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Hypoxia is a hallmark of tumor microenvironments, exerting wide-ranging impacts on key processes of tumor progression and metastasis. However, our understanding of how hypoxia regulates these processes has been based primarily on studying the effects of hypoxia within the primary tumor. Recently, an increasing number of studies have suggested the importance of hypoxic regulation within metastatic target organs, but hypoxic metastatic niches in the body are difficult to access with current imaging techniques, hampering detailed in vivo investigation of hypoxia at metastatic sites. Here, we report an engineered biomaterial scaffold that is able to establish an in vivo hypoxic metastatic niche in a readily accessible area, enabling the investigation of hypoxic regulation at a metastatic site. We engineered hypoxic environments within microporous poly(lactide-co-glycolide) (PLG) scaffolds, which have previously been shown to act as premetastatic niche mimics, via the addition of CoCl2, a hypoxia-mimetic agent...

Irreversible electroporation (IRE) is an emerging technology for non-thermal ablation of solid tumors. This study sought to integrate electrodes into microporous poly(caprolactone) (PCL) scaffolds previously shown to recruit metastasizing cancer cells in vivo in order to facilitate application of IRE to disseminating cancer cells. As the ideal parallel plate geometry would render much of the porous scaffold surface inaccessible to infiltrating cells, numerical modeling was utilized to predict the spatial profile of electric field strength within the scaffold for alternative electrode designs. Metal mesh electrodes with 0.35 mm aperture and 0.16 mm wire diameter established electric fields with similar spatial uniformity as the parallel plate geometry. Composite PCL-IRE scaffolds were fabricated by placing cylindrical porous PCL scaffolds between two PCL dip-coated stainless steel wire meshes. PCL-IRE scaffolds exhibited no difference in cell infiltration in vivo compared to PCL scaffolds. In addition, upon application of IRE in vivo, cells infiltrating the PCL-IRE scaffolds were successfully ablated, as determined by histological analysis 3 days post-treatment. The ability to establish homogeneous electric fields within a biomaterial that can recruit metastatic cancer cells, especially when combined with immunotherapy, may further advance IRE technology beyond solid tumors to the treatment of systemic cancer.

Advanced stage ovarian cancer is challenging to treat due to widespread seeding of tumor spheroids throughout the mesothelial lining of the peritoneal cavity. In this work, a therapeutic strategy using graphene nanoribbons (GNR) functionalized with 4-arm polyethylene glycol (PEG) and chlorin e6 (Ce6), a sonosensitizer, to target metastatic ovarian cancer spheroids is reported. GNR-PEG-Ce6 adsorbs onto the spheroids and disrupts their adhesion to extracellular matrix proteins or LP-9 mesothelial cells. Furthermore, for spheroids that do adhere, GNR-PEG-Ce6 delays spheroid disaggregation and spreading as well as mesothelial clearance, key metastatic processes following adhesion. Owing to the sonodynamic effects of Ce6 and its localized delivery via the biomaterial, GNR-PEG-Ce6 can kill ovarian cancer spheroids adhered to LP-9 cell monolayers when combined with mild ultrasound irradiation. The interaction with GNR-PEG-Ce6 also loosens cell-cell adhesions within the spheroids, rendering them more susceptible to treatment with the chemotherapeutic agents cisplatin and paclitaxel, which typically have difficulty in penetrating ovarian cancer spheroids. Thus, this material can facilitate effective chemotherapeutic and sonodynamic combination therapies. Finally, the adhesion inhibiting and sonodynamic effects of GNR-PEG-Ce6 are also validated with tumor spheroids derived from the ascites fluid of ovarian cancer patients, providing evidence of the translational potential of this biomaterial approach.

