Tracking Drug‐Induced Epithelial–Mesenchymal Transition in Breast Cancer by a Microfluidic Surface‐Enhanced Raman Spectroscopy Immunoassay

Epithelial–mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E‐cadherin and N‐cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed “SERS immunoassay”) based on sensitive and simultaneous detection of soluble E‐cadherin (sE‐cadherin) and soluble N‐cadherin (sN‐cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface‐enhanced Raman scattering readout to enable accurate detection of sE‐cadherin and sN‐cadherin from as low as 10 cells mL^?1. This assay enables tracking of a concurrent decrease in sE‐cadherin and increase in sN‐cadherin in breast cancer cells undergoing drug‐induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE‐cadherin and sN‐cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.     

The Architecture and Function of Monoclonal Antibody‐Functionalized Mesoporous Silica Nanoparticles Loaded with Mifepristone: Repurposing Abortifacient for Cancer Metastatic Chemoprevention

The circulating tumor cells (CTCs) existing in cancer survivors are considered the root cause of cancer metastasis. To prevent the devastating metastasis cascade from initiation, we hypothesize that a biodegradable nanomaterial loaded with the abortifacient mifepristone (MIF) and conjugated with the epithelial cell adhesion molecule antibody (aEpCAM) may serve as a safe and effective cancer metastatic preventive agent by targeting CTCs and preventing their adhesion‐invasion to vascular intima. It is demonstrated that MIF‐loaded mesoporous silica nanoparticles (MSN) coated with aEpCAM (aE‐MSN‐M) can specifically target and bind colorectal cancer cells in either cell medium or blood through EpCAM recognition proven by quantitative flow cytometric detection and free aEpCAM competitive assay. The specific binding results in downregulation of the captured cells and drives them into G0/G1 phase primarily attributed to the effect of aEpCAM. The functional nanoparticles significantly inhibit the heteroadhesion between cancer cells and endothelial cells, suggesting the combined inhibition effects of aEpCAM and MIF on E‐selectin and ICAM‐1 expression. The functionalized nanoparticles circulate in mouse blood long enough to deliver MIF and inhibit lung metastasis. The present proof‐of‐concept study shows that the aE‐MSN‐M can prevent cancer metastasis by restraining CTC activity and their adhesion‐invasion to vascular intima.     

Whole Genome Sequencing of Single Circulating Tumor Cells Isolated by Applying a Pulsed Laser to Cell‐Capturing Microstructures

Single cell analysis of heterogeneous circulating tumor cells (CTCs), by which the genomic profiles of rare single CTCs are connected to the clinical status of cancer patients, is crucial for understanding cancer metastasis and the clinical impact on patients. However, the heterogeneity in genotypes and phenotypes and rarity of CTCs have limited extensive single CTC genome research, further hindering clinical investigation. Despite recent efforts to build platforms that separate CTCs, the investigation on CTCs is difficult due to the lack of a retrieval process at the single cell level. In this study, laser‐induced isolation of microstructures on an optomechanically‐transferrable‐chip and sequencing (LIMO‐seq) is applied for whole genome sequencing of single CTCs. Also, the whole genome sequences and the molecular profiles of the isolated single cells from the whole blood of a breast cancer patient are analyzed.     

Reversible Cavitation‐Induced Junctional Opening in an Artificial Endothelial Layer

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation‐assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel‐on‐a‐chip is exploited to investigate and quantify the effect of ultrasound‐excited microbubbles—stable cavitation—on endothelial integrity. In the vessel‐on‐a‐chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation‐enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.     

Leukocyte‐Repelling Biomimetic Immunomagnetic Nanoplatform for High‐Performance Circulating Tumor Cells Isolation

Downstream studies of circulating tumor cells (CTCs), which may provide indicative evaluation information for therapeutic efficacy, cancer metastases, and cancer prognosis, are seriously hindered by the poor purity of enriched CTCs as large amounts of interfering leukocytes still nonspecifically bind to the isolation platform. In this work, biomimetic immunomagnetic nanoparticles (BIMNs) with the following features are designed: i) the leukocyte membrane camouflage, which could greatly reduce homologous leukocyte interaction and actualize high‐purity CTCs isolation, is easily extracted by graphene nanosheets; ii) facile antibody conjugation can be achieved through the “insertion” of biotinylated lipid molecules into leukocyte‐membrane‐coated nanoparticles and streptavidin conjunction; iii) layer‐by‐layer assembly techniques could integrate high‐magnetization Fe₃O₄ nanoparticles and graphene nanosheets efficiently. Consequently, the resulting BIMNs achieve a capture efficiency above 85.0% and CTCs purity higher than 94.4% from 1 mL blood with 20–200 CTCs after 2 min incubation. Besides, 98.0% of the isolated CTCs remain viable and can be directly cultured in vitro. Moreover, application of the BIMNs to cancer patients' peripheral blood shows good reproducibility (mean relative standard deviation 8.7 ± 5.6%). All results above suggest that the novel biomimetic nanoplatform may serve as a promising tool for CTCs enrichment and detection from clinical samples.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Topography‐Induced Cell Self‐Organization from Simple to Complex Aggregates

