Hierarchical integration of DNA nanostructures and NanoGold onto a microchip facilitates covalent chemistry-mediated purification of circulating tumor cells in head and neck squamous cell carcinoma.

It is well-established that the combined use of nanostructured substrates and immunoaffinity agents can enhance the cell-capture performance of the substrates, thus offering a practical solution to effectively capture circulating tumor cells (CTCs) in peripheral blood. Developing along this strategy, this study first demonstrated a top-down approach for the fabrication of tetrahedral DNA nanostructure (TDN)-NanoGold substrates through the hierarchical integration of three functional constituents at various length-scales: a macroscale glass slide, sub-microscale self-organized NanoGold, and nanoscale self-assembled TDN. The TDN-NanoGold substrates were then assembled with microfluidic chaotic mixers to give TDN-NanoGold Click Chips. In conjunction with the use of copper (Cu)-catalyzed azide-alkyne cycloaddition (CuAAC)-mediated CTC capture and restriction enzyme-triggered CTC release, TDN-NanoGold Click Chips allow for effective enumeration and purification of CTCs with intact cell morphologies and preserved molecular integrity. To evaluate the clinical utility of TDN-NanoGold Click Chips, we used these devices to isolate and purify CTCs from patients with human papillomavirus (HPV)-positive (+) head and neck squamous cell carcinoma (HNSCC). The purified HPV(+) HNSCC CTCs were then subjected to RT-ddPCR testing, allowing for detection of E6/E7 oncogenes, the characteristic molecular signatures of HPV(+) HNSCC. We found that the resulting HPV(+) HNSCC CTC counts and E6/E7 transcript copy numbers are correlated with the treatment responses in the patients, suggesting the potential clinical utility of TDN-NanoGold Click Chips for non-invasive diagnostic applications of HPV(+) HNSCC.

Prostate cancer extracellular vesicle digital scoring assay - a rapid noninvasive approach for quantification of disease-relevant mRNAs.

Optimizing outcomes in prostate cancer (PCa) requires precision in characterization of disease status. This effort was directed at developing a PCa extracellular vesicle (EV) Digital Scoring Assay (DSA) for detecting metastasis and monitoring progression of PCa. PCa EV DSA is comprised of an EV purification device (i.e., EV Click Chip) and reverse-transcription droplet digital PCR that quantifies 11 PCa-relevant mRNA in purified PCa-derived EVs. A Met score was computed for each plasma sample based on the expression of the 11-gene panel using the weighted Z score method. Under optimized conditions, the EV Click Chips outperformed the ultracentrifugation or precipitation method of purifying PCa-derived EVs from artificial plasma samples. Using PCa EV DSA, the Met score distinguished metastatic (n = 20) from localized PCa (n = 20) with an area under the receiver operating characteristic curve of 0.88 (95% CI:0.78-0.98). Furthermore, longitudinal analysis of three PCa patients showed the dynamics of the Met scores reflected clinical behavior even when disease was undetectable by imaging. Overall, a sensitive PCa EV DSA was developed to identify metastatic PCa and reveal dynamic disease states noninvasively. This assay may complement current imaging tools and blood-based tests for timely detection of metastatic progression that can improve care for PCa patients.

HCC EV ECG score: An extracellular vesicle-based protein assay for detection of early-stage hepatocellular carcinoma.

BACKGROUND AND AIMS: The 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 RESULTS: Tissue 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). CONCLUSION: HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Coupling Lipid Labeling and Click Chemistry Enables Isolation of Extracellular Vesicles for Noninvasive Detection of Oncogenic Gene Alterations.

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.

Discovery and characterization of circulating tumor cell clusters in neuroendocrine tumor patients using nanosubstrate-embedded microchips.

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.

Circulating trophoblast cell clusters for early detection of placenta accreta spectrum disorders.

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.