Silk-elastinlike protein-based hydrogels for drug delivery and embolization.

Silk-Elastinlike Protein-Based Polymers (SELPs) can form thermoresponsive hydrogels that allow for the generation of in-situ drug delivery matrices. They are produced by recombinant techniques, enabling exact control of monomer sequence and polymer length. In aqueous solutions SELP strands form physical crosslinks as a function of temperature increase without the addition of crosslinking agents. Gelation kinetics, modulus of elasticity, pore size, drug release, biorecognition, and biodegradation of SELP hydrogels can be controlled by placement of amino acid residues at strategic locations in the polymer backbone. SELP hydrogels have been investigated for delivery of a variety of bioactive agents including small molecular weight drugs and fluorescent probes, oligomers of glycosaminoglycans, polymeric macromolecules, proteins, plasmid DNA, and viral gene delivery systems. In this review we provide a background for use of SELPs in matrix-mediated delivery and summarize recent investigations of SELP hydrogels for controlled delivery of bioactive agents as well as their use as liquid embolics.

Protein-based polymer liquid embolics for cerebral aneurysms.

Cerebral aneurysms (CA), an abnormal bulge in the arteries that supply blood to the brain, are prone to rupture and can cause hemorrhagic stroke. Physicians can treat CA by blocking blood flow to the aneurysmal sac via clipping of the aneurysm neck via open procedure, or endovascular occlusion of the aneurysm with embolic materials to promote thrombus formation to prevent further inflow of blood into the aneurysm. Endovascular treatment options for CA still have significant limitations in terms of safety, usability in coagulopathic patients, and risks of device migration. Bioactive embolic therapies, consisting of non-toxic bioresorbable materials that encourage the growth of neointima across the aneurysm neck, are needed to improve the healing of CA. In this work, the bioinspired silk-elastinlike protein-based polymer (SELP 815K), was used to embolize aneurysms in a rabbit elastase model. SELP 815K effectively embolized the model aneurysms in vivo, achieving >90% occlusion, using commercial microcatheters. No device-associated adverse effects were observed in any of the animals, and SELP 815K showed no cytotoxicity. SELP embolization did not show any deleterious effects to local tissues, and features consistent with reendothelialization of the aneurysm neck were noted in histological examination one-month post-embolization. SELP 815K shows promise as an embolic treatment for unruptured CA. STATEMENT OF SIGNIFICANCE: Unruptured cerebral aneurysms are present in approximately 3% of the population, with a fatality rate of up to 65% upon rupture. In this work a silk-elastinlike protein polymer (SELP) is explored as a liquid embolic for occlusion of cerebral aneurysms. This embolic exists as a liquid at room temperature before rapidly forming a gel at physiological temperature. This shape filling property was used to successfully occlude cerebral aneurysms in rabbits, with stable occlusion persisting for over thirty days. SELP occlusions show evidence for reendothelialization of the aneurysm sac and provide an opportunity for delivery of bioactive agents to further improve treatments.

Liquid-cell transmission electron microscopy for imaging of thermosensitive recombinant polymers.

Various polymers used in controlled release applications exhibit solution-based thermal responses. Unfortunately, very few characterization and imaging techniques permit resolution of individual polymers during their thermally-triggered phase transitions. Here, we demonstrate the use of temperature-ramp liquid-cell transmission electron microscopy (LCTEM) for real-time evaluation of the solution and interfacial behavior of elastinlike polypeptides (ELPs) and their self-assembled nanostructures over a temperature range incorporating their intrinsic lower critical solution temperatures (LCSTs). Individual polymers and supramolecular assemblies were discriminated dependent upon solubility states. The recombinant polymers were shown to adsorb to the silicon-nitride chip window from the buffered saline solution and desorb in a temperature-dependent manner. Silk-elastinlike protein block copolymers (SELPs) (composed of repeat peptide motifs of silk and elastin) differed from ELPs in thermal behavior. While both polymers were shown to cluster, only SELPs formed robust amyloid-like fibers upon heating.

An Oligomeric Sulfated Hyaluronan and Silk-Elastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis.

