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Background & Aims Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. Methods We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. Results Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. Conclusions In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells due to lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS: We performed studies with LSL-Kras(G12D/+); Ptf1a(Cre/+) mice (KC, develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice, do not develop Tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice), and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasias (PanINs), 19 patients with intraductal papillary mucinous neoplasms (IPMN), and 197 patients with PDA. RESULTS: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D(2) than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells following administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of PGD(2), compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury, following administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and IPMN had gene expression signatures associated with tuft cells and increased expression of Hpgds mRNA compared with PDA. CONCLUSIONS: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of PGD(2). These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1–6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG–peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG–PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG–RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering.

Migration assays are a common tool to screen toxic effects of compounds. However, assays using monolayer cell cultures may not entirely represent a native in vivo environment and may result in misinterpretation of compound toxicity. Magnetic 3D bioprinting in combination with automated kinetic imaging provides an easy and robust high-throughput screening approach of compound effects on cell migration in a 3D environment.

The recapitulation of the material properties and structure of the native aortic valve leaflet, specifically its anisotropy and laminate structure, is a major design goal for scaffolds for heart valve tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for this purpose as they are biocompatible, can be modified for their mechanical and biofunctional properties, and can be laminated. This study investigated augmenting PEG hydrogels with polycaprolactone (PCL) as an analog to the fibrosa to improve strength and introduce anisotropic mechanical behavior. However, due to its hydrophobicity, PCL must be modified prior to embedding within PEG hydrogels. In this study, PCL was electrospun (ePCL) and modified in three different ways, by protein adsorption (pPCL), alkali digestion (hPCL), and acrylation (aPCL). Modified PCL of all types maintained the anisotropic elastic moduli and yield strain of unmodified anisotropic ePCL. Composites of PEG and PCL (PPCs) maintained anisotropic elastic moduli, but aPCL and pPCL had isotropic yield strains. Overall, PPCs of all modifications had elastic moduli of 3.79±0.90 MPa and 0.46±0.21 MPa in the parallel and perpendicular directions, respectively. Valvular interstitial cells seeded atop anisotropic aPCL displayed an actin distribution aligned in the direction of the underlying fibers. The resulting scaffold combines the biocompatibility and tunable fabrication of PEG with the strength and anisotropy of ePCL to form a foundation for future engineered valve scaffolds.

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

When diseased, aortic valves are typically replaced with bioprosthetic heart valves (BPHVs), either porcine valves or bovine pericardium that are fixed in glutaraldehyde. These replacements fail within 10–15 years due to calcification and fatigue, and their failure coincides with a loss of glycosaminoglycans (GAGs). This study investigates this relationship between GAG concentration and the tensile and viscoelastic properties of aortic valve leaflets. Aortic valve leaflets were dissected from porcine hearts and digested in hyaluronidase in concentrations ranging from 0 to 5 U/mL for 0–24 h, yielding a spectrum of GAG concentrations that was measured using the uronic acid assay and confirmed by Alcian Blue staining. Digested leaflets with varying GAG concentrations were then tested in tension in the circumferential and radial directions with varying strain rate, as well as in stress relaxation. The GAG concentration of the leaflets was successfully reduced using hyaluronidase, although water content was not affected. Elastic modulus, the maximum stress, and hysteresis significantly increased with decreasing GAG concentration. Extensibility and the radius of transition curvature did not change with GAG concentration. The stress relaxation behavior and strain-rate independent nature of the leaflet did not change with GAG concentration. These results suggest that GAGs in the spongiosa lubricate tissue motion and reduce stresses experienced by the leaflet. This study forms the basis for predictive models of BPHV mechanics based on GAG concentration, and guides the rational design of future heart valve replacements.

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives.

determined diabetic rats randomly got intracavernous (IC) injection of phosphate buffer solution (PBS), ADSCs or MTs. Another eight normal rats equally received IC injection of PBS. MTs were generated with a hanging drop method and the injected cells were tracked in ADSCs and MTs injected rats. Four weeks after the treatments, intracavernous pressure (ICP), histopathological changes in corpus cavernosum (CC), and functional proteins were measured. Rat cytokine antibody array was used to detect ADSCs or MTs lysate. RESULTS: MTs expressed vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and tumour necrosis factor-stimulated gene 6 (TSG-6). MTs injection had a higher retention than ADSCs injection and MTs treatment better improved ICP, neuronal nitric oxide synthase (nNOS) expression, smooth muscle and endothelial contents in diabetic rats, ameliorated local inflammation in CC. CONCLUSIONS: IC injection of MTs improves the erectile function and histopathological changes in streptozotocin-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.

