Dendrimer-Peptide Conjugates for Effective Blockade of the Interactions between SARS-CoV-2 Spike Protein and Human ACE2 Receptor.

The coronavirus disease 2019 (COVID-19) pandemic has threatened the stability of global healthcare, which is becoming an endemic issue. Despite the development of various treatment strategies to fight COVID-19, the currently available treatment options have shown varied efficacy. Herein, we have developed an avidity-based SARS-CoV-2 antagonist using dendrimer-peptide conjugates (DPCs) for effective COVID-19 treatment. Two different peptide fragments obtained from angiotensin-converting enzyme 2 (ACE2) were integrated into a single sequence, followed by the conjugation to poly(amidoamine) (PAMAM) dendrimers. We hypothesized that the strong multivalent binding avidity endowed by dendrimers would help peptides effectively block the interaction between SARS-CoV-2 and ACE2, and this antagonist effect would be dependent upon the generation (size) of the dendrimers. To assess this, binding kinetics of the DPCs prepared from generation 4 (G4) and G7 PAMAM dendrimers to spike protein of SARS-CoV-2 were quantitatively measured using surface plasmon resonance. The larger dendrimer-based DPCs exhibited significantly enhanced binding strength by 3 orders of magnitude compared to the free peptides, whereas the smaller one showed a 12.8-fold increase only. An in vitro assay using SARS-CoV-2-mimicking microbeads also showed the improved SARS-CoV-2 blockade efficiency of the G7-peptide conjugates compared to G4. In addition, the interaction between the DPCs and SARS-CoV-2 was analyzed using molecular dynamics (MD) simulation, providing an insight into how the dendrimer-mediated multivalent binding effect can enhance the SARS-CoV-2 blockade. Our findings demonstrate that the DPCs having strong binding to SARS-CoV-2 effectively block the interaction between ACE2 and SARS-CoV-2, providing a potential as a high-affinity drug delivery system to direct anti-COVID payloads to the virus.

Size-Dependent Drug Loading, Gene Complexation, Cell Uptake, and Transfection of a Novel Dendron-Lipid Nanoparticle for Drug/Gene Co-delivery.

Dendron micelles have shown promising results as a multifunctional delivery system, owing to their unique molecular architecture. Herein, we have prepared a novel poly(amidoamine) (PAMAM) dendron-lipid hybrid nanoparticle (DLNP) as a nanocarrier for drug/gene co-delivery and examined how the dendron generation of DLNPs impacts their cargo-carrying capabilities. DLNPs, formed by a thin-layer hydration method, were internally loaded with chemo-drugs and externally complexed with plasmids. Compared to generation 2 dendron DLNP (D2LNPs), D3LNPs demonstrated a higher drug encapsulation efficiency (31% vs 87%) and better gene complexation (minimal N/P ratio of 20:1 vs 5:1 for complexation) due to their smaller micellar aggregation number and higher charge density, respectively. Furthermore, D3LNPs were able to avoid endocytosis and subsequent lysosomal degradation and demonstrated a higher cellular uptake than D2LNPs. As a result, D3LNPs exhibited significantly enhanced antitumor and gene transfection efficacy in comparison to D2LNPs. These findings provide design cues for engineering multifunctional dendron-based nanotherapeutic systems for effective combination cancer treatment.

Immunoavidity-Based Capture of Tumor Exosomes Using Poly(amidoamine) Dendrimer Surfaces.

Tumor-derived blood-circulating exosomes have potential as a biomarker to greatly improve cancer treatment. However, effective isolation of exosomes remains a tremendous technical challenge. This study presents a novel nanostructured polymer surface for highly effective capture of exosomes through strong avidity. Various surface configurations, consisting of multivalent dendrimers, PEG, and tumor-targeting antibodies, were tested using exosomes isolated from tumor cell lines. We found that a dual layer dendrimer configuration exhibited the highest efficiency in capturing cultured exosomes spiked into human serum. Importantly, the optimized surface captured a > 4-fold greater amount of tumor exosomes from head and neck cancer patient plasma samples than that from healthy donors. Nanomechanical analysis using atomic force microscopy also revealed that the enhancement was attributed to multivalent binding (avidity) and augmented short-range adhesion mediated by dendrimers. Our results support that the dendrimer surface detects tumor exosomes at high sensitivity and specificity, demonstrating its potential as a new cancer liquid biopsy platform.

