Mutation analysis using cell-free DNA for endocrine therapy in patients with HR+ metastatic breast cancer.

We prospectively evaluated the utility of ESR1 and PIK3CA mutation analysis with cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) for the efficacy of endocrine therapy (ET) in hormone receptive positive (HR+) metastatic breast cancer (MBC) patients. CfDNA was analyzed just before the start of ET for MBC. E380Q, Y537N, Y537S, and D538G were assessed for ESR1 mutations and H1047R, E545K, and E542K were assessed for PIK3CA mutations. A total of 75 patients were enrolled. Of those, 31 (41.3%) received letrozole with palbociclib, and 28 (37.3%) received exemestane and everolimus (EverX). ESR1 mutations were found in 36 (48.0%) patients, of which 16 (21.3%) had more than one variant. Seventeen (23.6%) patients had one PIK3CA mutation and 8 (11.1%) had two. In the total population, time to progression of the first ET after enrollment (TTP1) decreased significantly as the number of ESR1 mutations increased (p < 0.001). PIK3CA mutations were also significantly associated with shorter TTP1 (median TTP1: 16.2 months vs. 10.9 months, p = 0.03). In contrast, PIK3CA mutations were significantly associated with longer TTP in patients receiving EverX treatment (median TTP of EverX: 15.9 months vs. 5.2 months, p = 0.01) and remained a significant factor in multivariable analysis for TTP of EverX in this subgroup (hazard ratio = 0.2, 95% CI = 0.1- 0.8, p = 0.03). ESR1 and PIK3CA mutations in cfDNA were associated with clinical efficacies of ET in HR+ MBC patients.

Anti-tumor activity of BET inhibitors in androgen-receptor-expressing triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease comprising several subtypes. Androgen-receptor (AR) signaling has been targeted by several investigational agents in luminal AR subtype TNBCs. Bromodomain (BRD) and extra-terminal motif (BET) protein inhibitors have been shown to attenuate AR signaling in metastatic castration-resistant prostate cancer and to overcome enzalutamide resistance. We demonstrated potent anti-tumor effects of the BET inhibitor JQ1 against AR-positive TNBC cell lines using cell viability and cell cycle analysis. To reveal the mechanisms of JQ1 effects, multiplex gene expression analysis and immunoblotting assays were used. We examined in vivo effects of JQ1 in a xenograft model of AR expressing TNBC. JQ1 exhibited its anti-proliferative activity by inducing apoptosis and cell cycle arrest. JQ1 activity was not mediated by MYC downregulation. Instead, JQ1 blocked the interactions among the ATPase-family AAA-domain-containing 2 protein (ATAD2), BRD2, BRD4, and AR; effectively suppressing the expression of AR associated targets. In addition, JQ1 showed significant anti-tumor activity in vivo in TNBC xenograft mouse models as a monotherapy and in combination with anti-AR therapy. Taken together, our results showed that the BET inhibitor JQ1 is a promising therapeutic agent for the treatment of AR-positive TNBC.

Heparin-bound transforming growth factor-beta3 enhances neocartilage formation by rabbit mesenchymal stem cells.

BACKGROUND: Heparin binds growth factors to form a stable complex that maintains the biological activity and can retard the release pattern. To differentiate the embedded the stem cells, heparin-bind transforming growth factor (TGF)-beta3 was mixed with rabbit mesenchymal stem cells encapsulated with thermo-reversible hydrogel. It is suggested that the heparin-bound TGF-beta3 would help to increase the chondrogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel. METHODS: To determine the optimal condition for neocartilage formation, we characterized hydrogel constructs with growth factor release profiles, confocal laser microscopy, reverse transcription-polymerase chain reaction analysis, and histology. RESULTS: Reverse transcription-polymerase chain reaction analysis of the resultant cartilage tissue revealed that a thermo-reversible hydrogels with a heparin-bound TGF-beta3 was optimal for cartilage tissue formation as measured by production of collagen Type II, aggrecan, and SOX9, and cartilage oligomeric matrix protein gene expression. Additionally, the proliferation rate and cartilage specific ECM production were both significantly greater in the presence of heparin-bound TGF-beta3 than in the control. The amount of cartilage-associated ECM proteins was examined by immunohistochemical staining (collagen type II), Safranin-O staining, and Alcian blue staining. CONCLUSIONS: These data indicate the potential use of heparin-binding TGFbeta-3 for reconstruction of neocartilage formation.

