Retraction of "Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial".

[This retracts the article DOI: 10.1021/acscentsci.8b00050.].

cPLA2 blockade attenuates S100A7-mediated breast tumorigenicity by inhibiting the immunosuppressive tumor microenvironment.

BACKGROUND: Molecular mechanisms underlying inflammation-associated breast tumor growth are poorly studied. S100A7, a pro-inflammatory molecule has been shown to enhance breast cancer growth and metastasis. However, the S100A7-mediated molecular mechanisms in enhancing tumor growth and metastasis are unclear. METHODS: Human breast cancer tissue and plasma samples were used to analyze the expression of S100A7, cPLA2, and PGE2. S100A7-overexpressing or downregulated human metastatic breast cancer cells were used to evaluate the S100A7-mediated downstream signaling mechanisms. Bi-transgenic mS100a7a15 overexpression, TNBC C3 (1)/Tag transgenic, and humanized patient-derived xenograft mouse models and cPLA2 inhibitor (AACOCF3) were used to investigate the role of S100A7/cPLA2/PGE2 signaling in tumor growth and metastasis. Additionally, CODEX, a highly advanced multiplexed imaging was employed to delineate the effects of S100A7/cPLA2 inhibition on the recruitment of various immune cells. RESULTS: In this study, we found that S100A7 and cPLA2 are highly expressed and correlate with decreased overall survival in breast cancer patients. Further mechanistic studies revealed that S100A7/RAGE signaling promotes the expression of cPLA2 to mediate its oncogenic effects. Pharmacological inhibition of cPLA2 suppressed S100A7-mediated tumor growth and metastasis in multiple pre-clinical models including transgenic and humanized patient-derived xenograft (PDX) mouse models. The attenuation of cPLA2 signaling reduced S100A7-mediated recruitment of immune-suppressive myeloid cells in the tumor microenvironment (TME). Interestingly, we discovered that the S100A7/cPLA2 axis enhances the immunosuppressive microenvironment by increasing prostaglandin E2 (PGE2). Furthermore, CO-Detection by indEXing (CODEX) imaging-based analyses revealed that cPLA2 inhibition increased the infiltration of activated and proliferating CD4+ and CD8+ T cells in the TME. In addition, CD163+ tumor associated-macrophages were positively associated with S100A7 and cPLA2 expression in malignant breast cancer patients. CONCLUSIONS: Our study provides new mechanistic insights on the cross-talk between S100A7/cPLA2 in enhancing breast tumor growth and metastasis by generating an immunosuppressive TME that inhibits the infiltration of cytotoxic T cells. Furthermore, our studies indicate that S100A7/cPLA2 could be used as novel prognostic marker and cPLA2 inhibitors as promising drugs against S100A7-overexpressing aggressive breast cancer.

Oxidation and RGD Modification Affect the Early Neural Differentiation of Murine Embryonic Stem Cells Cultured in Core-Shell Alginate Hydrogel Microcapsules.

Directed neural differentiation of embryonic stem cells (ESCs) has been studied extensively to improve the treatment of neurodegenerative disorders. This can be done through stromal-cell derived inducing activity (SDIA), by culturing ESCs directly on top of a layer of feeder stromal cells. However, the stem cells usually become mixed with the feeder cells during the differentiation process, making it difficult to obtain a pure population of the differentiated cells for further use. To address this issue, a non-planar microfluidic device is used here to encapsulate murine ESCs (mESCs) in the 3D liquid core of microcapsules with an alginate hydrogel shell of different sizes for early neural differentiation through SDIA, by culturing mESC-laden microcapsules over a feeder layer of PA6 cells. Furthermore, the alginate hydrogel shell of the microcapsules is modified via oxidation or RGD peptide conjugation to examine the mechanical and chemical effects on neural differentiation of the encapsulated mESC aggregates. A higher expression of Nestin is observed in the aggregates encapsulated in small (∼300 µm) microcapsules and cultured over the PA6 cell feeder layer. Furthermore, the modification of the alginate with RGD facilitates early neurite extension within the microcapsules. This study demonstrates that the presence of the RGD peptide, the SDIA effect of the PA6 cells, and the absence of leukemia inhibition factor from the medium can lead to the early differentiation of mESCs with extensive neurites within the 3D microenvironment of the small microcapsules. This is the first study to investigate the effects of cell adhesion and degradation of the encapsulation materials for directed neural differentiation of mESCs. The simple modifications (i.e., oxidation and RGD incorporation) of the miniaturized 3D environment for improved early neural differentiation of mESCs may potentially enhance further downstream differentiation of the mESCs into more specialized neurons for therapeutic use and drug screening.