An Avidity-Based PD-L1 Antagonist Using Nanoparticle-Antibody Conjugates for Enhanced Immunotherapy.

Upregulation of programmed death ligand 1 (PD-L1) allows cancer cells to evade antitumor immunity. Despite tremendous efforts in developing PD-1/PD-L1 immune checkpoint inhibitors (ICIs), clinical trials using such ICIs have shown inconsistent benefits. Here, we hypothesized that the ICI efficacy would be dictated by the binding strength of the inhibitor to the target proteins. To assess this, hyperbranched, multivalent poly(amidoamine) dendrimers were employed to prepare dendrimer-ICI conjugates (G7-aPD-L1). Binding kinetics measurements using SPR, BLI, and AFM revealed that G7-aPD-L1 exhibits significantly enhanced binding strength to PD-L1 proteins, compared to free aPD-L1. The binding avidity of G7-aPD-L1 was translated into in vitro efficiency and in vivo selectivity, as the conjugates improved the PD-L1 blockade effect and enhanced accumulation in tumor sites. Our results demonstrate that the dendrimer-mediated multivalent interaction substantially increases the binding avidity of the ICIs and thereby improves the antagonist effect, providing a novel platform for cancer immunotherapy.

Nanoparticle Conjugation Stabilizes and Multimerizes ß-Hairpin Peptides To Effectively Target PD-1/PD-L1 ß-Sheet-Rich Interfaces.

ß-Hairpin peptides present great potential as antagonists against ß-sheet-rich protein surfaces, of which wide and flat geometries are typically "undruggable" with small molecules. Herein, we introduce a peptide-dendrimer conjugate (PDC) approach that stabilizes the ß-hairpin structure of the peptide via intermolecular forces and the excluded volume effect as well as exploits the multivalent binding effect. Because of the synergistic advantages, the PDCs based on a ß-hairpin peptide isolated from an engineered programmed death-1 (PD-1) protein showed significantly higher affinity (avidity) to their binding counterpart, programmed death-ligand 1 (PD-L1), as compared to free peptides (by up to 5 orders of magnitude). The enhanced binding kinetics with high selectivity was translated into an improved immune checkpoint inhibitory effect in vitro, at a level comparable to (if not better than) that of a full-size monoclonal antibody. The results demonstrate the potential of the PDC system as a novel class of inhibitors targeting ß-strand-rich protein surfaces, such as PD-1 and PD-L1, displaying its potential as a new cancer immunotherapy platform.

Programmed Death 1 and Cytotoxic T-Lymphocyte-Associated Protein 4 Gene Expression in Peripheral Blood Mononuclear Cells Can Serve as Prognostic Biomarkers for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly aggressive form of liver cancer with poor prognosis. The lack of reliable biomarkers for early detection and accurate diagnosis and prognosis poses a significant challenge to its effective clinical management. In this study, we investigated the diagnostic and prognostic potential of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in peripheral blood mononuclear cells (PBMCs) in HCC. PD-1 and CTLA-4 gene expression was analyzed comparatively using PBMCs collected from HCC patients and healthy individuals. The results revealed higher PD-1 gene expression levels in patients with multifocal tumors, lymphatic invasion, or distant metastasis than those in their control counterparts. However, conventional serum biomarkers of liver function do not exhibit similar correlations. In conclusion, PD-1 gene expression is associated with OS and PFS and CTLA-4 gene expression is associated with OS, whereas the serum biomarkers analyzed in this study show no significant correlation with survival in HCC. Hence, PD-1 and CTLA-4 expressed in PBMCs are considered potential prognostic biomarkers for patients with HCC that can facilitate prediction of malignancy, response to currently available HCC treatments, and overall survival.