The use of chondrogenic differentiation drugs to induce stem cell differentiation using double bead microsphere structure.

A double bead microsphere device that functions as a 3D scaffold and also delivers bioactive molecules to cells has been developed. The system we developed consists of large polymeric microspheres that were loaded with dexamethasone (DEXA) and coated with DHEA, which were physically immobilized using a layer-by-layer (LbL) system. The initial step in this strategy involves the creation of microparticles that contain DHEA, which were simply produced using a water-in-oil-in-water (W/O/W) emulsion method. In the second step, small sized microparticles containing DHEA were coated onto positively-charged poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres that contained DEXA pretreated with poly(ethyleneimmine) (PEI). Microsphere constructs that contain DEXA and DHEA showed a significantly higher number of specific lacuna phenotypes at the end of a 4-week study in vitro and at the end of a 6-week study in vivo, irrespective of the presence of DEXA and DHEA. Therefore, the dual delivery of DEXA and DHEA can be used to engineer inflammation-free tissue in the vicinity of the implant. These double beaded PLGA microsphere constructs containing DEXA and DHEA show promise as coatings for implantable biomedical devices to improve biocompatibility and ensure in vivo performance.

PLGA microsphere construct coated with TGF-beta 3 loaded nanoparticles for neocartilage formation.

Polymeric microsphere system has been widely used in tissue-regeneration matrix and drug delivery systems. To apply these biomaterials as novel cell supporting matrix for stem cell delivery, we have devised a novel method for the fabrication of nanostructured 3D scaffolds that growth factor loaded heparin/poly(L-lysine) nanoparticles were physically attached on the positively charged surface of PLGA microspheres precoated with low molecular weight of poly(ethyleneimmine) (PEI) via a layer-by-layer (LbL) system. Based on a previous study, we have prepared poly(lactide-co-glycolide) (PLGA) microspheres harboring heparin/poly(L-lysine) loaded with growth factors. Growth factor loaded heparin/poly(L-lysine) nanoparticles, which were simply produced as polyion complex micelles (PICM) with diameters of 50-150 nm, were fabricated in the first step. Microsphere matrix (size, 20 approximately 80 nm) containing TGF-beta 3 showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study in vitro culture of mesenchymal stem cells. Thus, growth factor delivery of PLGA microsphere can be used to engineer synthetic extracellular matrix. This PLGA microsphere matrix containing TGF-beta 3 showed promise as coatings for implantable biomedical devices to improve biocompatibility and ensure in vivo performance.

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2).

The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Synergistic effect of TGFbeta-3 on chondrogenic differentiation of rabbit chondrocytes in thermo-reversible hydrogel constructs blended with hyaluronic acid by in vivo test.

In this study, a hydrogel composite, based on the thermo-reversible hydrogel of p(NiPAAm-co-AAc) and hyaluronic acid (HA) was used as an injectable cell and growth factor carrier for cartilage tissue engineering applications. Rabbit chondrocytes were embedded in blended hydrogel composites co-encapsulated with the transforming growth factor beta-3 (TGFbeta-3). The blended hydrogel with the embedded chondrocytes and HA co-encapsulating unloaded growth factors and those with the thermo-reversible hydrogel were used as the controls to examine the effects of TGFbeta-3 on neocartilage formation. The blended hydrogel loaded with TGFbeta-3 embedded with chondrocytes were injected subcutaneously into the nude mice. The mice were monitored for 8 weeks after the injection. Both the differentiation and level of cartilage-specific ECM production were significantly higher in the presence of HA and growth factor than in the control without the growth factor. The level of cartilage associated ECM proteins was examined by immunohistochemical staining (collagen types II and X) as well as by Safranin-O and Alcian blue (GAG) staining. The results showed the potential application of blended hydrogel mixed with the growth factor to neocartilage formation.