Biophysical cues, such as topography, and electrical cues can provide external stimulation for the promotion of stem cell neurogenesis. Here, we demonstrate an electroconductive surface nanotopography for enhancing neuronal differentiation and the functional maturation of human neural stem cells (hNSCs). The electroconductive nanopatterned substrates were prepared by depositing a thin layer of titanium (Ti) with nanograting topographies (150 to 300 nm groove/ridge, the thickness of the groove - 150 μm) onto polymer surfaces. The Ti-coated nanopatterned substrate (TNS) induced cellular alignment along the groove pattern via contact guidance and promoted focal adhesion and cytoskeletal reorganization, which ultimately led to enhanced neuronal differentiation and maturation of hNSCs as indicated by significantly elevated neurite extension and the upregulated expression of the neuronal markers Tuj1 and NeuN compared with the Ti-coated flat substrate (TFS) and the nanopatterned substrate (NS) without Ti coating. Mechanosensitive cellular events, such as β1-integrin binding/clustering and myosin-actin interaction, and the Rho-associated protein kinase (ROCK) and mitogen-activated protein kinase/extracellular signal regulated kinase (MEK-ERK) pathways, were found to be associated with enhanced focal adhesion and neuronal differentiation of hNSCs by the TNS. Among the neuronal subtypes, differentiation into dopaminergic and glutamatergic neurons was promoted on the TNS. Importantly, the TNS increased the induction rate of neuron-like cells exhibiting electrophysiological properties from hNSCs. Finally, the application of pulsed electrical stimulation to the TNS further enhanced neuronal differentiation of hNSCs due probably to calcium channel activation, indicating a combined effect of topographical and electrical cues on stem cell neurogenesis, which postulates the novelty of our current study. The present work suggests that an electroconductive nanopatterned substrate can serve as an effective culture platform for deriving highly mature, functional neuronal lineage cells from stem cells.

In vitro culture systems for primary neurons have served as useful tools for neuroscience research. However, conventional in vitro culture methods are still plagued by challenging problems with respect to applications to neurodegenerative disease models or neuron-based biosensors and neural chips, which commonly require long-term culture of neural cells. These impediments highlight the necessity of developing a platform capable of sustaining neural activity over months. Here, we designed a series of polymeric bilayers composed of poly(glycidyl methacrylate) (pGMA) and poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), designated pGMA:pDMAEMA, using initiated chemical vapor deposition (iCVD). Harnessing the surface-growing characteristics of iCVD polymer films, we were able to precisely engraft acetylcholine-like functionalities (tertiary amine and quaternary ammonium) onto cell culture plates. Notably, pGD3, a pGMA:pDMAEMA preparation with the highest surface composition of quaternary ammonium, fostered the most rapid outgrowth of neural cells. Clear contrasts in neural growth and survival between pGD3 and poly-l-lysine (PLL)-coated surfaces became apparent after 30 days in vitro (DIV). Moreover, brain-derived neurotrophic factor level continuously accumulated in pGD3-cultured neurons, reaching a 3-fold increase at 50 DIV. Electrophysiological measurements at 30 DIV revealed that the pGD3 surface not only promoted healthy maturation of hippocampal neurons but also enhanced the function of hippocampal ionotropic glutamate receptors in response to synaptic glutamate release. Neurons cultured long-term on pGD3 also maintained their characteristic depolarization-induced Ca

There is an urgent need for development of alternative treatment strategies for advanced-stage ovarian cancer. During metastatic spread, single cells or spheroids detach from the primary tumor and are carried by the peritoneal fluid until attaching to the mesothelial cells lining the abdominal cavity. While attachment of ovarian tumor cells and spheroids to the mesothelial layer is known to be mediated by β-integrins and CD44, efforts to block these receptors with antibodies have been insufficient to inhibit disease progression. As graphene oxide nanosheets have previously been shown bind to β-integrins on the surface of mesenchymal stem cells, we hypothesized that they would be able to attach to ovarian cancer spheroids, forming a physical barrier around the spheroids and thus inhibiting their adhesion to the mesothelium. SKOV-3 human ovarian cancer cells were used in this study, as this cell line was derived from ascites fluid. SKOV-3 cells were placed in poly(hydroxyethyl methacrylate) (polyHEMA)-coated plates for 24 hours to form spheroids, followed by 48-hour treatment with the nanosheets. The graphene oxide nanosheets were functionalized with 4-arm polyethylene glycol (PEG) to enhance biocompatibility and provide additional steric hindrance to prevent spheroid adhesion. Scanning electron microscopy confirmed that the nanosheets were able to bind to the tumor spheroids. Association of the nanosheets with the SKOV-3 spheroids reduced their adhesion to extracellular matrix proteins collagen and fibronectin as well as LP-9 human mesothelial cells over a 7-day time period, with the PEG-functionalized nanosheets showing the greatest reduction in adhesion. The nanomaterials did not exhibit cytotoxicity to LP-9 cells at the range of concentrations used to prevent adhesion. Receptor blocking studies confirmed that the nanosheets were predominantly engaging with the tumor spheroids via β-integrins. Finally, for tumor spheroids that did bind to the mesothelial cells, the presence of nanosheets on the surface of the spheroids decreased the rate of mesothelial layer clearance by the tumor cells. Taken together, these results demonstrate that graphene oxide nanomaterials can bind to ovarian tumor spheroids and inhibit their attachment to matrix proteins and mesothelial cells, while also reducing their ability to clear mesothelial cells once bound. Future efforts are focused on testing these materials with human ascites fluid and further modifying the nanosheets to enable ablation of tumor cells bound to the nanosheets to enhance their therapeutic potential. Citation Format: Hak Rae Lee, Melissa A. Geller, Samira M. Azarin. Inhibition of ovarian cancer spheroid adhesion using graphene oxide nanomaterials [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B52.