A Quick and Efficient Method for the Generation of Immunomodulatory Mesenchymal Stromal Cell from Human Induced Pluripotent Stem Cell.

Mesenchymal stromal cells (MSCs) have been widely investigated for their regenerative capacity, anti-inflammatory properties and beneficial immunomodulatory effects across multiple clinical indications. Nevertheless, their widespread clinical utilization is limited by the variability in MSC quality, impacted by donor age, metabolism, and disease. Human induced pluripotent stem cells (hiPSCs) generated from readily accessible donor tissues, are a promising source of stable and rejuvenated MSC but differentiation methods generally require prolonged culture and result in low frequencies of stable MSCs. To overcome this limitation, we have optimized a quick and efficient method for hiPSC differentiation into footprint-free MSCs (human induced MSCs [hiMSCs]) in this study. This method capitalizes on the synergistic action of growth factors Wnt3a and Activin A with bone morphogenetic protein-4 (BMP4), leading to an enrichment of MSC after only 4 days of treatment. These hiMSCs demonstrate a significant upregulation of mesenchymal stromal markers (CD105+, CD90+, CD73, and cadherin 11) compared with bone marrow-derived MSCs (bmMSCs), with reduced expression of the pluripotency genes (octamer-binding transcription factor [Oct-4], cellular myelocytomatosis oncogene [c-Myc], Klf4, and Nanog homebox [Nanog]) compared with hiPSC. Moreover, they show improved proliferation capacity in culture without inducing any teratoma formation in vivo. Osteogenesis, chondrogenesis, and adipogenesis assays confirmed the ability of hiMSCs to differentiate into the three different lineages. Secretome analyses showed cytokine profiles compared with bmMSCs. Encapsulated hiMSCs in alginate beads cocultured with osteoarthritic (OA) cartilage explants showed robust immunomodulation, with stimulation of cell growth and proteoglycan production in OA cartilage. Our quick and efficient protocol for derivation of hiMSC from hiPSC, and their encapsulation in microbeads, therefore, presents a reliable and reproducible method to boost the clinical applications of MSCs.

Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture.

Organoids are lumen-containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine-glycine-aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes-associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

Correction to Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial.

[This corrects the article DOI: 10.1021/acscentsci.8b00050.].

A dysfunctional TRPV4-GSK3ß pathway prevents osteoarthritic chondrocytes from sensing changes in extracellular matrix viscoelasticity.

Changes in the composition and viscoelasticity of the extracellular matrix in load-bearing cartilage influence the proliferation and phenotypes of chondrocytes, and are associated with osteoarthritis. However, the underlying molecular mechanism is unknown. Here we show that the viscoelasticity of alginate hydrogels regulates cellular volume in healthy human chondrocytes (with faster stress relaxation allowing cell expansion and slower stress relaxation restricting it) but not in osteoarthritic chondrocytes. Cellular volume regulation in healthy chondrocytes was associated with changes in anabolic gene expression, in the secretion of multiple pro-inflammatory cytokines, and in the modulation of intracellular calcium regulated by the ion-channel protein transient receptor potential cation channel subfamily V member 4 (TRPV4), which controls the phosphorylation of glycogen synthase kinase 3ß (GSK3ß), an enzyme with pleiotropic effects in osteoarthritis. A dysfunctional TRPV4-GSK3ß pathway in osteoarthritic chondrocytes rendered the cells unable to respond to environmental changes in viscoelasticity. Our findings suggest strategies for restoring chondrocyte homeostasis in osteoarthritis.