Self‐organization is a fundamental and indispensable process in a living system. To understand cell behavior in vivo such as tumorigenesis, 3D cellular aggregates, instead of 2D cellular sheets, have been employed as a vivid in vitro model for self‐organization. However, most focus on the macroscale wetting and fusion of cellular aggregates. In this study, it is reported that self‐organization of cells from simple to complex aggregates can be induced by multiscale topography through confined templates at the macroscale and cell interactions at the nanoscale. On the one hand, macroscale templates are beneficial for the organization of individual cells into simple and complex cellular aggregates with various shapes. On the other hand, the realization of these macro‐organizations also depends on cell interactions at the nanoscale, as demonstrated by the intimate contact between nanoscale pseudopodia stretched by adjacent frontier cells, much like holding hands and by the variation in the intermolecular interactions based on E‐cadherin. Therefore, these findings may be very meaningful for clarifying the organizational mechanism of tumor development, tissue engineering and regenerative medicine.     

Nanotentacle‐Structured Magnetic Particles for Efficient Capture of Circulating Tumor Cells

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture efficiency and purity still remain challenges that limit clinical practice. Here, the construction of “nanotentacle”‐structured magnetic particles using M13‐bacteriophage and their application for the efficient capturing of CTCs is demonstrated. The M13‐bacteriophage to magnetic particles followed by modification with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle‐structured magnetic particles results in a high capture purity (>45%) and efficiency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high efficiency and purity for the diagnosis and monitoring of cancer status.     

Continuous Flow Deformability‐Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label‐free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label‐free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10⁴‐fold enrichment of target cells relative to leukocytes. In patients with metastatic castration‐resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.     

Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip

Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface‐enhanced Raman scattering (SERS)‐coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report‐molecule 4‐aminothiophenol through introducing the SERS‐coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs.     

Visual Quantitative Detection of Circulating Tumor Cells with Single‐Cell Sensitivity Using a Portable Microfluidic Device

Immunocytological technologies, molecular technologies, and functional assays are widely used for detecting circulating tumor cells (CTCs) after enrichment from patients' blood sample. Unfortunately, accessibility to these technologies is limited due to the need for sophisticated instrumentation and skilled operators. Portable microfluidic devices have become attractive tools for expanding the access and efficiency of detection beyond hospitals to sites near the patient. Herein, a volumetric bar chart chip (V‐Chip) is developed as a portable platform for CTC detection. The target CTCs are labeled with aptamer‐conjugated nanoparticles (ACNPs) and analyzed by V‐Chip through quantifying the byproduct (oxygen) of the catalytic reaction between ACNPs and hydrogen peroxide, which results in the movement of an ink bar to a concentration‐dependent distance for visual quantitative readout. Thus, the CTC number is decoded into visually quantifiable information and a linear correlation can be found between the distance moved by the ink and number of cells in the sample. This method is sensitive enough that a single cell can be detected. Furthermore, the clinical capabilities of this system are demonstrated for quantitative CTC detection in the presence of a high leukocyte background. This portable detection method shows great potential for quantification of rare cells with single‐cell sensitivity for various applications.     

Circulating Tumor Cell Phenotyping via High‐Throughput Acoustic Separation

The study of circulating tumor cells (CTCs) offers pathways to develop new diagnostic and prognostic biomarkers that benefit cancer treatments. In order to fully exploit and interpret the information provided by CTCs, the development of a platform is reported that integrates acoustics and microfluidics to isolate rare CTCs from peripheral blood in high throughput while preserving their structural, biological, and functional integrity. Cancer cells are first isolated from leukocytes with a throughput of 7.5 mL h⁻¹, achieving a recovery rate of at least 86% while maintaining the cells' ability to proliferate. High‐throughput acoustic separation enables statistical analysis of isolated CTCs from prostate cancer patients to be performed to determine their size distribution and phenotypic heterogeneity for a range of biomarkers, including the visualization of CTCs with a loss of expression for the prostate specific membrane antigen. The method also enables the isolation of even rarer, but clinically important, CTC clusters.     

