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Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis. Transcriptome analysis of circulating tumor cells (CTCs) provides insights into monitoring target therapeutics and underlying tumor metastasis. Here the authors present Hydro-Seq, a contamination-free high-throughput hydrodynamic scRNA-seq barcoding technique for rare CTCs.

Circulating tumor cells (CTCs) have become an established biomarker for prognosis in patients with various carcinomas. However, current ex vivo CTC isolation technologies rely on small blood volumes from a single venipuncture limiting the number of captured CTCs. This produces statistical variability and inaccurate reflection of tumor cell heterogeneity. Here, we describe an in vivo indwelling intravascular aphaeretic CTC isolation system to continuously collect CTCs directly from a peripheral vein. The system returns the remaining blood products after CTC enrichment, permitting interrogation of larger blood volumes than classic phlebotomy specimens over a prolonged period of time. The system is validated in canine models showing capability to screen 1–2% of the entire blood over 2 h. Our result shows substantial increase in CTC capture, compared with serial blood draws. This technology could potentially be used to analyze large number of CTCs to facilitate translation of analytical information into future clinical decisions. Ex vivo methods of circulating tumor cell (CTC) isolation use small blood volumes, limiting sensitivity and introducing analytical inaccuracies. The authors describe a proof-of-concept study of an in vivo aphaeresis system that continuously collects CTCs from a peripheral vein over several hours.

Preclinical studies demonstrated that radiation up-regulates PD-L1 expression in tumor cells, providing a rationale for combining PD-1/PD-L1 inhibitors with radiation. However this has not been validated in patients with non-small cell lung cancer due to the difficulty to obtain serial biopsies. Measuring PD-L1 expression in circulating tumor cells (CTCs), may allow real-time monitoring of immune activation in tumor. In this study, whole blood from non-metastatic NSCLC patients was collected before, during, and after radiation or chemoradiation using a microfluidic chip. PD-L1 expression in CTCs was assessed by immunofluorescence and qPCR and monitored through the course of treatment. Overall, PD-L1(+) CTCs were detected in 25 out of 38 samples (69.4%) with an average of 4.5 cells/ml. After initiation of radiation therapy, the proportion of PD-L1(+) CTCs increased significantly (median 0.7% vs. 24.7%, P < 0.01), indicating up-regulation of PD-L1 in tumor cells in response to radiation. In addition, patients positive for PD-L1 (≥5% of CTCs positive for PD-L1) at baseline had shorter PFS. Gene expression analysis revealed that higher levels of PD-L1 were associated with poor prognosis. Therefore, CTCs can be used to monitor dynamic changes of PD-L1 during radiation therapy which is potentially prognostic of response to treatment.

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or “HB-Chip,” which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

As the recognition between natural killer (NK) cells and cancer cells does not require antigen presentation, NK cells are being actively studied for use in adoptive cell therapies in the rapidly evolving armamentarium of cancer immunotherapy. In addition to utilizing NK cells, recent studies have shown that exosomes derived from NK cells also exhibit antitumor properties. Furthermore, these NK cell‐derived exosomes exhibit higher stability, greater modification potentials and less immunogenicity compared to NK cells. Therefore, technologies that allow highly sensitive and specific isolation of NK cells and NK cell‐derived exosomes can enable personalized NK‐mediated cancer therapeutics in the future. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes is proposed. In a small cohort of non‐small cell lung cancer (NSCLC) patients, both NK cells and circulating tumor cells (CTCs) were isolated, and it is found NSCLC patients have high numbers of NK and NK‐exosomes compared with healthy donors, and these concentrations show a trend of positive and negative correlations with bloodborne CTC numbers, respectively. It is further demonstrated that the NK‐exosomes harvested from NK‐graphene oxide chip exhibit cytotoxic effect on CTCs. This versatile system is expected to be used for patient‐specific NK‐based immunotherapies along with CTCs for potential prognostic/diagnostic applications. Natural killer (NK) cells are lymphocytes that have the ability to destroy cancer cells without antigen presentation. The NK cell‐derived exosomes exhibit higher stability, greater modification potentials, and less immunogenicity compared to NK cells. Here, a novel microfluidic system to collect patient‐specific NK cells and on‐chip biogenesis of NK‐exosomes for diagnostic and therapeutic use of NK‐exosomes is proposed.

Melanoma is among the most aggressive cancers, and its rate of incidence continues to grow. Early detection of melanoma has been hampered due to the lack of promising markers for testing. Recent advances in liquid biopsy have proposed noninvasive alternatives for cancer diagnosis and monitoring. Circulating tumor cells (CTCs) and cancer‐exosomes are gaining influence as promising biomarkers because of their cancer‐associated molecular markers and signatures. However, technologies that offer the dual‐isolation of CTCs and exosomes using a single sample have not been thoroughly developed. The dual‐utilization OncoBean (DUO) device is conjugated with melanoma specific antibodies, MCAM and MCSP, enabling simultaneous CTC and exosome isolations. Using blood samples from patients, CTCs and exosomes are specifically isolated from a single sample and then undergo molecular profiling for comprehensive study. Melanoma patients have 0–17CTCs mL−1 and 299 µg exosomal protein mL−1 while healthy donors display fewer than 2CTCs and 75.6 µg of exosomes mL−1, respectively. It is also demonstrated that both markers express melanoma‐associated genes using multiplex qRT‐PCR to test for expression pattern of a 96 gene panel. The dual isolation and molecular characterization will allow for further research into melanoma to identify viable markers for disease progression and treatment efficacy. Circulating tumor cells (CTCs) and cancer exosomes are gaining influence as promising biomarkers in liquid biopsy, however, technologies that offer the dual‐isolation of CTCs and exosomes have not been developed. Here, a microfluidic device is devised conjugated with melanoma cell adhesion molecule (MCAM) and melanoma‐associated chondroitin sulfate proteoglycan (MCSP) for isolation and molecular profiling of both melanoma CTCs and exosomes.