Precision medicine holds the promise of designing patient-specific therapies to improve therapeutic efficiency. However, the scarcity of tumor and biopsy tissue is a limiting factor in the development of diagnostic assays. Cells isolated from these tissues could be used to overcome these issues, while serving as the basis for assays to diagnose and guide treatment. It is critical that the in vitro culture of these cells be performed in three-dimensional (3D) environments that can better replicate the native tumor microenvironment. However, currently available 3D cell culture platforms, like Matrigel, suffer from technical limitations in reproducibility and handling that make the development of such assays difficult. Towards that end, this study isolates cells from human prostate cancer (PC) and renal cell carcinoma (RCC) tumor biopsies and patient-derived xenografts (PDX) and prints them into spheroids using magnetic 3D bioprinting. The core principle of magnetic 3D bioprinting is the magnetization of cells and their aggregation using mild magnetic forces. Once aggregated, these cells form spheroids that mimic native tumor environments in extracellular matrix and cell-cell and cell-ECM interactions. This technique can be used to actively magnetize cells and generate spheroids from a scarce cell source, while overcoming the limitations of other 3D cell culture platforms. In this study, we demonstrated our ability to print spheroids from cells isolated from human tumor biopsies and PDX. Isolation techniques ranging from simple mincing and filtration to enzymatic digestion were employed. Next, these cells were magnetized by incubation with a biocompatible magnetic nanoparticle assembly, NanoShuttle. Once magnetized, these cells were printed into spheroids of varying sizes, from 1,000-20,000 cells, in 384-well plates. These cells were cultured for days, after which viability was measured using CellTiter-Glo. Our preliminary studies demonstrated our ability to isolate cells and print them into spheroids. Isolation was best with either mincing and filtration alone or collagenase II (400 U/mL) digestion for 1 h. These cells were then successfully magnetized and printed into spheroids, which remained viable after 72 h. Spheroids of 10,000-20,000 cells were the most successful, and further optimization is needed to reduce the size needed for viable spheroids to take full advantage of scarce resources such as tumor biopsies. We also demonstrated the ability to assay compound toxicity, showing a dose-dependent toxicity on spheroids derived from PDX tumors. In all, we demonstrated our ability to isolate cells from human tumor biopsies and PDX models and print them into spheroids with high throughput. These preliminary results will serve as a platform for the further development of precision medicine assays to optimize PC and RCC treatment. Citation Format: Hubert Tseng, Jacob A. Gage, Pujan K. Desai, Reynolds Brobey, Sheri Skinner, Mehdi Dehghani, Kevin P. Rosenblatt, Wenliang Li, Robert J. Amato, Glauco R. Souza. Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4251.

584 Background: Tumor genotyping using fluid samples such as blood can potentially allow tracking of dynamic changes in mutational profiles over time and allow better access than biopsies. We present a method to detect somatic mutations from a blood draw, where circulating tumor cell (CTC) enrichment above 10% of total cell numbers allows the use of standard gene panels typically used to analyze tissue-based biopsies. Methods: Analytical samples were obtained from 9 prostate cancer (PC) patients and 6 renal cell cancer (RCC) patients, followed by CTC enrichment using the IsoFlux System. Cells were lysed and DNA amplified by whole genome amplification (WGA) using the NGS Kit (Fluxion Biosciences) and quantified via qPCR. Samples were enumerated to determine CTC load, with CTCs defined as CK+, CD45- nucleated cells (DAPI+). Next-generation sequencing was performed using 3 targeted cancer panels on the Ion torrent PGM platform: the Ion ampliseq cancer hotspot panel (50 genes; 6 PC samples), the Oncomine (143 genes; 3 PC samples), and a 29-gene panel of actionable mutations in RCC (6 samples). Data was analyzed using a customized variant calling/filtering pipeline based on standard alignment and variant calling tools. Variant filtering and functional interpretation was performed using VarSeq. All data was analyzed in a blinded manner. Results: Our method was able to isolate CTCs from all patient samples. Whole genome amplified DNA concentration was at a range of 25-164 ng/µL (median, 69) in PC and 29-180 ng/µL (median, 69) in RCC. CTCs were recovered in 2.9-33.7% (median, 10.5%) of PC samples and 1.9-33% (median, 14.5%) of RCC samples After WGA, we found 1 variant/sample using hotpot, 12/sample using Oncomine, and 3/sample using the RCC panel. Conclusions: Our assay consistently detected somatic variants from blood draw using standard gene panels in both PC and RCC. Obtaining repeat tumor biopsies from patients during treatment and/or at time of progression is both challenging and impractical from a clinical perspective. Our assay provides molecular characterization using standard blood draws and will be prospectively validated in clinical trials.

Purpose: To evaluate pentagalloyl-glucose (PGG) stabilized elastinderived vascular grafts (EDVGs) in terms of their suitability as vascular substitutes. Methods: EDVGs were prepared by alkaline decellularization (0.1 M NaOH at 37°C for 3 h), rinsing and sterilization in 0.1% peracetic acid. Batches of EDVG scaffolds were treated with sterile 0.1% PGG (pH 5.5) containing 20% isopropanol for 24 h, rinsed and stored in sterile PBS. A subset of PGG and non-PGG samples were additionally covalently heparinized via diamine coupling and reductive amination using nitrous acid degraded heparin. Grafts were implanted in a rat infrarenal aortic model for 4 and 8 weeks. Remodeling, patency, endothelialization and healing of the explants were determined by gross and histological evaluation. Results: While PGG treatment did not significantly affect the denaturation temperature (DT) of the tissue, heparinization resulted in significant increases in DT (from 52°C to 81–82°C) and heparin content (from baseline noise levels of b10 mg/g to N100 mg/g). Overall the nonheparinized groups showed strong evidence of remodeling and recellularization, with a high patency rate of 82%. At 8 weeks, only small fragments of the original grafts were preserved with good neovessel formation and moderate intimal hyperplasia (IH). The heparinized groups showed 100% patency, but with little remodeling, presumably due to the increased crosslink density resulting from the amination of the tissue prior to heparin attachment. Some surface endothelialization was observed in all treatment groups. Conclusions: EDVGs are promising candidates as vascular grafts, either as substrates for remodeling, or in more highly cross-linked and heparinized forms.