Although cancer stem cells (CSC) are thought to be responsible for tumor recurrence and resistance to chemotherapy, CSC-related research and drug development have been hampered by the limited supply of diverse, patient-derived CSC. Here, we present a functional polymer thin film (PTF) platform that promotes conversion of cancer cells to highly tumorigenic three-dimensional (3D) spheroids without the use of biochemical or genetic manipulations. Culturing various human cancer cells on the specific PTF, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), gave rise to numerous multicellular tumor spheroids within 24 hours with high efficiency and reproducibility. Cancer cells in the resulting spheroids showed a significant increase in the expression of CSC-associated genes and acquired increased drug resistance compared with two-dimensional monolayer-cultured controls. These spheroids also exhibited enhanced xenograft tumor-forming ability and metastatic capacity in nude mice. By enabling the generation of tumorigenic spheroids from diverse cancer cells, the surface platform described here harbors the potential to contribute to CSC-related basic research and drug development. Significance: A new cell culture technology enables highly tumorigenic 3D spheroids to be easily generated from various cancer cell sources in the common laboratory.

In this study, we present a method for assembling biofunctionalized paper into a multiform structured scaffold system for reliable tissue regeneration using an origami-based approach. The surface of a paper was conformally modified with a poly(styrene-co-maleic anhydride) layer via initiated chemical vapor deposition followed by the immobilization of poly-l-lysine (PLL) and deposition of Ca(2+). This procedure ensures the formation of alginate hydrogel on the paper due to Ca(2+) diffusion. Furthermore, strong adhesion of the alginate hydrogel on the paper onto the paper substrate was achieved due to an electrostatic interaction between the alginate and PLL. The developed scaffold system was versatile and allowed area-selective cell seeding. Also, the hydrogel-laden paper could be folded freely into 3D tissue-like structures using a simple origami-based method. The cylindrically constructed paper scaffold system with chondrocytes was applied into a three-ring defect trachea in rabbits. The transplanted engineered tissues replaced the native trachea without stenosis after 4 wks. As for the custom-built scaffold system, the hydrogel-laden paper system will provide a robust and facile method for the formation of tissues mimicking native tissue constructs.

Initiated chemical vapor deposition (iCVD) was utilized to generate a 200nm thick, uniform, functionalized polymer nanolayer comprised of glycidyl methacrylate (GMA) on the surface of titanium implants as a means to improve cellular attachment. Dot-patterned GMA-coated specimens were prepared as well as fully coated specimens. In vitro cellular responses, including cell morphology, protein adsorption, cell proliferation assays, alkaline phosphate activity (ALP) assays, and calcium deposition assays were studied using adipose derived stem cells. The mechanical stability of the thin film was investigated by XPS and FE-SEM analysis of the GMA-coated implant after implantation to an extracted bone from a pig. The GMA-coated specimens displayed increased protein adsorption, higher alkaline phosphatase activities, and higher calcium deposition as compared to control sample with no cytotoxicity. Additionally, no defect was observed in the test of mechanical stability. Notably, dot-patterned GMA-coated samples displayed higher alkaline phosphatase activities than others. Functionalized polymer nanolayer deposition via iCVD is a flexible and robust technique capable of mass production of biocompatible layers. These properties make this technique very suitable for implant applications in a variety of ways.