Prospective Characterization of Circulating Tumor Cell Kinetics in Patients With Oligometastatic Disease Receiving Definitive Intent Radiation Therapy.

PURPOSE: There are currently no predictive molecular biomarkers to identify patients with oligometastatic disease (OMD) who will benefit from definitive-intent radiation therapy (RT). We prospectively characterized circulating tumor cell (CTC) kinetics in patients with OMD undergoing definitive-intent RT. METHODS: This prospective correlative biomarker study included patients with any solid malignancy ≤5 metastatic sites in ≤3 anatomic organ systems undergoing definitive-intent RT to all disease sites. Circulating tumor cells (CTCs) were captured and enumerated using a biomimetic cell rolling and nanotechnology-based assay functionalized with antibodies against epithelial cell adhesion molecule, against human epidermal growth factor receptor 2, and against epidermal growth factor receptor before and during RT and at follow-up visits up to 2 years post-RT. RESULTS: We enrolled 43 patients with a median follow-up of 14.3 months. The pretreatment CTC level (cells captured/mL) was not associated with the number of disease sites (median one metastatic site/patient, range 1-5) or metastasis location (bone, brain, visceral) on Wilcoxon signed-rank test, P > .05. Post-RT, 56% of patients received systemic therapy, and 72% of patients experienced subsequent local or systemic progression. For 90% of patients, a CTC level <15 within 130 days post-RT corresponded to a durable control of irradiated lesions. Patients with a favorable versus an unfavorable clearance profile experienced significantly longer progression-free survival after RT (median 13 v 4 months, log-rank test, P = .0011). On logistic regression, CTC level >15 at a given time point was associated with clinical disease progression within the subsequent 6 months (odds ratio 3.31, P = .007). In 26% of patients with disease progression, a CTC level >15 preceded radiographic or clinical progression. CONCLUSION: CTCs may serve as a biomarker for disease control in OMD and may predict disease progression before standard assessments for patients receiving diverse cancer-directed therapies.

Nanotechnology and machine learning enable circulating tumor cells as a reliable biomarker for radiotherapy responses of gastrointestinal cancer patients.

A highly sensitive, circulating tumor cell (CTC)-based liquid biopsy was used to monitor gastrointestinal cancer patients during treatment to determine if CTC abundance was predictive of disease recurrence. The approach used a combination of biomimetic cell rolling on recombinant E-selectin and dendrimer-mediated multivalent immunocapture at the nanoscale to purify CTCs from peripheral blood mononuclear cells. Due to the exceptionally high numbers of CTCs captured, a machine learning algorithm approach was developed to efficiently and reliably quantify abundance of immunocytochemically-labeled cells. A convolutional neural network and logistic regression model achieved 82.9% true-positive identification of CTCs with a false positive rate below 0.1% on a validation set. The approach was then used to quantify CTC abundance in peripheral blood samples from 27 subjects before, during, and following treatments. Samples drawn from the patients either prior to receiving radiotherapy or early in chemotherapy had a median 50 CTC ml-1 whole blood (range 0.6-541.6). We found that the CTC counts drawn 3 months post treatment were predictive of disease progression (p = .045). This approach to quantifying CTC abundance may be a clinically impactful in the timely determination of gastrointestinal cancer progression or response to treatment.

Circulating tumor cell abundance in head and neck squamous cell carcinoma decreases with successful chemoradiation and cetuximab treatment.