Combination material delivery of dexamethasone and growth factor in hydrogel blended with hyaluronic acid constructs for neocartilage formation.

The aim of this study was to assess the efficacy of poly(NiPAAm-co-AAc) blended with hyaluronic acid (HA) as an injectable cell vehicle and a cell therapeutic agent in the form of a supporting matrix for the chondrogenic differentiation of rabbit chondrocytes. Specially, rabbit chondrocytes were embedded in blended hydrogels co-encapsulation with dexamethasone (Dex) and growth factors for enhancing the chondrogenic differentiation. Blended hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 or unloaded Dex and sTGF beta-3 served as controls to assess the effects of Dex on chondrogenic differentiation. Hydrogel constructs consisting of embedded cells co-encapsulating Dex and TGF beta-3 on chondrogenic differentiation. The hydrogel constructs were injected subcutaneously into the nude mice and monitored for 1, 4, and 8 weeks after the injection. The level of the cartilage-associated ECM proteins was determined by immunohistochemical (collagen type II; specific marker for chondrogenic differentiation), Safranin-O, and Alcian blue (GAG) staining. Over the same time period, the glycosamingoglycan content per cell remained constant for all formulations, indicating that the dramatic increase in cell number for samples with Dex and TGF beta-3 loaded in hydrogel constructs was accompanied by maintenance of the cell phenotypes.

Tumor-derived IL-18 induces PD-1 expression on immunosuppressive NK cells in triple-negative breast cancer.

PURPOSE: While the inflammatory cytokine interleukin-18 (IL-18) is known to activate natural killer (NK) cells, its precise role in cancer is controversial. In this study, we investigated the role of tumor-derived IL-18 on peripheral blood NK cells in breast cancer patients. RESULTS: In breast cancer cell lines, IL-18 was expressed and secreted in the triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and HCC-70 but not in MCF-7 cells. The immature and non-cytotoxic CD56dimCD16dim/- NK cell fraction was increased following co-culture with MDA-MB-231 cells, and this increase was not observed with tumor cells transfected with siRNA for IL-18 or in MCF-7 cells. In addition, tumor-derived IL-18 increased PD-1 expression on CD56dimCD16dim/- NK cells, although no effect on PD-L1 expression in tumor cells was observed. Among EBC patients, serum IL-18 levels were significantly increased in those with a TNBC subtype compared to levels from patients with other subtypes, and the IL-18 levels were strongly associated with poor survival. Similarly, serum IL-18 and CD56dimCD16dim/- NK cells were also increased in patients with metastatic TNBC who had progressive disease following cytotoxic chemotherapy. EXPERIMENTAL DESIGN: We performed in vitro experiments in breast cancer cell lines, measured cytokine levels by RT-qPCR, western blot, and ELISA, and analyzed NK cell subsets by flow cytometry. For clinical validation, we collected and analyzed blood sample from patients with early breast cancer (EBC, N = 545) and metastatic breast cancer (MBC, N = 42). CONCLUSIONS: Our data revealed that tumor-derived IL-18 is associated with bad prognosis in patients with TNBC. Tumor-derived IL-18 increased the immunosuppressive CD56dimCD16dim/- NK cell fraction and induced PD-1 expression on these NK cells.

Neoangiogenesis of human mesenchymal stem cells transfected with peptide-loaded and gene-coated PLGA nanoparticles.