Hyaluronic Acid‐Functionalized Electrospun Polyvinyl Alcohol/Polyethyleneimine Nanofibers for Cancer Cell Capture Applications

Capturing circulating tumor cells (CTCs) with sufficient sensitivity and specificity in vitro is of paramount importance for early cancer diagnosis. Here a facile approach to immobilizing hyaluronic acid (HA) onto electrospun polyvinyl alcohol/polyethyleneimine (PVA/PEI) nanofibers for capturing cancer cells overexpressing CD44 receptors is reported. In this study, electrospun PVA/PEI nanofibers were crosslinked using glutaraldehyde vapor, covalently conjugated with HA via N‐(3‐dimethy‐laminopropyl)‐N′‐ethylcarbodiimide/N‐hydroxysuccinimide coupling reaction, followed by neutralization of the remaining fiber surface PEI amines via acetylation. The formed nanofibers were characterized using different techniques. It is shown that the HA‐modified PVA/PEI nanofibers with a mean diameter of 459.7 nm possess a smooth and uniform fibrous morphology, similar to the PVA/PEI nanofibers without HA modification. The HA‐modified PVA/PEI nanofibers display good cytocompatibility and hemocompatibility as confirmed by cell viability, hemolysis, and anticoagulant assays. Importantly, with the modified HA, the nanofibers exhibit superior capability to capture CD44 receptor‐overexpressing cancer cells. The developed HA‐modified PVA/PEI nanofibers may hold a great promise to be applied for capturing CTCs for cancer diagnosis applications.     

Light‐Responsive Biodegradable Nanorattles for Cancer Theranostics

Cancer nanotheranostics, integrating both diagnostic and therapeutic functions into nanoscale agents, are advanced solutions for cancer management. Herein, a light‐responsive biodegradable nanorattle‐based perfluoropentane‐(PFP)‐filled mesoporous‐silica‐film‐coated gold nanorod (GNR@SiO₂‐PFP) is strategically designed and prepared for enhanced ultrasound (US)/photoacoustic (PA) dual‐modality imaging guided photothermal therapy of melanoma. The as‐prepared nanorattles are composed of a thin mesoporous silica film as the shell, which endows the nanoplatform with flexible morphology and excellent biodegradability, as well as large cavity for PFP filling. Upon 808 nm laser irradiation, the loaded PFP will undergo a liquid–gas phase transition due to the heat generation from GNRs, thus generating nanobubbles followed by the coalescence into microbubbles. The conversion of nanobubbles to microbubbles can improve the intratumoral permeation and retention in nonmicrovascular tissue, as well as enhance the tumor‐targeted US imaging signals. This nanotheranostic platform exhibits excellent biocompatibility and biodegradability, distinct gas bubbling phenomenon, good US/PA imaging contrast, and remarkable photothermal efficiency. The results demonstrate that the GNR@SiO₂‐PFP nanorattles hold great potential for cancer nanotheranostics.     

An Ultra pH‐Sensitive and Aptamer‐Equipped Nanoscale Drug‐Delivery System for Selective Killing of Tumor Cells

Nanotechnology has often been applied in the development of targeted drug‐delivery systems for the treatment of cancer. An ideal nanoscale system for drug delivery should be able to selectively deliver and rapidly release the carried therapeutic drug(s) in cancer cells and, more importantly, not react to off‐target cells so as to eliminate unwanted toxicity on normal tissues. To reach this goal, a selective chemotherapeutic is formulated using a hollow gold nanosphere (HAuNS) equipped with a biomarker‐specific aptamer (Apt), and loaded with the chemotherapy drug doxorubicin (DOX). The formed Apt‐HAuNS‐Dox, approximately 42 nm in diameter, specifically binds to lymphoma tumor cells and does not react to control cells that do not express the biomarker. Through aptamer‐mediated selective cell binding, the Apt‐HAuNS‐Dox is internalized exclusively into the targeted tumor cells, and then released the DOX intracellularly. Of note, although the formed Apt‐HAuNS‐Dox is stable under normal biological conditions (pH 7.4), it appears ultrasensitive to pH change and rapidly releases 80% of the loaded DOX within 2 h at pH 5.0, a condition seen in cell lysosomes. Functional assays using cell mixtures show that the Apt‐HAuNS‐Dox selectively kills lymphoma tumor cells, but has no effect on the growth of the off‐target cells in the same cultures, indicating that this ultra pH‐sensitive Apt‐HAuNS‐Dox can selectively treat cancer through specific aptamer guidance, and will have minimal side effects on normal tissue.     

Antibody‐Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody‐free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC‐7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 10⁵ SMMC‐7721 cells are incubated on a 4.5 cm² hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC‐7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC‐7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.     

Scaffold‐Free Liver‐On‐A‐Chip with Multiscale Organotypic Cultures

The considerable advances that have been made in the development of organotypic cultures have failed to overcome the challenges of expressing tissue‐specific functions and complexities, especially for organs that require multitasking and complex biological processes, such as the liver. Primary liver cells are ideal biological building blocks for functional organotypic reconstruction, but are limited by their rapid loss of physiological integrity in vitro. Here the concept of lattice growth used in material science is applied to develop a tissue incubator, which provides physiological cues and controls the 3D assembly of primary cells. The cues include a biological growing template, spatial coculture, biomimetic radial flow, and circulation in a scaffold‐free condition. The feasibility of recapitulating a multiscale physiological structural hierarchy, complex drug clearance, and zonal physiology from the cell to tissue level in long‐term cultured liver‐on‐a‐chip is demonstrated. These methods are promising for future applications in pharmacodynamics and personal medicine.     

