Hand-held electron spin resonance scanner for subcutaneous oximetry using OxyChip.

PURPOSE: Electron spin resonance (ESR) is used to measure oxygen partial pressure (pO2) in biological media with many clinical applications. Traditional clinical ESR involves large magnets that encompass the subject of measurement. However, certain applications might benefit from a scanner operating within local static magnetic fields. Our group recently developed such a compact scanner for transcutaneous (surface) pO2 measurements of skin tissue. Here we extend this capability to subsurface (subcutaneous) pO2 measurements and verify it using an artificial tissue emulating (ATE) phantom. METHODS: We introduce a new scanner, tailored for subcutaneous measurements up to 2 mm beneath the skin's surface. This scanner captures pulsed ESR signals from embedded approximate 1-mm oxygen-sensing solid paramagnetic implant, OxyChip. The scanner features a static magnetic field source, producing a uniform region outside its surface, and a compact microwave resonator, for exciting and receiving ESR signals. RESULTS: ESR readings derived from an OxyChip, positioned approximately 1.5 mm from the scanner's surface, embedded in ATE phantom, exhibited a linear relation of 1/T2 versus pO2 for pO2 levels at 0, 7.6, 30, and 160 mmHg, with relative reading accuracy of about 10%. CONCLUSION: The compact ESR scanner can report pO2 data in ATE phantom from an external position relative to the scanner. Implementing this scanner in preclinical and clinical applications for subcutaneous pO2 measurements is a feasible next phase for this development. This innovative design also has the potential to operate in conjunction with artificial skin graft for wound healing, combining therapeutic and pO2 diagnostic features.

Porcine arterial ECM hydrogel: Designing an in vitro angiogenesis model for long-term high-throughput research.

The field of angiogenesis research provides deep understanding regarding this important process, which plays fundamental roles in tissue development and different abnormalities. In vitro models offer the advantages of low-cost high-throughput research of angiogenesis while sparing animal lives, and enabling the use of human cells. Nevertheless, prevailing in vitro models lack stability and are limited to a few days' assays. This study, therefore, examines the hypothesis that closely mimicking the vascular microenvironment can more reliably support longer angiogenesis processes in vitro. To this end, porcine arterial extracellular matrix (paECM)- a key component of blood vessels-was isolated and processed into a thermally induced hydrogel and characterized in terms of composition, structure, and mechanical properties, thus confirming the preservation of important characteristics of arterial extracellular matrix. This unique hydrogel was further tailored into a three-dimensional model of angiogenesis using endothelial cells and supporting cells, in a configuration that allows high-throughput quantitative analysis of cell viability and proliferation, cell migration, and apoptosis, thus revealing the advantages of paECM over frequently used biomaterials. Markedly, when applied with well-known effectors of angiogenesis, the model measures reflected the expected response, hence validating its efficacy and establishing its potential as a promising tool for the research of angiogenesis.

Nano-Ghosts: Biomimetic membranal vesicles, technology and characterization.

Currently, nano-carriers for anti-cancer drug delivery are complex systems, which struggle with immunogenicity and enhanced permeability effect (EPR)-related problems that halt the clinical translation of many therapeutics. Consequently, a rapidly growing field of research has been focusing on biomimetic nano-vesicles (BNVs) as an effective delivery alternative. Nevertheless, the translation of many BNVs is limited due to scalability problems, inconsistent production process, and insufficient loading efficiency. Here we discuss the process of our previously published BNVs, termed Nano-Ghosts (NGs), which are produced from the membrane of mesenchymal stem cells. We demonstrate the flexibility of the process, while alternating physical methodologies (sonication or extrusion) to produce the NGs while preserving their desired characteristics. We also show that our NGs can be labeled using multiple methods (fluorescence, radiolabeling, and genetic engineering) for tracking and diagnostic purposes. Lastly, we demonstrate that the loading efficiency can be improved by using electroporation to accommodate a range of therapeutics (small molecules, peptides and DNA) that can be delivered by the NGs. Our results emphasize the robustness of the NGs technology, its versatility and a vast range of applications, differentiating it from other BNVs and leading the way towards clinical translation.

Extracellular Matrix Hydrogels Originated from Different Organs Mediate Tissue-Specific Properties and Function.