Head and neck squamous cell carcinoma (HNSCC) is a common and deadly cancer. Circulating tumor cell (CTC) abundance may a valuable, prognostic biomarker in low- and intermediate-risk patients. However, few technologies have demonstrated success in detecting CTCs in these populations. We prospectively collected longitudinal CTC counts from two cohorts of patients receiving treatments at our institution using a highly sensitive device that purifies CTCs using biomimetic cell rolling and dendrimer-conjugated antibodies. In patients with intermediate risk human papillomavirus (HPV)-positive HNSCC, elevated CTC counts were detected in 13 of 14 subjects at screening with a median of 17 CTC/ml (range 0.2-2986.5). A second cohort of non-metastatic, HPV- HNSCC subjects received cetuximab monotherapy followed by surgical resection. In this cohort, all subjects had elevated baseline CTC counts median of 73 CTC/ml (range 5.4-332.9) with statistically significant declines during treatment. Interestingly, two patients with recurrent disease had elevated CTC counts during and following treatment, which also correlated with growth of size and ki67 expression in the primary tumor. The results suggest that our device may be a valuable tool for evaluating the success of less intensive treatment regimens.

Branched, dendritic, and hyperbranched polymers in liquid biopsy device design.

The development of minimally invasive tests for cancer diagnosis and prognosis will aid in the research of new treatments and improve survival rates. Liquid biopsies seek to derive actionable information from tumor material found in routine blood samples. The relative scarcity of tumor material in this complex mixture makes isolating and detecting cancerous material such as proteins, circulating tumor DNA, exosomes, and whole circulating tumor cells a challenge for device engineers. This review describes the chemistry and applications of branched and hyperbranched to improve the performance of liquid biopsy devices. These polymers can improve the performance of a liquid biopsy through several mechanisms. For example, polymers designed to increase the affinity of capture enhance device sensitivity. On the other hand, polymers designed to increase binding avidity or repel nonspecific adsorption enhance device specificity. Branched and hyperbranched polymers can also be used to amplify the signal from small amounts of detected material. The further development of hyperbranched polymers in liquid biopsy applications will enhance device capabilities and help these critical technologies reach the oncology clinic where they are sorely needed. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices.

Dendrimers for cancer immunotherapy: Avidity-based drug delivery vehicles for effective anti-tumor immune response.

Cancer immunotherapy, or the utilization of a patient's own immune system to treat cancer, has shifted the paradigm of cancer treatment. Despite meaningful responses being observed in multiple studies, currently available immunotherapy platforms have only proven effective to a small subset of patients. To address this, nanoparticles have been utilized as a novel carrier for immunotherapeutic drugs, achieving robust anti-tumor effects with increased adaptive and durable responses. Specifically, dendrimer nanoparticles have attracted a great deal of scientific interest due to their versatility in various therapeutic applications, resulting from their unique physicochemical properties and chemically well-defined architecture. This review offers a comprehensive overview of dendrimer-based immunotherapy technologies, including their formulations, biological functionalities, and therapeutic applications. Common formulations include: (1) modulators of cytokine secretion of immune cells (adjuvants); (2) facilitators of the recognition of tumorous antigens (vaccines); (3) stimulators of immune effectors to selectively attack cells expressing specific antigens (antibodies); and (4) inhibitors of immune-suppressive responses (immune checkpoint inhibitors). On-going works and prospects of dendrimer-based immunotherapies are also discussed. Overall, this review provides a critical overview on rapidly growing dendrimer-based immunotherapy technologies and serves as a guideline for researchers and clinicians who are interested in this field. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Hierarchically Multivalent Peptide-Nanoparticle Architectures: A Systematic Approach to Engineer Surface Adhesion.

The multivalent binding effect has been the subject of extensive studies to modulate adhesion behaviors of various biological and engineered systems. However, precise control over the strong avidity-based binding remains a significant challenge. Here, a set of engineering strategies are developed and tested to systematically enhance the multivalent binding of peptides in a stepwise manner. Poly(amidoamine) (PAMAM) dendrimers are employed to increase local peptide densities on a substrate, resulting in hierarchically multivalent architectures (HMAs) that display multivalent dendrimer-peptide conjugates (DPCs) with various configurations. To control binding behaviors, effects of the three major components of the HMAs are investigated: i) poly(ethylene glycol) (PEG) linkers as spacers between conjugated peptides; ii) multiple peptides on the DPCs; and iii) various surface arrangements of HMAs (i.e., a mixture of DPCs each containing different peptides vs DPCs cofunctionalized with multiple peptides). The optimized HMA configuration enables significantly enhanced target cell binding with high selectivity compared to the control surfaces directly conjugated with peptides. The engineering approaches presented herein can be applied individually or in combination, providing guidelines for the effective utilization of biomolecular multivalent interactions using DPC-based HMAs.