Encapsulated Mesenchymal Stromal Cell Microbeads Promote Endogenous Regeneration of Osteoarthritic Cartilage Ex Vivo.

The anti-inflammatory secretome of mesenchymal stromal cells (MSCs) is lucrative for the treatment of osteoarthritis (OA), a disease characterized by low-grade inflammation. However, the precise effects of the MSC secretome on patient-derived OA tissue is lacking. To investigate these effects, alginate encapsulated MSCs are co-cultured with patient-derived OA cartilage explants for 8 days. Proteoglycan distribution in OA cartilage explants examined by Safranin O staining is markedly improved when cultured with MSC microbeads as compared to control OA explants cultured alone. Total sulfated glycosaminoglycan (sGAG) content in OA explants is significantly increased upon co-culture with MSC microbeads on day 8. The sGAG released into the culture media is unchanged by the presence of MSC microbeads, suggesting de novo sGAG synthesis in OA explants. Co-culture with MSC microbeads increased the DNA content and Ki67+ cells in OA explants, indicating proliferation. An increase in secreted cytokines IL-10, HGF, and sFAS assessed by multiplex cytokine assay, increased TIMP1 levels, and reduction in percent apoptotic cells in OA explants is noted. Together, data demonstrates that paracrine factors secreted by alginate encapsulated MSCs microbeads in response to OA cartilage, create an anabolic, proliferative, and anti-apoptotic microenvironment inducing endogenous regeneration in clinically relevant, patient-derived OA cartilage.

Bioinspired One Cell Culture Isolates Highly Tumorigenic and Metastatic Cancer Stem Cells Capable of Multilineage Differentiation.

Cancer stem cells (CSCs) are rare cancer cells that are postulated to be responsible for cancer relapse and metastasis. However, CSCs are difficult to isolate and poorly understood. Here, a bioinspired approach for label-free isolation and culture of CSCs, by microencapsulating one cancer cell in the nanoliter-scale hydrogel core of each prehatching embryo-like core-shell microcapsule, is reported. Only a small percentage of the individually microencapsulated cancer cells can proliferate into a cell colony. Gene and protein expression analyses indicate high stemness of the cells in the colonies. Importantly, the colony cells are capable of cross-tissue multilineage (e.g., endothelial, cardiac, neural, and osteogenic) differentiation, which is not observed for "CSCs" isolated using other contemporary approaches. Further studies demonstrate the colony cells are highly tumorigenic, metastatic, and drug resistant. These data show the colony cells obtained with the bioinspired one-cell-culture approach are truly CSCs. Significantly, multiple pathways are identified to upregulate in the CSCs and enrichment of genes related to the pathways is correlated with significantly decreased survival of breast cancer patients. Collectively, this study may provide a valuable method for isolating and culturing CSCs, to facilitate the understanding of cancer biology and etiology and the development of effective CSC-targeted cancer therapies.

Targeted Heating of Mitochondria Greatly Augments Nanoparticle-Mediated Cancer Chemotherapy.

Cancer is the second leading cause of mortality globally. Various nanoparticles have been developed to improve the efficacy and safety of chemotherapy, photothermal therapy, and their combination for treating cancer. However, most of the existing nanoparticles are low in both subcellular precision and drug loading content (<≈5%), and the effect of targeted heating of subcellular organelles on the enhancement of chemotherapy has not been well explored. Here, a hybrid Py@Si-TH nanoparticle is reported to first target cancer cells overexpressed with the variant CD44 via its natural ligand HA on the outermost surface of the nanoparticle before cellular uptake, and then target mitochondria after they are taken up inside cells. In addition, the nanoparticle is ultraefficient for encapsulating doxorubicin hydrochloride (DOX) to form Py@Si-TH-DOX nanoparticle. The encapsulation efficiency is ≈100% at the commonly used low feeding ratio of 1:20 (DOX:empty nanoparticle), and >80% at an ultrahigh feeding ratio of 1:1. In combination with near infrared (NIR, 808 nm) laser irradiation, the tumor weight in the Py@Si-TH-DOX treatment group is 8.5 times less than that in the Py@Si-H-DOX (i.e., DOX-laden nanoparticles without mitochondrial targeting) group, suggesting targeted heating of mitochondria is a valuable strategy for enhancing chemotherapy to combat cancer.