Porcine extracellular matrix (pECM)-derived hydrogels were introduced, in recent years, aiming to benefit the pECM's microstructure and bioactivity, while controlling the biomaterial's physical and mechanical properties. The use of pECM from different tissues, however, offers tissue-specific features that can better serve different applications. In this study, pECM hydrogels derived from cardiac, artery, pancreas, and adipose tissues were compared in terms of composition, structure, and mechanical properties. While major similarities were demonstrated between all the pECM hydrogels, their distinctive attributes were also identified, and their substantial effects on cell-ECM interactions were revealed. Furthermore, through comprehensive protein and gene expression analyses, we show, for the first time, that each pECM hydrogel supports the spontaneous differentiation of induced pluripotent stem cells towards the resident cells of its origin tissue. These findings imply that the origin of ECM should be carefully considered when designing a biomedical platform, to achieve a maximal bioactive impact.

Nanoghosts as a Novel Natural Nonviral Gene Delivery Platform Safely Targeting Multiple Cancers.

Nanoghosts derived from mesenchymal stem cells and retaining their unique surface-associated tumor-targeting capabilities were redesigned as a selective and safe universal nonviral gene-therapy platform. pDNA-loaded nanoghosts efficiently targeted and transfected diverse cancer cells, in vitro and in vivo, in subcutaneous and metastatic orthotopic tumor models, leading to no adverse effects. Nanoghosts loaded with pDNA encoding for a cancer-toxic gene inhibited the growth of metastatic orthotopic lung cancer and subcutaneous prostate cancer models and dramatically prolonged the animals' survival.

PLGA-Listeriolysin O microspheres: Opening the gate for cytosolic delivery of cancer antigens.

Strategies for cancer protein vaccination largely aim to activate the cellular arm of the immune system against cancer cells. This approach, however, is limited since protein vaccines mostly activate the system's humoral arm instead. One way to overcome this problem is to enhance the cross-presentation of such proteins by antigen-presenting cells, which may consequently lead to intense cellular response. Here we examined the ability of listeriolysin O (LLO) incorporated into poly-lactic-co-glycolic acid (PLGA) microspheres to modify the cytosolic delivery of low molecular weight peptides and enhance their cross-presentation. PLGA microspheres were produced in a size suitable for uptake by phagocytic cells. The peptide encapsulation and release kinetics were improved by adding NaCl to the preparation. PLGA microspheres loaded with the antigenic peptide and incorporated with LLO were readily up-taken by phagocytic cells, which exhibited an increase in the expression of peptide-MHC-CI complexes on the cell surface. Furthermore, this system enhanced the activation of a specific T hybridoma cell line, thus simulating cytotoxic T cells. These results establish, for the first time, a proof of concept for the use of PLGA microspheres incorporated with a pore-forming agent and the antigen peptide of choice as a unique cancer protein vaccination delivery platform.

The effect of AZD2171- or sTRAIL/Apo2L-loaded polylactic-co-glycolic acid microspheres on a subcutaneous glioblastoma model.

Studies with AZD2171-a new anti-angiogenic inhibitor of tyrosine kinases associated with VEGF signaling-have shown great promise for treating glioblastoma. Unfortunately, AZD2171 success is limited by low permeability through the blood-brain barrier. Due to AZD2171's short half-life and high toxicity, its local administration will require multiple intracranial procedures, making this approach clinically unfeasible. In this study, we investigated the potential of the highly hydrophobic AZD2171, released from modified polylactic-co-glycolic acid microspheres (PLGA-MS), to treat glioblastoma. To further demonstrate the versatile loading capacity of this system, the same PLGA formulation, which was found optimal for the loading and release of AZD2171, was tested with sTRAIL/Apo2L-a biologic drug that is very different than AZD2171 in its molecular weight, solubility, and charge. AZD2171 released from PLGA-MS was at least effective as the free drug in inhibiting endothelial growth and proliferation (in vitro), and, surprisingly, had a profound cytotoxic effect also towards in vitro cultured glioblastoma cell-lines (U87 and A172). Complete tumor inhibition was achieved following a single treatment with AZD2171-loaded PLGA-MS (6 (mg)/kg) administered locally adjacent to human U87 glioma tumors inoculated subcutaneously in nude mice. This improved effect, compared to other therapeutic approaches involving AZD2171, was shown to affect both tumor vasculature and the glioma cells. sTRAIL-loaded microspheres, administered at very low doses (0.3 (mg)/kg), led to 35 % inhibition of tumor growth in 2 weeks. Collectively, our results provide pre-clinical evidence for the potential of PLGA formulations of AZD2171 and sTRAIL to serve as an effective treatment for glioblastoma.

Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking.