Machine-Learning-Based Clinical Biomarker Using Cell-Free DNA for Hepatocellular Carcinoma (HCC).

(1) Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Although various serum enzymes have been utilized for the diagnosis and prognosis of HCC, the currently available biomarkers lack the sensitivity needed to detect HCC at early stages and accurately predict treatment responses. (2) Methods: We utilized our highly sensitive cell-free DNA (cfDNA) detection system, in combination with a machine learning algorithm, to provide a platform for improved diagnosis and prognosis of HCC. (3) Results: cfDNA, specifically alpha-fetoprotein (AFP) expression in captured cfDNA, demonstrated the highest accuracy for diagnosing malignancies among the serum/plasma biomarkers used in this study, including AFP, aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase, and bilirubin. The diagnostic/prognostic capability of cfDNA was further improved by establishing a cfDNA score (cfDHCC), which integrated the total plasma cfDNA levels and cfAFP-DNA expression into a single score using machine learning algorithms. (4) Conclusion: The cfDHCC score demonstrated significantly improved accuracy in determining the pathological features of HCC and predicting patients' survival outcomes compared to the other biomarkers. The results presented herein reveal that our cfDNA capture/analysis platform is a promising approach to effectively utilize cfDNA as a biomarker for the diagnosis and prognosis of HCC.

Bimodal liquid biopsy for cancer immunotherapy based on peptide engineering and nanoscale analysis.

Despite its high potential, PD-L1 expressed by tumors has not been successfully utilized as a biomarker for estimating treatment responses to immunotherapy. Circulating tumor cells (CTCs) and tumor-derived exosomes that express PD-L1 can potentially be used as biomarkers; however, currently available assays lack clinically significant sensitivity and specificity. Here, a novel peptide-based capture surface is developed to effectively isolate PD-L1-expressing CTCs and exosomes from human blood. For the effective targeting of PD-L1, this study integrates peptide engineering strategies to enhance the binding strength and specificity of a ß-hairpin peptide derived from PD-1 (pPD-1). Specifically, this study examines the effect of poly(ethylene glycol) spacers, the secondary peptide structure, and modification of peptide sequences (e.g., removal of biologically redundant amino acid residues) on capture efficiency. The optimized pPD-1 configuration captures PD-L1-expressing tumor cells and tumor-derived exosomes with 1.5-fold (p = 0.016) and 1.2-fold (p = 0.037) higher efficiencies, respectively, than their whole antibody counterpart (aPD-L1). This enhanced efficiency is translated into more clinically significant detection of CTCs (1.9-fold increase; p = 0.035) and exosomes (1.5-fold increase; p = 0.047) from patients' baseline samples, demonstrating stronger correlation with patients' treatment responses. Additionally, we confirmed that the clinical accuracy of our system can be further improved by co-analyzing the two biomarkers (bimodal CTC/exosome analysis). These data demonstrate that pPD-1-based capture is a promising approach for capturing PD-L1-expressing CTCs and exosomes, which can be used as a reliable biomarker for cancer immunotherapy.

Chemically and Biologically Engineered Bacteria-Based Delivery Systems for Emerging Diagnosis and Advanced Therapy.