Semisynthetic glycosaminoglycan ethers (SAGEs) are short, sulfated hyaluronans which combine the natural properties of hyaluronan with chemical sulfation. In a murine model, SAGEs provide protection against radiation induced proctitis (RIP), a side effect of lower abdominal radiotherapy for cancer. The anti-inflammatory effects of SAGE have been studied in inflammatory diseases at mucosal barrier sites; however, few mechanisms have been uncovered necessitating high throughput methods. SAGEs were combined with silk-elastinlike polymers (SELPs) to enhance rectal accumulation in mice. After high radiation exposure to the lower abdominal area, mice were followed for 3 days or until they met humane endpoints, before evaluation of behavioral pain responses and histological assessment of rectal inflammation. RNA sequencing was conducted on tissues from the 3-day cohort to determine molecular mechanisms of SAGE-SELP. After 3 days, mice receiving the SAGE-SELP combination yielded significantly lowered pain responses and amelioration of radiation-induced rectal inflammation. Mice receiving the drug-polymer combination survived 60% longer than other irradiated mice, with a fraction exhibiting long term survival. Sequencing reveals varied regulation of toll like receptors, antioxidant activities, T-cell signaling, and pathways associated with pain. This investigation elucidates several molecular mechanisms of SAGEs and exhibits promising measures for prevention of RIP.

Translational Development of a Silk-Elastinlike Protein Polymer Embolic for Transcatheter Arterial Embolization.

Locally blocking blood flow to tumors with embolic materials is the key to transcatheter arterial embolization for treating hepatocellular carcinoma. Current microparticle agents do not deeply penetrate target tissues and are compatible with a very limited selection of therapeutic agents. Silk-elastinlike protein polymers (SELPs) combine the solubility of elastin and the strength of silk to create an easily injected liquid embolic that transition into a solid depot amenable to loading with drugs, gene therapy agents, or biologics. SELP, injected as liquid solution, penetrates the vasculature before transitioning to a solid hydrogel. The objective of this manuscript is to evaluate SELP embolization, stability, and biocompatibility at 7-, 30-, and 90-day survival intervals in a porcine model. SELP embolics selectively block blood flow in the kidneys and livers, with no off-target infarctions. As assessed with angiography, SELP renal embolization exhibits decreasing persistence for the duration of the 90-day study period. There is an increased presence of microscopic SELP emboli in the renal setting, compared to Embosphere. Histologically scored inflammatory reactions to SELP are decreased in both the renal and hepatic implantations compared to Embosphere. In conclusion, a bioresorbable SELP liquid embolic system deeply penetrates target tissue and selectively embolizes blood vessels in vivo.

A dual-functional Embolization-Visualization System for Fluorescence image-guided Tumor Resection.

Rationale: Intraoperative bleeding impairs physicians' ability to visualize the surgical field, leading to increased risk of surgical complications and reduced outcomes. Bleeding is particularly challenging during endoscopic-assisted surgical resection of hypervascular tumors in the head and neck. A tool that controls bleeding while marking tumor margins has the potential to improve gross tumor resection, reduce surgical morbidity, decrease blood loss, shorten procedure time, prevent damage to surrounding tissues, and limit postoperative pain. Herein, we develop and characterize a new system that combines pre-surgical embolization with improved visualization for endoscopic fluorescence image-guided tumor resection. Methods: Silk-elastinlike protein (SELP) polymers were employed as liquid embolic vehicles for delivery of a clinically used near-infrared dye, indocyanine green (ICG). The biophysical properties of SELP, including gelation kinetics, modulus of elasticity, and viscosity, in response to ICG incorporation using rheology, were characterized. ICG release from embolic SELP was modeled in tissue phantoms and via fluorescence imaging. The embolic capability of the SELP-ICG system was then tested in a microfluidic model of tumor vasculature. Lastly, the cytotoxicity of the SELP-ICG system in L-929 fibroblasts and human umbilical vein endothelial cells (HUVEC) was assessed. Results: ICG incorporation into SELP accelerated gelation and increased its modulus of elasticity. The SELP embolic system released 83 ± 8% of the total ICG within 24 hours, matching clinical practice for pre-surgical embolization procedures. Adding ICG to SELP did not reduce injectability, but did improve the gelation kinetics. After simulated embolization, ICG released from SELP in tissue phantoms diffused a sufficient distance to deliver dye throughout a tumor. ICG-loaded SELP was injectable through a clinical 2.3 Fr microcatheter and demonstrated deep penetration into 50-µm microfluidic-simulated blood vessels with durable occlusion. Incorporation of ICG into SELP improved biocompatibility with HUVECs, but had no effect on L-929 cell viability. Principle Conclusions: We report the development and characterization of a new, dual-functional embolization-visualization system for improving fluorescence-imaged endoscopic surgical resection of hypervascular tumors.