Liquid biopsies hold promise to improve the diagnosis, assessment of response to therapy, and ultimately guide the management of cancer patients. However, implementation of this approach in brain tumors has proven challenging due to the limited passage of molecules across the blood-brain barrier (BBB). We recently reported results from a phase I clinical trial in which the BBB was transiently opened in glioblastoma (GBM) patients using skull-implantable low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB). In this study, treatment and BBB opening was performed every 3 weeks with paclitaxel administration until disease progression or up to 6 cycles (NCT04528680). As an exploratory objective of this trial, here we investigate extracellular vesicles and particles (EVPs/EPs) released into circulation in the context of tumor cell death as a potential biomarker for response to treatment. We develop and validate a microfluidic device designed to capture tumor-derived EVPs in glioblastoma patients ( Glio ExoChip). This approach leverages GBM-based expression of phosphatidylserine and Annexin-V chemistry that is traditionally used to measure apoptosis. EVPs are characterized using nanoparticle tracking analysis, proteomics, western blot, and scanning electron microscopy. Proteomic analysis of circulating EVPs isolated from GBM patients reveals distinct expression patterns to that of healthy individuals, and scRNA-seq analysis of these genes supported their tumoral origin within the GBM microenvironment. In vitro, paclitaxel-susceptible glioma cells treated with this drug exhibit apoptosis and dose-dependent EVP release. In concordance, we find changes in EVP release following the initiation of paclitaxel with LIPU/MB correlated with overall survival in GBM patients. Thus, our study introduces an efficient microfluidic platform for the capture of circulating GBM EVPs and demonstrates that release upon BBB opening is predictive of outcomes following paclitaxel treatment. This approach represents a real-time surrogate biomarker for treatment response for a disease where imaging-based assessment of response has not been shown to be reliable. Future prospective validation is warranted. Recently published results from a Phase I trial showed the blood brain barrier could be transiently opened in glioblastoma patients using low-intensity ultrasound and microbubbles. Here, the authors develop a microfluidic chip to capture tumour-derived extracellular vesicles and particles in response to paclitaxel treatment.

(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45−) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 ± 1023 CTCs/mL). EpCAM− CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients—of which, 75% were EpCAM−. CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1, and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM−, suggesting that these would have been missed using traditional antibody-based capture methods.

Amyotrophic lateral sclerosis (ALS) is a terminalneurodegenerative disease. Clinical and molecular observations suggest that ALS pathology originates at a single site and spreads in an organized and prion-like manner, possibly driven by extracellular vesicles. Extracellular vesicles (EVs) transfer cargo molecules associated with ALS pathogenesis, such as misfolded and aggregated proteins and dysregulated microRNAs (miRNAs). However, it is poorly understood whether altered levels of circulating extracellular vesicles or their cargo components reflect pathological signatures of the disease. In this study, we used immuno-affinity-based microfluidic technology, electron microscopy, and NanoString miRNA profiling to isolate and characterize extracellular vesicles and their miRNA cargo from frontal cortex, spinal cord, and serum of sporadic ALS ( n = 15) and healthy control ( n = 16) participants. We found larger extracellular vesicles in ALS spinal cord versus controls and smaller sized vesicles in ALS serum. However, there were no changes in the number of extracellular vesicles between cases and controls across any tissues. Characterization of extracellular vesicle-derived miRNA cargo in ALS compared to controls identified significantly altered miRNA levels in all tissues; miRNAs were reduced in ALS frontal cortex and spinal cord and increased in serum. Two miRNAs were dysregulated in all three tissues: miR-342-3p was increased in ALS, and miR-1254 was reduced in ALS. Additional miRNAs overlapping across two tissues included miR-587, miR-298, miR-4443, and miR-450a-2-3p. Predicted targets and pathways associated with the dysregulated miRNAs across the ALS tissues were associated with common biological pathways altered in neurodegeneration, including axon guidance and long-term potentiation. A predicted target of one identified miRNA (N-deacetylase and N-sulfotransferase 4; NDST4) was likewise dysregulated in an in vitro model of ALS, verifying potential biological relevance. Together, these findings demonstrate that circulating extracellular vesicle miRNA cargo mirror those of the central nervous system disease state in ALS, and thereby offer insight into possible pathogenic factors and diagnostic opportunities.

Recurrent loss-of-function deletions cause frequent inactivation of tumour suppressor genes but often also involve the collateral deletion of essential genes in chromosomal proximity, engendering dependence on paralogues that maintain similar function. Although these paralogues are attractive anticancer targets, no methodology exists to uncover such collateral lethal genes. Here we report a framework for collateral lethal gene identification via metabolic fluxes, CLIM, and use it to reveal MTHFD2 as a collateral lethal gene in UQCR11 -deleted ovarian tumours. We show that MTHFD2 has a non-canonical oxidative function to provide mitochondrial NAD + , and demonstrate the regulation of systemic metabolic activity by the paralogue metabolic pathway maintaining metabolic flux compensation. This UQCR11–MTHFD2 collateral lethality is confirmed in vivo, with MTHFD2 inhibition leading to complete remission of UQCR11 -deleted ovarian tumours. Using CLIM’s machine learning and genome-scale metabolic flux analysis, we elucidate the broad efficacy of targeting MTHFD2 despite distinct cancer genetic profiles co-occurring with UQCR11 deletion and irrespective of stromal compositions of tumours. Achreja et al. develop a framework to identify collateral lethalities in cancer, uncovering MTHFD2 as a collateral lethal gene in UQCR11 -deficient ovarian tumours.