In the last decade, titanium has been effectively used in the dental field for oral surgery as an implant material. However, disinfected Ti can be easily re-infected by the surrounding environment. Thus, a novel anti-fouling treatment for Ti implants is currently necessary. In this study, we designed an anti-fouling surface comprised of poly N-isopropylacylamide (PIPAAM) grafted Ti by introducing poly glycidyl methacrylate (pGMA) coating via an initiated chemical vapor deposition (iCVD) system to prevent bacterial infection. The results indicate that pristine Ti was well coated with pGMA with a film thickness of approximately 60 nm and uniformly grafted with PIPAAM. The bacteria were effectively detached after rinsing with a buffer solution at room temperature, while hADSCs were well attached on the surface treated Ti surface at oral temperature. All tests clearly confirm that our strategy may be a useful means of imparting anti-fouling characteristics to Ti in order to prevent bacterial adhesion and resultant peri-implantitis.

Over the past few decades, titanium (Ti) implants have been widely used to repair fractured bones. To promote osteogenesis, immobilization of osteoinductive agents, such as recombinant human bone morphogenic protein-2 (rhBMP2), onto the Ti surface is required. In this study, we prepared rhBMP2 immobilized on glycidyl methacrylate (GMA) deposited Ti surface through initiated chemical vapor deposition (iCVD) technique. After preparation, the bio-functionalized Ti surface was characterized by physicochemical analysis. For in vitro analysis, the developed Ti was evaluated by cell proliferation, alkaline phosphatase activity, calcium deposition, and real-time polymerase chain reaction to verify their osteogenic activity against human adipose-derived stem cells (hASCs). The GMA deposited Ti surface was found to effectively immobilize a large dose of rhBMP2 as compared to untreated Ti. Additionally, rhBMP2 immobilized on Ti showed significantly enhanced osteogenic differentiation and increased calcium deposition with nontoxic cell viability. These results clearly confirm that our strategy may provide a simple, solvent-free strategy to prepare an osteoinductive Ti surface for bone tissue engineering applications.

Various biophysical and biochemical factors are important for determining the fate of neural stem cells (NSCs). Among biophysical signals, topographical stimulation by micro/nanopatterns has been applied to control NSC differentiation. In this study, we developed a hierarchically patterned substrate (HPS) platform that can synergistically enhance the differentiation of human NSCs (hNSCs) by simultaneously providing microscale and nanoscale spatial controls to facilitate the alignment of the cytoskeleton and the formation of focal adhesions. The multiscale HPS was fabricated by combining microgroove patterns (groove size: 1.5 μm), prepared by a conventional photolithographic process, and nanopore patterns (pore diameter: 10 nm), prepared from cylinder-forming block copolymer thin films. The hNSCs grown on the HPS exhibited not only a highly aligned, elongated morphology, but also a greatly enhanced differentiation into neuronal and astrocyte lineages, compared to hNSCs on a flat substrate (FS) or single-type patterned substrates [microgroove patterned substrate (MPS) and nanopore patterned substrate (NPS)]. Interestingly, the application of the HPS directed hNSC differentiation toward neurons rather than astrocytes. The expression of focal adhesion proteins in hNSCs was also significantly increased on the HPS compared to the FS, MPS, and NPS, likely a result of the presence of more focal contact points provided by nanopore structures. Inhibition of both β1 integrin-mediated binding and the intracellular Rho-associated protein kinase pathway of hNSCs eliminated the beneficial effects of the HPS on focal adhesion formation and actin filament alignment, which subsequently reduced hNSC differentiation. More importantly, hNSCs on the HPS differentiated into functional neurons exhibiting sodium currents and action potentials. The multiscale, hierarchically patterned topography would be useful for the design of functional biomaterial scaffolds to potentiate NSC therapeutic efficacy.