Location of stimuli-responsive peptide sequences within silk-elastinlike protein-based polymers affects nanostructure assembly and drug-polymer interactions.

Silk-elastinlike protein polymers (SELPs) self-assemble into nanostructures when designed with appropriate silk-to-elastin ratios. Here, we investigate the effect of insertion of a matrix metalloproteinase-responsive peptide sequence, GPQGIFGQ, into various locations within the SELP backbone on supramolecular self-assembly. Insertion of the hydrophilic, enzyme-degradable sequence into the elastin repeats allows the formation of dilution-stable nanostructures, while insertion into the hydrophobic silk motifs inhibited self-assembly. The SELP assemblies retained their lower critical solution temperature (LCST) thermal response, allowing up to eightfold volumetric changes due to temperature-induced size change. A model hydrophobic drug was incorporated into SELP nanoassemblies utilising a combination of precipitation, incubation and tangential flow filtration. While the nanoconstructs degraded in response to MMP activity, drug release kinetics was independent of MMP concentration. Drug release modelling suggests that release is driven by rates of water penetration into the SELP nanostructures and drug dissolution. In vitro testing revealed that SELP nanoassemblies reduced the immunotoxic and haemolytic side effects of doxorubicin in human blood while maintaining its cytotoxic activity.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome.

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

Self-Assembly of Thermoresponsive Recombinant Silk-Elastinlike Nanogels.

Recombinant silk-elastinlike protein polymers (SELPs) combine the biocompatibility and thermoresponsiveness of human tropoelastin with the strength of silk. Direct control over structure of these monodisperse polymers allows for precise correlation of structure with function. This work describes the fabrication of the first SELP nanogels and evaluation of their physicochemical properties and thermoresponsiveness. Self-assembly of dilute concentrations of SELPs results in nanogels with enhanced stability over micelles due to physically crosslinked beta-sheet silk segments. The nanogels respond to thermal stimuli via size changes and aggregation. Modifying the ratio and sequence of silk to elastin in the polymer backbone results in alterations in critical gel formation concentration, stability, aggregation, size contraction temperature, and thermal reversibility. The nanogels sequester hydrophobic compounds and show promise in delivery of bioactive agents.

Silk-elastinlike protein polymers enhance the efficacy of a therapeutic glycosaminoglycan for prophylactic treatment of radiation-induced proctitis.

Radiation-induced proctitis (RIP) is the most common clinical adverse effect for patients receiving radiotherapy as part of the standard course of treatment for ovarian, prostate, colon, and bladder cancers. RIP limits radiation dosage, interrupts treatment, and lowers patients' quality of life. A prophylactic treatment that protects the gastrointestinal tract from deleterious effects of radiotherapy will significantly improve patient quality of life and may allow for higher and more regular doses of radiation therapy. Semi-synthetic glycosaminoglycan (GAG), generated from the sulfation of hyaluronic acid, are anti-inflammatory but have difficulty achieving therapeutic levels in many tissues. To enhance the delivery of GAG, we created an in situ gelling rectal delivery system using silk-elastinlike protein polymers (SELPs). Using solutions of SELP 815K (which contains 6 repeats of blocks comprised of 8 silk-like units, 15 elastin-like units, and 1 lysine-substituted elastin-like unit) with GAG GM-0111, we created an injectable delivery platform that transitioned in <5min from a liquid at room temperature to a hydrogel at body temperature. The hydrogels released 50% of their payload within 30min and enhanced the accumulation of GAG in the rectum compared to traditional enema-based delivery. Using a murine model of radiation-induced proctitis, the prophylactic delivery of a single dose of GAG from a SELP matrix administered prior to irradiation significantly reduced radiation-induced pain after 3, 7, and 21days by 53±4%, 47±10%, and 12±6%, respectively. Matrix-mediated delivery of GAG by SELP represents an innovative method for more effective treatment of RIP and promises to improve quality of life of cancer patients by allowing higher radiotherapy doses with improved safety.

