Combined Molecular and Morphological Imaging of CTCs for HER2-Targeted Chemotherapy Guidance.

Since biomolecules change dynamically with tumor evolution and drug treatment, it is necessary to confirm target molecule expression in real time for effective guidance of subsequent chemotherapy treatment. However, current methods to confirm target proteins require complex processing steps and invasive tissue biopsies, limiting their clinical utility for targeted treatment monitoring. Here, CTCs, as a promising liquid biopsy source, were used to molecularly characterize the target protein HER2. To accurately identify CTCs, we specifically proposed a combined molecular and morphological imaging method, rather than using specific biomarker alone or morphology analysis, we identified CTCs as CK19+/CD45-/HE+. On the basis of the accurate identification of CTCs, we further analyzed the target protein HER2 in clinical patients at the single-CTC level. Comparative analysis of the clinical results of patient pathological tissue and paired blood samples showed that CTCs had a heterogeneous HER2 expression at the single-cell level and showed results inconsistent with the immunohistochemistry results in some cases. CTC-based analysis could help clinicians have a more comprehensive understanding of patient target protein expression. We believe that CTC-based target protein studies are of great significance for the precise management of targeted therapy.

Separation and single-cell analysis for free gastric cancer cells in ascites and peritoneal lavages based on microfluidic chips.

BACKGROUNDS: Detecting free cancer cells from ascites and peritoneal lavages is crucial for diagnosing gastric cancer (GC). However, traditional methods are limited for early-stage diagnosis due to their low sensitivity. METHODS: A label-free, rapid, and high-throughput technique was developed for separating cancer cells from ascites and peritoneal lavages using an integrated microfluidic device, taking advantage of dean flow fractionation and deterministic lateral displacement. Afterward, separated cells were analyzed using a microfluidic single-cell trapping array chip (SCTA-chip). In situ immunofluorescence for EpCAM, YAP-1, HER-2, CD45 molecular expressions, and Wright-Giemsa staining were performed for cells in SCTA-chips. At last, YAP1 and HER-2 expression in tissues was analyzed by immunohistochemistry. FINDINGS: Through integrated microfluidic device, cancer cells were successfully separated from simulated peritoneal lavages containing 1/10,000 cancer cells with recovery rate of 84.8% and purity of 72.4%. Afterward, cancer cells were isolated from 12 patients' ascites samples. Cytological examinations showed cancer cells were efficiently enriched with background cells excluded. Afterwards, separated cells from ascites were analyzed by SCTA-chips, and recognized as cancer cells through EpCAM+/CD45- expression and Wright-Giemsa staining. Interestingly, 8 out of 12 ascites samples showed HER-2+ cancer cells. At last, the results through a serial expression analysis showed that YAP1 and HER-2 have discordant expression during metastasis. INTERPRETATION: Microfluidic Chips developed in our study could not only rapidly detect label-free free GC cells in ascites and peritoneal lavages with high-throughput, they could also analyze ascites cancer cells at the single-cell level, improving peritoneal metastasis diagnosis and investigation of therapeutic targets. FUNDING: This research was supported by National Natural Science Foundation of China (22134004, U1908207, 91859111); Natural Science Foundation of Shandong Province of China (ZR2019JQ06); Taishan Scholars Program of Shandong Province tsqn (201909077); Local Science and Technology Development Fund Guided by the Central Government (YDZX20203700002568); Applied Basic Research Program of Liaoning Province (2022020284-JH2/1013).

Recent advances in microfluidic technologies for circulating tumor cells: enrichment, single-cell analysis, and liquid biopsy for clinical applications.

Circulating tumor cells (CTCs) detach from primary or metastatic lesions and circulate in the peripheral blood, which is considered to be the cause of distant metastases. CTC analysis in the form of liquid biopsy, enumeration and molecular analysis provide significant clinical information for cancer diagnosis, prognosis and therapeutic strategies. Despite the great clinical value, CTC analysis has not yet entered routine clinical practice due to lack of efficient technologies to perform CTC isolation and single-cell analysis. Taking the rarity and inherent heterogeneity of CTCs into account, reliable methods for CTC isolation and detection are in urgent demand for obtaining valuable information on cancer metastasis and progression from CTCs. Microfluidic technology, featuring microfabricated structures, can precisely control fluids and cells at the micrometer scale, thus making itself a particularly suitable method for rare CTC manipulation. Besides the enrichment function, microfluidic chips can also realize the analysis function by integrating multiple detection technologies. In this review, we have summarized the recent progress in CTC isolation and detection using microfluidic technologies, with special attention to emerging direct enrichment and enumeration in vivo. Further, few insights into single CTC molecular analysis are also demonstrated. We have provided a review of potential clinical applications of CTCs, ranging from early screening and diagnosis, tumor progression and prognosis, treatment and resistance monitoring, to therapeutic evaluation. Through this review, we conclude that the clinical utility of CTCs will be expanded as the isolation and analysis techniques are constantly improving.

Phenotype-related drug sensitivity analysis of single CTCs for medicine evaluation.