Bacteria are one of the main groups of organisms, which dynamically and closely participate in human health and disease development. With the integration of chemical biotechnology, bacteria have been utilized as an emerging delivery system for various biomedical applications. Given the unique features of bacteria such as their intrinsic biocompatibility and motility, bacteria-based delivery systems have drawn wide interest in the diagnosis and treatment of various diseases, including cancer, infectious diseases, kidney failure, and hyperammonemia. Notably, at the interface of chemical biotechnology and bacteria, many research opportunities have been initiated, opening a promising frontier in biomedical application. Herein, the current synergy of chemical biotechnology and bacteria, the design principles for bacteria-based delivery systems, the microbial modulation, and the clinical translation are reviewed, with a special focus on the emerging advances in diagnosis and therapy.

Cytochalasin B Treatment and Osmotic Pressure Enhance the Production of Extracellular Vesicles (EVs) with Improved Drug Loading Capacity.

Extracellular vesicles (EVs) have been highlighted as novel drug carriers due to their unique structural properties and intrinsic features, including high stability, biocompatibility, and cell-targeting properties. Although many efforts have been made to harness these features to develop a clinically effective EV-based therapeutic system, the clinical translation of EV-based nano-drugs is hindered by their low yield and loading capacity. Herein, we present an engineering strategy that enables upscaled EV production with increased loading capacity through the secretion of EVs from cells via cytochalasin-B (CB) treatment and reduction of EV intravesicular contents through hypo-osmotic stimulation. CB (10 µg/mL) promotes cells to extrude EVs, producing ~three-fold more particles than through natural EV secretion. When CB is induced in hypotonic conditions (223 mOsm/kg), the produced EVs (hypo-CIMVs) exhibit ~68% less intravesicular protein, giving 3.4-fold enhanced drug loading capacity compared to naturally secreted EVs. By loading doxorubicin (DOX) into hypo-CIMVs, we found that hypo-CIMVs efficiently deliver their drug cargos to their target and induce up to ~1.5-fold more cell death than the free DOX. Thus, our EV engineering offers the potential for leveraging EVs as an effective drug delivery vehicle for cancer treatment.

Enhanced detection of cell-free DNA (cfDNA) enables its use as a reliable biomarker for diagnosis and prognosis of gastric cancer.

Although circulating cell-free DNA (cfDNA) is a promising biomarker for the diagnosis and prognosis of various tumors, clinical correlation of cfDNA with gastric cancer has not been fully understood. To address this, we developed a highly sensitive cfDNA capture system by integrating polydopamine (PDA) and silica. PDA-silica hybrids incorporated different molecular interactions to a single system, enhancing cfDNA capture by 1.34-fold compared to the conventional silica-based approach (p = 0.001), which was confirmed using cell culture supernatants. A clinical study using human plasma samples revealed that the diagnostic accuracy of the new system to be superior than the commercially available cfDNA kit, as well as other serum antigen tests. Among the cancer patients, plasma cfDNA levels exhibited a good correlation with the size of a tumor. cfDNA was also predicative of distant metastasis, as the median cfDNA levels of metastatic cancer patients were ~60-fold higher than those without metastasis (p = 0.008). Furthermore, high concordance between tissue biopsy and cfDNA genomic analysis was found, as HER2 expression in cfDNA demonstrated an area under ROC curve (AUC) of 0.976 (p <0.001) for detecting patients with HER2-positive tumors. The new system also revealed high prognostic capability of cfDNA, as the concentration of cfDNA was highly associated with the survival outcomes. Our novel technology demonstrates the potential to achieve efficient detection of cfDNA that may serve as a reliable biomarker for gastric tumor.

Surface engineering for efficient capture of circulating tumor cells in renal cell carcinoma: From nanoscale analysis to clinical application.