Silk-Elastinlike Protein Polymer Liquid Chemoembolic for Localized Release of Doxorubicin and Sorafenib.

Locoregional therapies for cancer are minimally invasive procedures in which the treatment is administered directly into cancerous tissue. Transarterial chemoembolization (TACE) is used to treat intermediate stage hepatocellular carcinoma (HCC). TACE uses an embolic material to block blood flow while coadministering a chemotherapeutic to the neoplastic tissue. Liquid embolics capable of drug loading are at the forefront of development as they allow for deeper permeation of tumor vasculature, increase neoplasm exposure to therapeutics, and resist revascularization by occupying both large and small diameter vessels. In this work, two chemotherapeutics used in the treatment of HCC, doxorubicin and sorafenib, were incorporated into the in situ gelling liquid embolic composed of a silk-elastinlike protein polymer (SELP-815 K). The base forms of the drugs had no significant effect on the viscosity, the gelation kinetics, and the gel stiffness of the SELP: all properties essential for the successful performance of an injectable liquid embolic. In vitro release studies indicated that the SELP liquid embolic delivered doxorubicin and sorafenib, either alone or in combination, at therapeutically relevant concentrations for a minimum of 14 and 30 days, respectively.

Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

BACKGROUND: Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. METHODS: Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. RESULTS: GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral ß-galactosidase expression as compared to PBS. CONCLUSION: The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes.

Drug-Encoded Biomarkers for Monitoring Biological Therapies.

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.

In vivo evaluation of matrix metalloproteinase responsive silk-elastinlike protein polymers for cancer gene therapy.

Silk-elastinlike protein polymers (SELPs) have been effectively used as controlled release matrices for the delivery of viruses for cancer gene therapy in preclinical models. However, the degradability of these polymers needs to be tuned for improved localized intratumoral gene delivery. Using recombinant techniques, systematic modifications in distinct regions of the polymer backbone, namely, within the elastin blocks, silk blocks, and adjacent to silk and elastin blocks, have been made to impart sensitivity to specific matrix metalloproteinases (MMPs) known to be overexpressed in the tumor environment. In this report we investigated the structure-function relationship of MMP-responsive SELPs for viral mediated gene therapy of head and neck cancer. These polymers showed significant degradation in vitro in the presence of MMPs. Their degradation rate was a function of the location of the MMP-responsive sequence in the polymer backbone when in hydrogel form. Treatment efficacy of the adenoviral vectors released from the MMP responsive SELP analogs in a xenograft mouse model of head and neck squamous cell carcinoma (HNSCC) was shown to be polymer structure dependent. These results demonstrate the tunable nature of MMP-responsive SELPs for localized matrix-mediated gene delivery.

In situ gelling silk-elastinlike protein polymer for transarterial chemoembolization.