Due to the heterogeneous and variable drug sensitivity of tumor cells, real-time monitoring of a patient's drug response is desirable for implementing personalized and dynamic therapy. Although considerable efforts have been directed at drug screening in living cells, performing repeated drug sensitivity analysis using patient-derived primary tumor cells at the single-cell level remains challenging. Here, we present an efficient approach to assess phenotype-related drug sensitivity at the single-cell level using patient-derived circulating tumor cells (CTCs) based on a drug sensitivity microfluidic chip (DS-Chip). The DS-Chip consists of a drug gradient generator and parallel cell traps, achieving continuous single CTC capture, drug gradient distributions, drug stimulation, fluorescent probe labeling and three-color fluorescence imaging. Based on the established DS-Chip, we investigated the drug sensitivity of single cells by simultaneously monitoring epithelial-mesenchymal transition (EMT) biomarkers and apoptosis in living cells, and verified the correlation between EMT gradients and drug sensitivity. Using the new approach, we further tested the optimal drug response dose in individual CTCs isolated from 5 cancer patients through fluorescence analysis of EMT and apoptosis. The DS-Chip allows noninvasive and real-time measurements of the drug sensitivity of a patient's tumor cells during therapy. This developed approach has practical significance and can effectively guide drug selection and therapeutic evaluation for personalized medicine.

Single-Cell Phenotypic Profiling of CTCs in Whole Blood Using an Integrated Microfluidic Device.

Single-cell phenotypic profiling of circulating tumor cells (CTCs) in the blood of cancer patients can reveal vital tumor biology information. Even though various approaches have been provided to enrich and detect CTCs, it remains challenging for consecutive CTC sorting, enumeration, and single-cell characterizations. Here, we report an integrated microfluidic device (IMD) for single-cell phenotypic profiling of CTCs that enables automated CTCs sorting from whole blood following continuous single-cell phenotypic analysis while satisfying the requirements of both high purity (92 ± 3%) of cell sorting and high-throughput processing capacity (5 mL whole blood/3 h). Using this new technique we test the phenotypes of individual CTCs collected from xenograft tumor-bearing mice and colorectal (CRC) patients at different tumor stages. We obtained a correlation between CTC characterization and clinical tumor stage and treatment response. The developed IMD offers a high-throughput, convenient, and rapid strategy to study individual CTCs toward minimally invasive cancer therapy prediction and disease monitoring and has the potential to be translated to clinic for liquid biopsy.

Niche nanoparticle-based FRET assay for bleomycin detection via DNA scission.

We describe a highly sensitive nanoparticle-based fluorescence resonance energy transfer (FRET) probe developed without using molecular fluorophores as donors and acceptors. The success of this work relies on the strategy that DNA scission was designed to occur to the probe when target presented, which enabled the fluorescence signal "turn-on" of graphene quantum dots (GQDs) and thus quantitative analysis. In particular, amino-modified SiO2 NPs were initially coated by GQDs to form highly emitting SiO2/GQDs, followed by conjunction with DNA functionalized gold nanoparticles (Au NPs-DNA) to form SiO2/GQDs-DNA-Au NPs composite. Owing to the FRET interactions between the GQDs and Au NPs, the fluorescence of GQDs was effectively quenched by Au NPs. When bleomycin (BLM), a model analyte, was mixed with the probe, the fluorescence signal of GQDs would be restored due to the removal of Au NPs from the SiO2/GQDs surface by DNA scission treatment with BLM in the presence of Fe (II). The current FRET probe shows a good linear relationship between the fluorescence intensity and the concentration of BLM in the range from 0.5nM to 1µM with a detection limit of 0.2nM. The probe also shows satisfactory results for the analysis of clinical serum samples. This method provides versatility to the application of GQDs in FRET biosensing and could be potentially extended to other similar systems by replacing the linker between the GQDs and Au NPs.

Graphene oxide quantum dots@silver core-shell nanocrystals as turn-on fluorescent nanoprobe for ultrasensitive detection of prostate specific antigen.

We report a fluorescent turn-on nanoprobe for ultrasensitive detection of prostate specific antigen (PSA) based on graphene oxide quantum dots@silver (GQDs@Ag) core-shell nanocrystals. The success of this work relies on the assembly of quantities of GQDs in one GQDs@Ag probe, which makes the ratio of probe to target significantly increased and thus enables the fluorescent signal enhancement. When the silver shell was removed via oxidative etching using hydrogen peroxide (H2O2), the incorporated GQDs could be readily released and the whole process caused little change to their fluorescence performance. We tested the probe for the ultrasensitive detection of PSA based on the sandwich protocol of immunosensors. In particular, magnetic beads (MBs) were employed to immobilize anti-PSA antibody (Ab1) and acted as a separable capture probe, while GQDs@Ag was used as detection probe by linking antibody (Ab2). The developed immunosensor showed a good linear relationship between the fluorescence intensity and the concentration of PSA in the range from 1 pg/mL to 20 ng/mL with a detection limit of 0.3 pg/mL. The immunosensor used for the analysis of clinical serum samples exhibited satisfactory results, which demonstrated its potential for practical diagnostic applications. This method provides a possible solution to the application of GQDs in immunosensing and could be potentially extended to other similar systems.