Considerable interest has arisen in precision fabrication of cell bearing scaffolds and structures by free form fabrication. Gelatin is an ideal material for creating cell entrapping constructs, yet its application in free form fabrication remains challenging. We demonstrate the use of gelatin, crosslinked with microbial transglutaminase (mTgase), as a material to print cell bearing hydrogels for both 2-dimensional (2-D) precision patterns and 3-dimensional (3-D) constructs. The precision patterning was attained with 3 % gelatin and 2 % high molecular weight poly (ethylene oxide) (PEO) whereas 3-D constructs were obtained using a 5 % gelatin solution. These hydrogels, referred to as "bioinks" supported entrapped cell growth, allowing cell spreading and proliferation for both HEK293 cells and Human Umbilical Vein Endothelial Cells (HUVECs). These bioinks were shown to be dispensable by robotic precision, forming patterns and constructs that were insoluble and of suitable stiffness to endure post gelation handling. The two bioinks were further characterized for fabrication parameters and mechanical properties.

Reconstructed stem cell nanoghosts: a natural tumor targeting platform.

The ultimate goal in cancer therapy is achieving selective targeting of cancer cells. We report a novel delivery platform, based on nanoghosts (NGs) produced from the membranes of mesenchymal stem cells (MSCs). Encompassing MSC surface molecules, the MSC-NGs retained MSC-specific in vitro and in vivo tumor targeting capabilities and were cleared from blood-filtering organs. MSC-NGs were found to be biocompatible. Systemic administration of drug loaded MSC-NGs demonstrated 80% inhibition of human prostate cancer.

Functionalized PLGA-doped zirconium oxide ceramics for bone tissue regeneration.

Bone tissue engineering is an alternative approach to bone grafts. In our study we aim to develop a composite scaffold for bone regeneration made of doped zirconium oxide (ZrO2) conjugated with poly(lactic-co-glycolic acid) (PLGA) particles for the delivery of growth factors. In this composite, the PLGA microspheres are designed to release a crucial growth factor for bone formation, bone morphogenetic protein-2 (BMP2). We found that by changing the polymer's molecular weight and composition, we could control microsphere loading, release and size. The BMP2 released from PLGA microspheres retained its biological activity and increased osteoblastic marker expression in human mesenchymal stem cells (hMSCs). Uncapped PLGA microspheres were conjugated to ZrO2 scaffolds using carbodiimide chemistry, and the composite scaffold was shown to support hMSCs growth. We also demonstrated that human umbilical vein endothelial cells (HUVECs) can be co-cultured with hMSCs on the ZrO2 scaffold for future vascularization of the scaffold. The ZrO2 composite scaffold could serve as a bone substitute for bone grafting applications with the added ability of releasing different growth factors needed for bone regeneration.

Collagen-cellulose composite thin films that mimic soft-tissue and allow stem-cell orientation.

Mechanical properties of collagen films are less than ideal for biomaterial development towards musculoskeletal repair or cardiovascular applications. Herein, we present a collagen-cellulose composite film (CCCF) compared against swine small intestine submucosa in regards to mechanical properties, cell growth, and histological analysis. CCCF was additionally characterized by FE-SEM, NMR, mass spectrometry, and Raman Microscopy to elucidate its physical structure, collagen-cellulose composition, and structure activity relationships. Mechanical properties of the CCCF were tested in both wet and dry environments, with anisotropic stress-strain curves that mimicked soft-tissue. Mesenchymal stem cells, human umbilical vein endothelial cells, and human coronary artery smooth muscle cells were able to proliferate on the collagen films with specific cell orientation. Mesenchymal stem cells had a higher proliferation index and were able to infiltrate CCCF to a higher degree than small intestine submucosa. With the underlying biological properties, we present a collagen-cellulose composite film towards forthcoming biomaterial-related applications.

ECM-based bioactive microencapsulation significantly improves islet function and graft performance.