Several factors are involved in angiogenesis. To form new blood vessels, we fabricated vehicles carrying an angiogenesis-related peptide (apelin) and gene (vascular endothelial growth factor (VEGF)165) that were internalized by human mesenchymal stem cells (hMSCs). These non-toxic poly-(DL)-lactic-co-glycolic acid (PLGA) nanoparticles (NPs) easily entered hMSCs without cytotoxicity. The negatively charged outer surface of PLGA NPs can be easily complexed with highly positively charged polyethylenimine (PEI) to deliver genes into cells. PLGA NPs complexed with PEI could be coated with negatively charged VEGF plasmid DNA and loaded with apelin. The physical characteristics of these PLGA NPs were determined by size distribution, gel retardation, and morphological analyses. Transfection of VEGF-coated apelin-loaded PLGA NPs resulted in the differentiation of hMSCs into endothelial cells and vascular formation in Matrigel in vitro. Following injection of hMSCs transfected with these PLGA NPs into an ischemic hind limb mouse model, these cells differentiated into endothelial cells and accelerated neovascularization.

Carboxymethylcellulose (CMC) formed nanogels with branched poly(ethyleneimine) (bPEI) for inhibition of cytotoxicity in human MSCs as a gene delivery vehicles.

Specific vehicles are necessary for safe and efficient gene transfection into cells. Nano-type hydrogels (nanogel) comprising carboxymethylcellulose (CMC) complexed with branched type cationic poly(ethleneimine) (bPEI) were used as gene delivery vehicles. When complexes of CMC and bPEI were used in vitro, CMC showed nano-gel type properties, as shown by the results of a viscosity test, and bPEI showed low cytotoxicity comparing to bPEI alone. Together, these properties are shown to maintain high gene transfection efficiency. In viability experiments using three types of adult stem cells, cell viability varied depending on the branch form of PEI and whether or not it is in a complex with CMC. The gene delivery efficacy showed that the CMC nanogel complexed with bPEI (CMC-bPEI) showed more uptaking and gene transfection ability in hMSCs comparing to bPEI alone. In osteogenesis, the CMC-bPEI complexed with OSX pDNA showed more easy internalization than bPEI alone complexed with OSX pDNA in hMSCs. Specific genes and proteins related in osteogenic differentiation were expressed in hMSCs when the CMC-bPEI complexed with OSX pDNA was used.

Neurogenesis Is Induced by Electrical Stimulation of Human Mesenchymal Stem Cells Co-Cultured With Mature Neuronal Cells.

For electrical stimulation of hMSCs, gold nanoparticles were coated onto polyethyleneimine coated glass cover slips. The effects of pulsed or constant electrical stimulation upon cytotoxicity and differentiation of hMSCs were examined. The effects of co culturing hMSCs with neuronal cells were also tested. The neuronal differentiation of the stem cells was evaluated by determining the expression of neuron-specific genes and proteins using RT-PCR and Western blotting. Morphological changes were evaluated by scanning electron microscopy. The hMSCs co-cultured with mature neuronal cells and stimulated with electrical shock showed the greatest level of neurite outgrowth (>150 mm) and smaller cell body sizes.

Aggrecan- and COMP-loaded poly-(lactic-co-glycolic acid) nanoparticles stimulate chondrogenic differentiation of human mesenchymal stem cells.

During embryogenesis, specific proteins expressed in cells have key roles in the formation of differentiated cells and tissues. Delivery of specific proteins into specific cells, both in vitro and in vivo, has proved to be exceedingly difficult. In this study, we developed a safe and efficient protein delivery system using encapsulation of proteins into biodegradable poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The PLGA NPs were used to deliver proteins into human mesenchymal stem cells (hMSCs). Fluorescent markers loaded into the PLGA NPs were used to verify the internalization of NPs into hMSCs using FACS analysis and confocal microscopy. With these methods, we demonstrated that the encapsulated model proteins are readily delivered into hMSCs, released from the NP vehicles, and, finally, moved into the cytosols. Using chondrogenesis-related proteins such as aggrecan and cartilage oligomeric matrix protein (COMP), chondrogenic differentiation of hMSCs treated with aggrecan and COMP encapsulated PLGA NPs was clearly observed and caused to differentiate into chondrocytes.