Author Correction: Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

The Supplementary Information originally published with this Article was an older version, in which 'IFN-γ' was misspelled 'INF-γ' in Supplementary Fig. 9 and the ß-Actin blot in Supplementary Fig. 13 was the wrong image. The Supplementary Information has now been replaced.

Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer.

TP53 is the most frequently mutated or deleted gene in triple negative breast cancer (TNBC). Both the loss of TP53 and the lack of targeted therapy are significantly correlated with poor clinical outcomes, making TNBC the only type of breast cancer that has no approved targeted therapies. Through in silico analysis, we identified POLR2A in the TP53-neighbouring region as a collateral vulnerability target in TNBC tumours, suggesting that its inhibition via small interfering RNA (siRNA) may be an amenable approach for TNBC targeted treatment. To enhance bioavailability and improve endo/lysosomal escape of siRNA, we designed pH-activated nanoparticles for augmented cytosolic delivery of POLR2A siRNA (siPol2). Suppression of POLR2A expression with the siPol2-laden nanoparticles leads to enhanced growth reduction of tumours characterized by hemizygous POLR2A loss. These results demonstrate the potential of the pH-responsive nanoparticle and the precise POLR2A targeted therapy in TNBC harbouring the common TP53 genomic alteration.

Enhanced cancer therapy with cold-controlled drug release and photothermal warming enabled by one nanoplatform.

Stimuli-responsive nanoparticles hold great promise for drug delivery to improve the safety and efficacy of cancer therapy. One of the most investigated stimuli-responsive strategies is to induce drug release by heating with laser, ultrasound, or electromagnetic field. More recently, cryosurgery (also called cryotherapy and cryoablation), destruction of diseased tissues by first cooling/freezing and then warming back, has been used to treat various diseases including cancer in the clinic. Here we developed a cold-responsive nanoparticle for controlled drug release as a result of the irreversible disassembly of the nanoparticle when cooled to below ∼10 °C. Furthermore, this nanoparticle can be used to generate localized heating under near infrared (NIR) laser irradiation, which can facilitate the warming process after cooling/freezing during cryosurgery. Indeed, the combination of this cold-responsive nanoparticle with ice cooling and NIR laser irradiation can greatly augment cancer destruction both in vitro and in vivo with no evident systemic toxicity.

Overcoming Ovarian Cancer Drug Resistance with a Cold Responsive Nanomaterial.

Drug resistance due to overexpression of membrane transporters in cancer cells and the existence of cancer stem cells (CSCs) is a major hurdle to effective and safe cancer chemotherapy. Nanoparticles have been explored to overcome cancer drug resistance. However, drug slowly released from nanoparticles can still be efficiently pumped out of drug-resistant cells. Here, a hybrid nanoparticle of phospholipid and polymers is developed to achieve cold-triggered burst release of encapsulated drug. With ice cooling to below ∼12 °C for both burst drug release and reduced membrane transporter activity, binding of the drug with its target in drug-resistant cells is evident, while it is minimal in the cells kept at 37 °C. Moreover, targeted drug delivery with the cold-responsive nanoparticles in combination with ice cooling not only can effectively kill drug-resistant ovarian cancer cells and their CSCs in vitro but also destroy both subcutaneous and orthotopic ovarian tumors in vivo with no evident systemic toxicity.

Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance.

Multidrug resistance is a major challenge to cancer chemotherapy. The multidrug resistance phenotype is associated with the overexpression of the adenosine triphosphate (ATP)-driven transmembrane efflux pumps in cancer cells. Here, we report a lipid membrane-coated silica-carbon (LSC) hybrid nanoparticle that targets mitochondria through pyruvate, to specifically produce reactive oxygen species (ROS) in mitochondria under near-infrared (NIR) laser irradiation. The ROS can oxidize the NADH into NAD+ to reduce the amount of ATP available for the efflux pumps. The treatment with LSC nanoparticles and NIR laser irradiation also reduces the expression and increases the intracellular distribution of the efflux pumps. Consequently, multidrug-resistant cancer cells lose their multidrug resistance capability for at least 5 days, creating a therapeutic window for chemotherapy. Our in vivo data show that the drug-laden LSC nanoparticles in combination with NIR laser treatment can effectively inhibit the growth of multidrug-resistant tumors with no evident systemic toxicity.