Despite advances in standard of care treatment, a significant number of ovarian cancer patients will exhibit disease recurrence, either due to the presence of chemoresistant cells or the reawakening of dormant tumor cells. Currently there are no methods to predict which patients are likely to recur, and the mechanisms that allow a unique subpopulation of cancer cells to enter dormancy and survive chemotherapy remain unclear. Since chemotherapy is more effective at destroying highly proliferative cells, it is theorized that tumor cells capable of entering a dormant state are also the same population able to evade initial treatment and ultimately engender chemoresistance. A silica gel encapsulation platform that uses physical confinement to inhibit proliferation has previously demonstrated the ability to distinguish between dormancy-capable and dormancy-incapable breast cancer cell lines. In this work, we sought to apply this platform to an ovarian cancer cell line and determine if it could be used to select for the dormancy-capable subset of cells and to detect differences upon enrichment of dormant or chemoresistant cell populations. Upon encapsulation in silica gels, surviving OVCAR-3 cells demonstrated hallmarks of dormancy, including reduction in Ki67 expression, modulation of key cell cycle regulatory genes and dormancy-associated signaling pathways, and ability to resume proliferation when the stress of physical confinement was removed. Moreover, enrichment of the dormant subpopulation using hypoxia treatment or serum starvation prior to silica gel encapsulation resulted in enhanced survival in gels relative to cells grown in standard culture conditions. These results suggest that silica gel encapsulation may be used as a simple method to isolate dormancy-capable cells for further applications such as gene or protein expression analysis and drug screening. Similarly, cells already in a dormant state demonstrated lower susceptibility to cisplatin drug treatment relative to actively proliferating cells, further supporting the idea that dormant cancer cells are less susceptible to chemotherapy and that chemoresistance may stem from a subpopulation of dormant cancer cells. After extraction from silica gels and subsequent return to a proliferative state, cells surviving prolonged encapsulation also demonstrated enhanced survival after cisplatin treatment relative to control populations. This suggests that the dormancy-capable cells selected for within silica gels are indeed more chemoresistant, even when no longer dormant, and that the ability to enter dormancy and chemoresistance in ovarian cancer are likely somehow connected. Lastly, when the drug-resistant phenotype was selected for through cisplatin drug treatment prior to encapsulation, a significant difference in viability was observed upon encapsulation between cisplatin-treated and untreated cells. Overall, these results indicate that the silica gel technology and the behavior of ovarian cancer cells after encapsulation may be an indicator of cells inherently capable of entering dormancy and less susceptible to platinum-based chemotherapies. In future applications, silica gel encapsulation of patient ovarian cancer cells could potentially be used as a predictive clinical tool to identify patients at risk of early recurrence or as a facile in vitro platform to investigate mechanistic links between dormancy, chemoresistance, and recurrence. Citation Format: Tiffany Lam, Hak Rae Lee, Melissa A. Geller, Alptekin Aksan, Samira Azarin. SILICA GEL ENCAPSULATION AS A POTENTIAL TOOL TO IDENTIFY DORMANT AND DRUG-RESISTANT CELL POPULATIONS IN OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-008.

Gold nanoparticles (GNPs) are quite attractive materials for use as osteogenic agents due to their potential effects on the stimulation of osteoblast differentiation. In this study, an osseo-integrated titanium (Ti) implant surface coated with GNPs was used for promotion of bone regeneration. We prepared a silanized Ti surface by chemical treatment of (3-Mercaptopropyl) trimethoxysilane (MPTMS) and immobilized the GNP layer (Ti-GNP) on their surfaces via Au-S bonding. The GNP layer is uniformly immobilized on the surface and the layer covers the titanium oxide surface well, as confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The Ti-GNP was used to investigate the effectiveness of this system both in vitro and in vivo. The in vitro results showed that the Ti-GNP significantly enhances the osteogenic differentiation with increased mRNA expression of osteogenic differentiation specific genes in human adipose-derived stem cells (ADSCs). Furthermore, the in vivo results showed that Ti-GNP had a significant influence on the osseous interface formation. Through these in vitro and vivo tests, we found that Ti-GNP can be useful as osseo-integration inducing dental implants for formation of an osseous interface and maintenance of nascent bone formation.