Holo‐Lactoferrin Modified Liposome for Relieving Tumor Hypoxia and Enhancing Radiochemotherapy of Cancer

Hypoxic microenvironments in the solid tumor play a negative role in radiotherapy. Holo‐lactoferrin (holo‐Lf) is a natural protein, which acts as a potential ligand of transferrin receptor (TfR). In this work, an anticancer drug, doxorubicin (Dox)‐loaded liposome‐holo‐Lf nanocomposites, is developed for tumor targeting and imaging guided combined radiochemotherapy. Dox‐loaded liposome‐holo‐Lf (Lf‐Liposome‐Dox) nanocomposites exhibit significant cellular uptake likely owing to the TfR receptor‐mediated targeting accumulation of Lf‐Liposome‐Dox nanocomposites. Additionally, the nanocomposites exhibit high accumulation in the tumor site after intravenous injection as evidenced from in vivo fluorescence imaging. More importantly, it is found that the holo‐Lf has the ability to catalyze the conversion of hydrogen peroxide (H₂O₂) to oxygen for relieving the tumor hypoxic microenvironment. Photoacoustic imaging further confirms the abundant generation of oxygen in the presence of Lf‐Liposome‐Dox nanocomposites. Based on these findings, in vivo combined radiochemotherapy is performed using Lf‐Liposome‐Dox as therapeutic agent, achieving excellent cancer treatment effect. The study further promotes the potential biomedical application of holo‐Lf in cancer treatment.     

Rapid Light‐Triggered Drug Release in Liposomes Containing Small Amounts of Unsaturated and Porphyrin–Phospholipids

Prompt membrane permeabilization is a requisite for liposomes designed for local stimuli‐induced intravascular release of therapeutic payloads. Incorporation of a small amount (i.e., 5 molar percent) of an unsaturated phospholipid, such as dioleoylphosphatidylcholine (DOPC), accelerates near infrared (NIR) light‐triggered doxorubicin release in porphyrin–phospholipid (PoP) liposomes by an order of magnitude. In physiological conditions in vitro, the loaded drug can be released in a minute under NIR irradiation, while liposomes maintain serum stability otherwise. This enables rapid laser‐induced drug release using remarkably low amounts of PoP (i.e., 0.3 molar percent). Light‐triggered drug release occurs concomitantly with DOPC and cholesterol oxidation, as detected by mass spectrometry. In the presence of an oxygen scavenger or an antioxidant, light‐triggered drug release is inhibited, suggesting that the mechanism is related to singlet oxygen mediated oxidization of unsaturated lipids. Despite the irreversible modification of lipid composition, DOPC‐containing PoP liposome permeabilization is transient. Human pancreatic xenograft growth in mice is significantly delayed with a single chemophototherapy treatment following intravenous administration of 6 mg kg^?1 doxorubicin, loaded in liposomes containing small amounts of DOPC and PoP.     

Virus‐Mimicking Chimaeric Polymersomes Boost Targeted Cancer siRNA Therapy In Vivo

Small interfering RNA (siRNA) offers a highly selective and effective pharmaceutical for various life‐threatening diseases, including cancers. The clinical translation of siRNA is, however, challenged by its short plasma life, poor cell uptake, and cumbersome intracellular trafficking. Here, cNGQGEQc peptide‐functionalized reversibly crosslinked chimaeric polymersomes (cNGQ/RCCPs) is shown to mediate high‐efficiency targeted delivery of Polo‐like kinase1 specific siRNA (siPLK1) to orthotopic human lung cancer in nude mice. Strikingly, siRNA is completely and tightly loaded into the aqueous lumen of the polymersomes at an unprecedentedly low N/P ratio of 0.45. cNGQ/RCCPs loaded with firefly luciferase specific siRNA (siGL3) or siPLK1 are efficiently taken up by α₃β₁‐integrin‐overexpressing A549 lung cancer cells and quickly release the payloads to the cytoplasm, inducing highly potent and sequence‐specific gene silencing in vitro. The in vivo studies using nude mice bearing orthotopic A549 human lung tumors reveal that siPLK1‐loaded cNGQ/RCCPs boost long circulation, superb tumor accumulation and selectivity, effective suppression of tumor growth, and significantly improved survival time. These virus‐mimicking chimaeric polymersomes provide a robust and potent platform for targeted cancer siRNA therapy.