanoVelcro CTC assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.

Astrocyte elevated gene-1(AEG-1) induces epithelial-mesenchymal transition in lung cancer through activating Wnt/ß-catenin signaling.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited therapeutic options, so development of novel therapies that target NSCLC is needed. During the early stage of metastasis, the cancer cells undergo an epithelial-mesenchymal transition (EMT), a phase in which Wnt/ß-catenin signaling is known to be involved. Simultaneously, AEG-1 has been demonstrated to activate Wnt-mediated signaling in some malignant tumors. METHODS: Human NSCLC cell lines and xenograft of NSCLC cells in nude mice were used to investigate the effects of AEG-1 on EMT. EMT or Wnt/ß-catenin pathway-related proteins were characterized by western blot, immunofluorescence and immunohistochemistry. RESULTS: In the present study, we demonstrated that astrocyte elevated gene-1(AEG-1) ectopic overexpression promoted EMT, which resulted from the down-regulation of E-cadherin and up-regulation of Vimentin in lung cancer cell lines and clinical lung cancer specimens. Using an orthotopic xenograft-mouse model, we also observed that AEG-1 overexpression in human carcinoma cells led to the development of multiple lymph node metastases and elevated mesenchymal markers such as Vimentin, which is a characteristic of cells in EMT. Furthermore, AEG-1 functioned as a critical protein in the regulation of EMT by directly targeting multiple positive regulators of the Wnt/ß-catenin signaling cascade, including GSK-3ß and CKIδ. Notably, overexpression of AEG-1 in metastatic cancer tissues was closely associated with poor survival of NSCLC patients. CONCLUSIONS: These results reveal the critical role of AEG-1 in EMT and suggest that AEG-1 may be a prognostic biomarker and its targeted inhibition may be utilized as a novel therapy for NSCLC.

Controlling Intracellular Machinery via Polymer Pen Lithography Molecular Patterning.

The plasma membrane and the actomyosin cytoskeleton play key roles in controlling how cells sense and interact with their surrounding environment. Myosin, a force-generating actin network-associated protein, is a major regulator of plasma membrane tension, which helps control endocytosis. Despite the important link between plasma membranes and actomyosin (the actin-myosin complex), little is known about how the actomyosin arrangement regulates endocytosis. Here, nanoscopic ligand arrangements defined by polymer pen lithography (PPL) are used to control actomyosin contractility and examine cell uptake. Confocal microscopy, atomic force microscopy, and flow cytometry suggest that the cytoskeletal tension imposed by the nanoscopic ligand arrangement can actively regulate cellular uptake through clathrin- and caveolin-mediated pathways. Specifically, ligand arrangements that increase cytoskeletal tension tend to reduce the cellular uptakes of cholera toxin (CTX) and spherical nucleic acids (SNAs) by regulating endocytic budding and limiting the formation of clathrin- and caveolae-coated pits. Collectively, this work demonstrates how the cell endocytic fate is regulated by actomyosin mechanical forces, which can be tuned by subcellular cues defined by PPL.

Correction: High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis.

[This corrects the article DOI: 10.18632/oncotarget.5358.].

The role of a semi-automated NanoVelcro system in capturing circulating tumor cells and evaluating their prognostic value for gestational choriocarcinoma.

To investigate whether circulating tumor cells (CTCs) are detectable in patients with gestational choriocarcinoma (GC) and evaluate the prognostic value of CTC enumeration. In this multicenter study, the presence of CTCs was examined in 180 GC patients using a semi-automated NanoVelcro system, among whom 106 patients underwent CTC re-evaluation after one cycle of chemotherapy. Approximately 96% of the GC patients contained ≥2 CTCs in 7.5 mL of blood. The number of CTCs per 7.5 mL of blood was much higher in patients with distant metastases (n = 95; range, 0 to 104) than in patients without distant metastases (n = 85; range, 0 to 6). Applying a 90-patient training and 90-patient validation cohort, a cutoff value of ≥6 CTCs was defined as the prognostic threshold for progression-free survival (PFS) and overall survival (OS). The presence of ≥6 CTCs was significantly associated with worse PFS and OS (both P < 0.001). A multivariate analysis showed that the CTC number (≥6 CTCs) was the strongest predictor of OS (hazard ratio [HR], 15.8; 95% confidence interval [CI], 4.3-57.9; P < 0.001). The number of CTCs decreased after one cycle of chemotherapy; univariate analyses demonstrated that the CTC count after the first chemotherapy cycle was a strong predictor of OS (HR, 36.1; 95% CI, 4.8-271.5; P < 0.001). CTCs are a promising prognostic factor for GC. The absolute CTC count after one cycle of chemotherapy in the context of this disease is a strong predictor of chemotherapy response.

Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues.

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.

Development and Validation of a Novel Signature to Predict Overall Survival in "Driver Gene-negative" Lung Adenocarcinoma (LUAD): Results of a Multicenter Study.