The effect of quantum dot size and poly(ethylenimine) coating on the efficiency of gene delivery into human mesenchymal stem cells.

Quantum dot (QDs) have been employed as bioimaging agents and delivery vehicles for gene therapeutics in several types of cells. In this study, we fabricated multiple QD bundled nanoparticles (NPs) to investigate the effect of QD size and poly(ethylenimine) (PEI) coating on the efficiency of gene delivery into human mesenchymal stem cells (hMSCs). Several types of QDs, which exhibit different ranges of particle size and fluorescence when employed, were coated with PEI to alter their negative charges and to enable them to be bundled into larger particles. Using specific wavelengths of QDs for bioimaging, gene-complexed QD bundled NPs were easily detected in the hMSCs using several different methods such as fluorescence-activated cell sorter, confocal laser scanning microscopy, and in vivo optical imaging. These PEI-coated, bundled QD NPs exhibited significantly higher gene transfection efficacy than single-type QDs. Particularly, the largest QD bundled NPs examined, QD655, had a much higher uptake capability and greater gene expression ability than the other QD NPs (QD525, QD565, and QD605). We believe that our findings help to enrich knowledge of design considerations that will aid in the engineering of QD NPs for stem cell application in the future.

Multi-modal transfection agent based on monodisperse magnetic nanoparticles for stem cell gene delivery and tracking.

Directing the controlled differentiation and tracking of stem cells is essential to achieve successful stem cell therapy. In this work, we describe a multi-modal (MR/optical) transfection agent (MTA) for efficient gene delivery and cell tracking of human mesenchymal stem cells (hMSCs). The MTA was synthesized through a facile two-step approach with 1) ligand exchange of a catechol-functionalized polypeptide (CFP) and 2) chemical immobilization of fluorescence labelled cationic polymer via aminolysis reaction. Cationic polymer-immobilized MTAs with size of ~40 nm exhibit greatly enhanced colloidal stability in aqueous solution. In addition, the MTAs were capable of binding DNA molecules for transfection. The MTA/pDNA complex showed relatively good transfection efficiency in hMSCs (compared to the commercial transfection agent, Lipofectamine) and good biocompatibility. MTA-treated hMSCs were successfully visualized after transplantation via MR and optical imaging system over 14 days. These studies highlight the challenges associated with the potential advantages of designing multi-modal nanostructured materials as tools for genetic materials delivery and cell-tracking in stem cell therapy.

Differentiation of endothelial progenitor cells into endothelial cells by heparin-modified supramolecular pluronic nanogels encapsulating bFGF and complexed with VEGF165 genes.

Specific genes and growth factors are involved in stem cell differentiation. In this study, we fabricated a delivery carrier for both protein and gene delivery that was introduced into human endothelial progenitor cells (EPCs). The highly negative charge carried by the heparin-modified pluronic nanogels allowed for binding to growth factors and localization in the core of nanogels. The residues of negatively charged heparin can complex with positively charged cationic materials, making it suitable for gene delivery. Supramolecular nanogels can be easily encapsulated the hydrophilic drugs and highly positive surfaces can be complexed with negative charge carrying plasmid DNA (pDNA). The size distribution, gel retardation, and denaturation of encapsulated growth factors and supramolecular nanogels modified with heparin were evaluated. The supramolecular nanogels containing basic fibroblast growth factors and complexing VEGF165 pDNA internalized into EPCs have been well formed vascular formation in matrigel gels. Proteins and genes introduced into EPCs using nanogels promoted neovascularization in an animal model of limb ischemia. EPCs that differentiated into endothelial cells both in vitro and in vivo were tested.