Sensitive detection of circulating tumor cells (CTCs) from patients' peripheral blood facilitates on-demand monitoring of tumor progression. However, clinically significant capture of renal cell carcinoma CTCs (RCC-CTCs) remains elusive due to their heterogenous surface receptor expression. Herein, a novel capture platform is developed to detect RCC-CTCs through integration of dendrimer-mediated multivalent binding, a mixture of antibodies, and biomimetic cell rolling. The nanoscale binding kinetics measured using atomic force microscopy reveal that dendrimer-coated surfaces exhibit an order of magnitude enhancement in off-rate kinetics compared to surface without dendrimers, which translated into cell capture improvements by ~60%. Selectin-induced cell rolling facilitates surface recruitment of cancer cells, further improving cancer cell capture by up to 1.7-fold. Lastly, an antibody cocktail targeting four RCC-CTC surface receptors, which included epithelial cell adhesion molecule (EpCAM), carbonic anhydrase IX (CA9), epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (c-Met), improves the capture of RCC cells by up to 80%. The optimal surface configuration outperforms the conventional assay solely relying on EpCAM, as demonstrated by detecting significantly more CTCs in patients' samples (9.8 ± 5.1 vs. 1.8 ± 2.0 CTCs mL-1). These results demonstrate that the newly engineered capture platform effectively detects RCC-CTCs for their potential use as tumor biomarkers.

Multi-modal liquid biopsy platform for cancer screening: screening both cancer-associated rare cells and cancer cell-derived vesicles on the fabric filters for a reliable liquid biopsy analysis.

Circulating tumor cells (CTCs) are receiving a great amount of scientific interest as a diagnostic biomarker for various types of cancer. Despite the recent progress in the development of highly sensitive CTC isolation devices, post-capture analysis of CTCs is still hindered by technical challenges associated with their rarity. Herein, we present a multi-modal CTC screening platform which is capable to analyze CTCs and CTC-derived extracellular vesicles (EVs), simultaneously from a single sample. Cytochalasin B (CB) treatment promotes cells to release large number of EVs from their surface, as demonstrated by CB-treated cells (5 µg/mL for 3 h) secreting 3.5-fold more EVs, compared to the non-treated cells. CB further generates 1.7-fold more EVs from the cells captured on our CTC filtration device (the fabric filter), compared to those from the cell culture flasks, owing to its multiple pore structure design which reduces the non-specific binding of EVs. Both CB-treated cancer cells and CB-induced EVs are found to overexpress tumor-associated markers, demonstrating a potential for the development of CTC dual-screening platform. Collectively, the results presented in this study reveal that our multi-modal cancer screening platform can synergistically improve the reliability and efficacy of the current CTC analysis systems.

Post-debulking circulating tumor cell as a poor prognostic marker in advanced stage ovarian cancer: A prospective observational study.

Circulating tumor cells (CTCs) have received enormous attention as a novel biomarker in various malignant diseases. We investigated the clinical association between the presence of perioperative CTCs and survival outcomes in women with ovarian cancer. In a total of 30 women who were scheduled to undergo a surgical treatment for ovarian cancer, peripheral blood samples were obtained before and after surgery. CTCs were isolated and counted using the optimized tapered-slit filter (TSF) platform. The association between the presence of perioperative CTCs and tumor features was evaluated. The impact of the presence of perioperative CTCs on progression-free survival (PFS) and overall survival (OS) rates were analyzed using a Kaplan-Meier method. The median age was 58 (range, 24-77) years, and the median follow-up period was 31.5 (range, 1-41) months. Overall, the CTC detection rate was not significantly different before and after surgery (76.7% vs 57.1%, P = .673). The presence of postoperative CTCs was not significantly associated with 3-year PFS (29.1% vs 58.3%, P = .130) and OS (84.4% vs 80.0%, P = .559) rates in the whole study population. In advanced stage, PFS rate in patients with postoperative CTCs had lower PFS rates than those without postoperative CTCs, although there was no statistical significance (18.8% vs 57.1%, P = .077). Postoperative CTC was more frequently detected in women who had lymph node involvement than those who did not (7/7 [100%] vs 3/10 [30.0%], P = .010). The presence of postoperative CTCs as detected using the TSF platform seems to be associated with poorer PFS rates in women with ovarian cancer of advanced stage. Further study with a larger population is warranted to validate our study findings.