Hepatocellular carcinoma annually affects over 700,000 people worldwide and trends indicate increasing prevalence. Patients ineligible for surgery undergo loco-regional treatments such as transarterial chemoembolization (TACE) to selectively target tumoral blood supply. Using a microcatheter, chemotherapeutics are infused followed by an embolic agent, or the drug is encapsulated by the embolic moiety; simultaneously inducing stasis while delivering localized chemotherapy. Presently, several products are used, but no universally accepted system is promoted because very disparate limitations exist. The goal of this investigation was to design and develop in situ gelling recombinant silk-elastinlike protein polymers (SELPs) for TACE. Two SELP compositions, SELP-47K and SELP-815K, with varying lengths of silk and elastin blocks, were investigated to formulate a new embolic that was injectable through commercially available microcatheters. The goal was to develop a composition providing maximal permeation of tumor vasculature while exhibiting effective embolic activity. The SELPs evaluated remain soluble until reaching 37 °C, when irreversible transition ensues forming a solid hydrogel network. SELP-815K formulated at 12% w/w with shear processing demonstrated acceptable rheological properties and clear embolic capability under flow conditions in vitro. A rabbit model showed feasibility of embolization in vivo allowing selective occlusion of lobar hepatic arterial branches.

Effect of shear on physicochemical properties of matrix metalloproteinase responsive silk-elastinlike hydrogels.

Silk-elastinlike protein polymers (SELPs) have been fabricated as matrices for controlled delivery of bioactive agents. In this application the need for an environmentally responsive, degradable polymer has risen to improve treatment outcomes. To satisfy this need, we have designed, synthesized, and expressed SELPs with matrix metalloproteinase (MMP) degradable sequences inserted in distinct regions of the polymer backbone. Upon characterization of the physicochemical properties of newly synthesized analogs, it was determined that conditioning of the polymers was necessary for normalization of batch properties, and to generate a more robust polymer network suitable for delivery. In this report we have examined the use of shear stress to condition synthesized material prior to application as a controlled release matrix. The application of high shear to SELPs results in significant changes in physiochemical properties as assayed by swelling ratio, soluble fraction release, rate of gel formation, stiffness of hydrogels, and nanoparticle release from matrices. These observed changes in material characteristics may be caused by the removal of semi-stable secondary and tertiary structures from single polymer strands leading to a more robust hydrogel with greater intermolecular interaction.

Engineering aqueous fiber assembly into silk-elastin-like protein polymers.

Self-assembled peptide/protein nanofibers are valuable 1D building blocks for creating complex structures with designed properties and functions. It is reported that the self-assembly of silk-elastin-like protein polymers into nanofibers or globular aggregates in aqueous solutions can be modulated by tuning the temperature of the protein solutions, the size of the silk blocks, and the charge of the elastin blocks. A core-sheath model is proposed for nanofiber formation, with the silk blocks in the cores and the hydrated elastin blocks in the sheaths. The folding of the silk blocks into stable cores--affected by the size of the silk blocks and the charge of the elastin blocks--plays a critical role in the assembly of silk-elastin nanofibers. Furthermore, enhanced hydrophobic interactions between the elastin blocks at elevated temperatures greatly influence the nanoscale features of silk-elastin nanofibers.

Oncolytic virotherapy of canine and feline cancer.

Cancer is the leading cause of disease-related death in companion animals such as dogs and cats. Despite recent progress in the diagnosis and treatment of advanced canine and feline cancer, overall patient treatment outcome has not been substantially improved. Virotherapy using oncolytic viruses is one promising new strategy for cancer therapy. Oncolytic viruses (OVs) preferentially infect and lyse cancer cells, without causing excessive damage to surrounding healthy tissue, and initiate tumor-specific immunity. The current review describes the use of different oncolytic viruses for cancer therapy and their application to canine and feline cancer.

Ordering recombinant silk-elastin-like nanofibers on the microscale.

Self-assembled peptide/polypeptide nanofibers are appealing building blocks for creating complex three-dimensional structures. However, ordering assembled peptide/polypeptide nanofibers into three-dimensional structures on the microscale remains challenging and often requires the employment of top-down approaches. We report that silk-elastin-like protein polymers self-assemble into nanofibers in physiologically relevant conditions, the assembled nanofibers further form fiber clusters on the microscale, and the nanofiber clusters eventually coalesce into three-dimensional structures with distinct nanoscale and microscale features. It is believed that the interplay between fiber growth and molecular diffusion leads to the ordering of the assembled silk-elastin-like nanofibers at the microscale.

Optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.

Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.