Microencapsulation is a promising strategy to prolong the survival and function of transplanted pancreatic islets for diabetes therapy, albeit its translation has been impeded by incoherent graft performance. The use of decellularized ECM has lately gained substantial research momentum due to its innate capacity to augment the function of cells originating from the same tissue type. In the present study, the advantages of both these approaches are leveraged in a porcine pancreatic ECM (pECM)-based microencapsulation platform, thus significantly enhancing murine pancreatic islet performance. pECM-encapsulated islets sustain high insulin secretion levels in vitro, surpassing those of islets encapsulated in conventional alginate microcapsules. Moreover, pECM-encapsulated islet cells proliferate and produce an enriched intra-islet ECM framework, displaying a distinctive structural rearrangement. The beneficial effect of pECM encapsulation is further reinforced by the temporary protection against cytokine-induced cytotoxicity. In-vivo, this platform significantly improves glucose tolerance and achieves glycemic correction in 100% of immunocompetent diabetic mice without any immunosuppression, compared to only 50% mice achieved glycemic correction by alginate encapsulation. Altogether, the results presented herein reveal that pECM-based microencapsulation offers a natural pancreatic niche that can restore the function of isolated pancreatic islets and deliver them safely, avoiding the need for immunosuppression. STATEMENT OF SIGNIFICANCE: Aiming to improve pancreatic islet transplantation outcomes in diabetic patients, we developed a microencapsulation platform based on pancreatic extracellular matrix (pECM). In these microcapsules the islets are entrapped within a pECM hydrogel that mimics the natural pancreatic microenvironment. We show that pECM encapsulation supports the islets' viability and function in culture, and provides temporal protection against cytokine-induced stress. In a diabetic mouse model, pECM encapsulation significantly improved glucose tolerance and achieved glycemic correction without any immunosuppression. These results reveal the potential of pECM encapsulation as a viable treatment for diabetes, providing a solid scientific basis for more advanced preclinical studies.

Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute.

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

Alginate encapsulated cells secreting Fas-ligand reduce lymphoma carcinogenicity.

Fas ligand (CD95L/APO-1) is considered as a potent anti-tumor agent due to its mediated cell death properties. We have designed a polymeric microencapsulation system, which encapsulates soluble FasL secreting cells. The encapsulated cells continuously release soluble FasL (sFasL) at the tumor site, while the device protects the encapsulated cells from the host immune system. The potential and efficacy of this system are demonstrated in vitro and in vivo for tumor inhibition. Polymeric microcapsules composed of Alginate Poly-l-lysine were optimized to encapsulate L5 secreting sFasL cells. The expression and anti-tumor activities of the sFasL were confirmed in vitro and tumor inhibition was studied in vivo in SCID mice bearing subcutaneous lymphoma tumors. In vitro, sFasL secreted by the encapsulated L5-sFasL cells was biologically active, inhibited proliferation and induced apoptotic cell death in Fas sensitive tumor cells. Mice injected with encapsulated L5-sFasL cells on the day of tumor injection or 10 days after tumor injection showed significant reduction in tumor volume, of 87% and 95%, respectively. Our findings show that encapsulated cells expressing sFasL can be used as a local device and efficiently suppress malignant Fas sensitive tumors with no side effects.

Encapsulated human mesenchymal stem cells: a unique hypoimmunogenic platform for long-term cellular therapy.

Cell encapsulation is a promising approach for long-term delivery of therapeutic agents. Nonetheless, this system has failed to reach clinical settings, as the entrapped cells provoke a host immune reaction. Mesenchymal stem cells (MSCs), however, potentially may overcome this impediment and serve as a promising platform for cell-based microencapsulation. They are known to be hypoimmunogenic and can be genetically modified to express a variety of therapeutic factors. We have designed alginate-PLL microcapsules that can encapsulate human MSCs (hMSCs) for extended periods, as demonstrated by fluorescence and H(3)-thymidine assays. The encapsulated hMSCs maintained their mesenchymal surface markers and differentiated to all the typical mesoderm lineages. In vitro and in vivo immunogenicity studies revealed that encapsulated hMSCs were significantly hypoimmunogenic, leading to a 3-fold decrease in cytokine expression compared to entrapped cell lines. The efficacy of such systems was demonstrated by genetically modifying the cells to express the hemopexin-like protein (PEX), an inhibitor of angiogenesis. Live imaging and tumor measurements showed that encapsulated hMSC-PEX injected adjacent to glioblastoma tumors in nude mice led to a significant reduction in tumor volume (87%) and weight (83%). We clearly demonstrate that hMSCs are the cell of choice for microencapsulation cell based-therapy, thus bringing this technology closer to clinical application.

Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides.

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine.

Nanoghosts: Mesenchymal Stem cells derived nanoparticles as a unique approach for cartilage regeneration.