PURPOSE: Examining the role of developmental signaling pathways in "driver gene-negative" lung adenocarcinoma (patients with lung adenocarcinoma negative for EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1 were identified as "driver gene-negative") may shed light on the clinical research and treatment for this lung adenocarcinoma subgroup. We aimed to investigate whether developmental signaling pathways activation can stratify the risk of "driver gene-negative" lung adenocarcinoma. EXPERIMENTAL DESIGN: In the discovery phase, we profiled the mRNA expression of each candidate gene using genome-wide microarrays in 52 paired lung adenocarcinoma and adjacent normal tissues. In the training phase, tissue microarrays and LASSO Cox regression analysis were applied to further screen candidate molecules in 189 patients, and we developed a predictive signature. In the validation phase, one internal cohort and two external cohorts were used to validate our novel prognostic signature. RESULTS: Kyoto Encyclopedia of Genes and Genomes pathway analysis based on whole-genome microarrays indicated that the Wnt/ß-catenin pathway was activated in "driver gene-negative" lung adenocarcinoma. Furthermore, the Wnt/ß-catenin pathway-based gene expression profiles revealed 39 transcripts differentially expressed. Finally, a Wnt/ß-catenin pathway-based CSDW signature comprising 4 genes (CTNNB1 or ß-catenin, SOX9, DVL3, and Wnt2b) was developed to classify patients into high-risk and low-risk groups in the training cohort. Patients with high-risk scores in the training cohort had shorter overall survival [HR, 10.42; 6.46-16.79; P < 0.001) than patients with low-risk scores. CONCLUSIONS: The CSDW signature is a reliable prognostic tool and may represent genes that are potential drug targets for "driver gene-negative" lung adenocarcinoma.

AEG-1 activates Wnt/PCP signaling to promote metastasis in tongue squamous cell carcinoma.

Despite advances in therapy, survival among patients with locally advanced squamous cell carcinoma of tongue (TSCC) and cervical lymph node metastasis remains dismal. Here, we estimated the functional effect of AEG-1 on TSCC metastasis and explored the molecular mechanism by which AEG-1 stimulates epithelial-mesenchymal transition (EMT). We initially found that AEG-1 mRNA levels were much higher in metastatic TSCC than in non-metastatic TSCC and that AEG-1 expression strongly correlates with EMT status. Receiver operating characteristic analysis showed that the combined AEG-1 and EMT statuses are predictive of the survival rate among TSCC patients. In addition, AEG-1 knockdown inhibited EMT in cultured TSCC cell lines and in a xenograft-mouse model. Recombinant AEG-1 activated Wnt/PCP-Rho signaling, and its stimulatory effects on TSCC cell invasiveness and EMT were reversed by an anti-Wnt5a neutralizing antibody or by inhibition of Rac1 or ROCK. These results highlight the critical stimulatory effect of AEG-1 on cancer cell invasiveness and EMT and indicate that AEG-1 may be a useful prognostic biomarker for TSCC patients.

High expression of CTHRC1 promotes EMT of epithelial ovarian cancer (EOC) and is associated with poor prognosis.

Collagen triple helix repeat-containing 1 (CTHRC1) is aberrantly overexpressed in multiple malignant tumors. However, the expression characteristics and function of CTHRC1 in epithelial ovarian cancer (EOC) remain unclear. We found that CTHRC1 expression was up-regulated in the paraffin-embedded EOC tissues compared to borderline or benign tumor tissues. CTHRC1 expression was positively correlated with tumor size (p = 0.008), menopause (p = 0.037), clinical stage (p = 0.002) and lymph node metastasis (p < 0.001) and was also an important prognostic factor for the overall survival of EOC patients, as revealed by Kaplan-Meier analysis. CTHRC1 increased the invasive capabilities of EOC cells in vitro by activating the Wnt/ß-catenin signaling pathway. We showed that ectopic transfection of CTHRC1 in EOC cells up-regulated the expression of EMT markers such as N-cadherin and vimentin, and EMT-associated transcriptional factor Snail. Knockdown of CTHRC1 expression in EOC cells resulted in down-regulation of N-cadherin, vimentin, Snail and translocation of ß-catenin. Collectively, CTHRC1 may promote EOC metastasis through the induction of EMT process and serve as a potential biomarker for prognosis as well as a target for therapy.

A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer.

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.

Programming thermoresponsiveness of NanoVelcro substrates enables effective purification of circulating tumor cells in lung cancer patients.

Unlike tumor biopsies that can be constrained by problems such as sampling bias, circulating tumor cells (CTCs) are regarded as the "liquid biopsy" of the tumor, providing convenient access to all disease sites, including primary tumor and fatal metastases. Although enumerating CTCs is of prognostic significance in solid tumors, it is conceivable that performing molecular and functional analyses on CTCs will reveal much significant insight into tumor biology to guide proper therapeutic intervention. We developed the Thermoresponsive NanoVelcro CTC purification system that can be digitally programmed to achieve an optimal performance for purifying CTCs from non-small cell lung cancer (NSCLC) patients. The performance of this unique CTC purification system was optimized by systematically modulating surface chemistry, flow rates, and heating/cooling cycles. By applying a physiologically endurable stimulation (i.e., temperature between 4 and 37 °C), the mild operational parameters allow minimum disruption to CTCs' viability and molecular integrity. Subsequently, we were able to successfully demonstrate culture expansion and mutational analysis of the CTCs purified by this CTC purification system. Most excitingly, we adopted the combined use of the Thermoresponsive NanoVelcro system with downstream mutational analysis to monitor the disease evolution of an index NSCLC patient, highlighting its translational value in managing NSCLC.