Co-delivery of Cbfa-1-targeting siRNA and SOX9 protein using PLGA nanoparticles to induce chondrogenesis of human mesenchymal stem cells.

During stem cell differentiation, various cellular responses occur that are mediated by transcription factors and proteins. This study evaluated the abilities of SOX9, a crucial protein during the early stage of chondrogenesis, and siRNA targeting Cbfa-1, a transcription factor that promotes osteogenesis, to stimulate chondrogenesis. Non-toxic poly-(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) were coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. Coomassie blue staining and circular dichroism revealed that the loaded SOX9 protein maintained its stability and bioactivity. These NPs easily entered human mesenchymal stem cells (hMSCs) in vitro and caused them to differentiate into chondrocytes. Markers that are typically expressed in mature chondrocytes were examined. These markers were highly expressed at the mRNA and protein levels in hMSCs treated with PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. By contrast, these cells did not express osteogenesis-related markers. hMSCs were injected into mice following internalization of PLGA NPs coated with Cbfa-1-targeting siRNA and loaded with SOX9 protein. When the injection site was excised, markers of chondrogenesis were found to be highly expressed at the mRNA and protein levels, similar to the in vitro results. When hMSCs internalized these NPs and were then cultured in vitro or injected into mice, chondrogenesis-related extracellular matrix components were highly expressed.

Poly(N-isopropylacrylamide-co-acrylic acid) nanogels for tracing and delivering genes to human mesenchymal stem cells.

Drugs, proteins, and cells can be macro- and micro-encapsulated by unique materials that respond to specific stimuli. The phases and hydrophobic interactions of these materials are reversibly altered by environmental stimuli such as pH and temperature. These changes can lead to self-assembly of the materials, which enables controlled drug release and safe gene delivery into cells and tissues. The fate of stem cells delivered by such methods is of great interest. The formation of transgenic tissues requires genes to be delivered safely into stem cells. A cell tracing vehicle and a gene delivery carrier were simultaneously introduced into human mesenchymal stem cells (hMSCs). A thermo-sensitive hydrogel, poly(N-isopropylacrylamide-co-acrylic acid) (p(NiPAAm-co-AAc)), was created to generate self-assembled nanoparticles with nanogel characteristics. Hydrophobic interactions mediated the binding of the carboxyl group on the outside of p(NiPAAm-co-AAc) with the amine group of iron oxide. Nanogels carrying iron oxide and a fluorescent dye were complexed with specific genes. These nanogels could be internalized by hMSCs, and the transplantation of these cells into mice was monitored by in vivo imaging. Self-assembled p(NiPAAm-co-dAAc) nanogels complexed with green fluorescent protein were highly expressed in hMSCs and are a potential material for gene delivery.

Multilineage differentiation of human-derived dermal fibroblasts transfected with genes coated on PLGA nanoparticles plus growth factors.

Wounded tissues and cells may be treated with growth factors and specific genes for the purpose of tissue repair and regeneration. To deliver specific genes into tissues and cells, this study presents the use of fabricated poly (DL-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) complexed with the cationic polymer poly (ethleneimine) (PEI). Through complexation with PEI, several types of genes (SOX9, Cbfa1, and C/EBP-α) were coated into PLGA NPs, which enhanced gene uptake into normal human-derived dermal fibroblast cells (NFDHCs) in vitro and in vivo. Several cell types (293T, HeLa, and fibroblast cells) were transfected with fluorescence-tagged PEI/SOX9, PEI/Cbfa1, and PEI/C/EBP-α gene-complexed PLGA NPs. The gene and protein expression levels in the cells were evaluated by RT-PCR, real-time quantitative PCR, Western blotting, and confocal laser microscopy. Fibroblast cells encapsulated in fibrin gels were transfected with the gene-complexed NPs plus specific growth factors (TGF-ß3, BMP-2, or IGF/bFGF), which induced chondrogenesis, osteogenesis, or adipogenesis both in vitro and after transplantation into nude mouse.