Osteoarthritis (OA) is a chronic degenerative disease, which affects the joints and is characterized by inflammation, cartilage loss and bone changes. Nowadays, there are no treatments for OA, and current therapies are focused on relieving the symptoms. As a new therapy approach, micro and nanoparticles have been extensively explored and among all the studied particles, the use of cell-membrane-based particles is expanding. Another promising approach studied to treat OA, is the use of mesenchymal stem cells (MSCs) which play an important role modulating inflammation. We developed a novel kind of MSCs' cytoplasmic-membrane-based nanoparticles, termed nano-ghosts (NGs). Retaining MSCs' surface properties and lacking cells' internal machinery allow the NGs to have immunomodulatory capacity and to be immune-evasive while not susceptible to host-induced changes. In this study, we demonstrate NGs' ability to target cartilage tissues, in vitro and in vivo, while modulating the inflammatory process. In vivo studies demonstrated NGs ability to act as an immunomodulatory drug slowing down cartilage degeneration process. Our proof-of-concept experiments show that NGs system is a versatile nano-carrier system, capable of therapeutics loading, with targeting capabilities towards healthy and inflamed cartilage cells. Our results, along with previously published data, clearly reveal the NGs system as a promising nano-carrier platform and as a potential immunomodulatory drug for several inflammation-related diseases.

A fail-safe mechanism for negative selection of isotype-switched B cell precursors is regulated by the Fas/FasL pathway.

In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Igmicro-deficient microMT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and microMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

Local delivery of poly lactic-co-glycolic acid microspheres containing imatinib mesylate inhibits intracranial xenograft glioma growth.

PURPOSE: In an effort to develop new therapeutic strategies to treat malignant gliomas, we have designed poly (lactic-co-glycolic) acid (PLGA) microparticles that deliver imatinib mesylate, a small molecule tyrosine kinase inhibitor. The local continuous release of imatinib mesylate at the tumor site overcomes many obstacles associated with systemic delivery. EXPERIMENTAL DESIGN: Polymeric microspheres were prepared from various compositions of PLGA and loaded with imatinib mesylate. Imatinib release profiles, biological activity, and effect on PDGFR-B phosphorylation were confirmed in vitro. The therapeutic efficacy of imatinib microspheres was examined in two s.c. and orthotopic human glioblastoma xenograft models. RESULTS: A single local injection of PLGA microspheres loaded with a low concentration of imatinib mesylate led to 88% and 79% reduction in s.c. human (U87-MG) and murine (GL261) glioma tumors, respectively. PLGA-imatinib mesylate administered intracranially led to a 79% reduction in U87MG tumor volume. Immunohistochemical analysis showed a marked decrease in proliferation indices and tumor vessel density in the s.c. model and induction of apoptosis in an intracranial model. CONCLUSION: This is the first study to show the therapeutic efficacy of the local delivery of imatinib mesylate using a polymeric delivery system.

Extracellular Vesicles Reflect the Efficacy of Wheatgrass Juice Supplement in Colon Cancer Patients During Adjuvant Chemotherapy.

INTRODUCTION: Colorectal cancer (CC) is the third most common type of cancer, accounting for 10% of all cancer cases. Adjuvant chemotherapy is recommended in stages II-III CC. Wheatgrass juice (WGJ) from wheat seeds has high nutritional values, may induce synergistic benefits to chemotherapy and may attenuate chemotherapy-related side effects. Extracellular vesicles (EVs) are subcellular membrane blebs. EVs include exosomes (generated in the endosome, in size <150 nm) and microvesicles (shed from the plasma cell membrane) provide information on their parental cells and play a role in intercellular communication. We aimed to elucidate the effects of chemotherapy administration with supportive treatment of WGJ on CC patients' EVs characteristics. METHODS: EVs were isolated from the blood samples of 15 healthy controls (HCs) and 50 CC patients post-surgery, treated by chemotherapy, with or without additional daily WGJ. Blood samples were taken before, during, and at the end of chemotherapy. EVs were characterized by size, concentration, membrane antigens and cytokine content using nanoparticle-tracking analysis, western blot, flow cytometry, and protein array methods. RESULTS: EVs were found to be similar by size and concentration with reduced levels of exosome markers (CD81) on samples at the end of combined treatment (chemotherapy and WGJ). Higher levels of endothelial EVs, which may indicate impairment of the vascular endothelial cells during treatment, were found in CC patients treated by chemotherapy only compared to those with chemotherapy and daily WGJ. Also, EVs thrombogenicity was lower in patients added WGJ compared to patients who had only chemotherapy (levels of tissue factor p = 0.029 and endothelial protein C receptor p = 0.005). Following treatments, levels of vascular endothelial growth factor receptors (VEGFR-1) and the majority of growth-factors/pro-inflammatory cytokines were higher in EVs of patients treated by chemotherapy only than in EVs obtained from patients with the combined treatment. CONCLUSION: Daily consumption of WGJ during chemotherapy may reduce vascular damage and chemotherapy-related thrombogenicity, growth factors and cytokines, as reflected by the characteristics of patient's EVs.