A Nano-In-Micro System for Enhanced Stem Cell Therapy of Ischemic Diseases.

Stem cell therapy holds great potential for treating ischemic diseases. However, contemporary methods for local stem cell delivery suffer from poor cell survival/retention after injection. We developed a unique multiscale delivery system by encapsulating therapeutic agent-laden nanoparticles in alginate hydrogel microcapsules and further coentrapping the nano-in-micro capsules with stem cells in collagen hydrogel. The multiscale system exhibits significantly higher mechanical strength and stability than pure collagen hydrogel. Moreover, unlike nanoparticles, the nano-in-micro capsules do not move with surrounding body fluid and are not taken up by the cells. This allows a sustained and localized release of extracellular epidermal growth factor (EGF), a substance that could significantly enhance the proliferation of mesenchymal stem cells while maintaining their multilineage differentiation potential via binding with its receptors on the stem cell surface. As a result, the multiscale system significantly improves the stem cell survival at 8 days after implantation to ∼70% from ∼4-7% for the conventional system with nanoparticle-encapsulated EGF or free EGF in collagen hydrogel. After injecting into the ischemic limbs of mice, stem cells in the multiscale system facilitate tissue regeneration to effectively restore ∼100% blood perfusion in 4 weeks without evident side effects.

Microfluidics Enabled Bottom-Up Engineering of 3D Vascularized Tumor for Drug Discovery.

Development of high-fidelity three-dimensional (3D) models to recapitulate the tumor microenvironment is essential for studying tumor biology and discovering anticancer drugs. Here we report a method to engineer the 3D microenvironment of human tumors, by encapsulating cancer cells in the core of microcapsules with a hydrogel shell for miniaturized 3D culture to obtain avascular microtumors first. The microtumors are then used as the building blocks for assembling with endothelial cells and other stromal cells to create macroscale 3D vascularized tumor. Cells in the engineered 3D microenvironment can yield significantly larger tumors in vivo than 2D-cultured cancer cells. Furthermore, the 3D vascularized tumors are 4.7 and 139.5 times more resistant to doxorubicin hydrochloride (a commonly used chemotherapy drug) than avascular microtumors and 2D-cultured cancer cells, respectively. Moreover, this high drug resistance of the 3D vascularized tumors can be overcome by using nanoparticle-mediated drug delivery. The high-fidelity 3D tumor model may be valuable for studying the effect of microenvironment on tumor progression, invasion, and metastasis and for developing effective therapeutic strategy to fight against cancer.

The effect of RGD peptide on 2D and miniaturized 3D culture of HEPM cells, MSCs, and ADSCs with alginate hydrogel.

Advancements in tissue engineering require the development of new technologies to study cell behavior in vitro. This study focuses on stem cell behavior within various miniaturized three-dimensional (3D) culture conditions of alginate biomaterials modified with the Arg-Gly-Asp (RGD) peptide known for its role in cell adhesion/attachment. Human embryonic palatal mesenchyme (HEPM) cells, bone marrow derived mesenchymal stem cells (MSCs), and human adipose derived stem cells (ADSCs) were cultured on a flat hydrogel of different concentrations of alginate-RGD, and in the miniaturized 3D core of microcapsules with either a 2% alginate or 2% alginate-RGD shell. The core was made of 0%, 0.5%, or 2% alginate-RGD. Cell spreading was observed in all systems containing the RGD peptide, and the cell morphology was quantified by measuring the cell surface area and circularity. In all types of stem cells, there was a significant increase in the cell surface area (p < 0.05) and a significant decrease in cell circularity (p < 0.01) in alginate-RGD conditions, indicating that cells spread much more readily in environments containing the peptide. This control over the cell spreading within a 3D microenvironment can help to create the ideal biomimetic condition in which to conduct further studies on cell behavior.