Modular and adaptable tumor niche prepared from visible light-initiated thiol-norbornene photopolymerization

Photopolymerized biomimetic hydrogels with adaptable properties have been widely used for cell and tissue engineering applications. As a widely adopted gel crosslinking method, photopolymerization provides experimenters on-demand and spatial-temporal controls in gelation kinetics. Long wavelength ultraviolet (UV) light initiated photopolymerization is among the most popular methods in the fabrication of cell-laden hydrogels owing to its rapid and relatively mild gelation conditions. The use of UV light, however, still causes concerns regarding its potential negative impacts on cells. Alternatively, visible light based photopolymerization can be used to crosslink cell-laden hydrogels. The majority of visible light based gelation schemes involve photoinitiator, co-initiator, and co-monomer. This multi-component initiation system creates added challenges for optimizing hydrogel formulations. Here, we report a co-initiator/co-monomer-free visible light initiated thiol-norbornene photopolymerization scheme to prepare modular biomimetic hydrogels suitable for in situ cell encapsulation. Eosin-Y was used as the sole initiator to initiate modular gelation between synthetic macromers (e.g., thiolated poly(vinyl alcohol) or poly(ethylene glycol)) and functionalized extracellular matrices (ECM), including norbornene-functionalized gelatin (GelNB) and/or thiolated hyaluronic acid (THA). These components are modularly crosslinked to afford bio-inert (i.e., purely synthetic), bioactive (i.e., using gelatin), and biomimetic (i.e., using gelatin and hyaluronic acid) hydrogels. The stiffness of the hydrogels can be easily tuned without affecting the contents of the bioactive components. Furthermore, the use of naturally-derived biomacromolecules (e.g., gelatin and HA) renders these hydrogels susceptible to enzyme-mediated degradation. In addition to demonstrating efficient and tunable visible light mediated gelation, we also utilized this biomimetic modular gelation system to formulate artificial tumor niche and to study the effects of cell density and gel modulus on the formation of pancreatic ductal adenocarcinoma (PDAC) spheroids.

Comparative study of visible light polymerized gelatin hydrogels for 3D culture of hepatic progenitor cells

Photopolymerization techniques have been widely used to create hydrogels for biomedical applications. Visible light-based photopolymerizations are commonly initiated by type II (i.e., noncleavage-type) photoinitiator in conjunction with a coinitiator. On the other hand, type I photoinitiators (i.e., cleavage type) are rarely compatible with visible light-based initiation due to their limited molar absorbability in the visible light wavelengths. Here, we report visible light initiated orthogonal photoclick crosslinking to fabricate gelatin-norbornene and poly(ethylene glycol)-tetra-thiol hydrogels using either cleavage-type (i.e., lithium acylphosphinate, LAP) or noncleavage-type photoinitiator (i.e., eosin-Y, EY) without the use of a coinitiator. Regardless of the initiator type, the step-growth gelatin-PEG hybrid hydrogels crosslinked and degraded similarly. While both systems exhibited similar cytocompatibility for hepatic progenitor HepaRG cells, gelation initiated by noncleavage-type initiator EY afforded slightly higher degree of hepatic gene expression

Enzyme-mediated stiffening hydrogels for probing activation of pancreatic stellate cells

The complex network of biochemical and biophysical cues in the pancreatic desmoplasia not only presents challenges to the fundamental understanding of tumor progression, but also hinders the development of therapeutic strategies against pancreatic cancer. Residing in the desmoplasia, pancreatic stellate cells (PSCs) are the major stromal cells affecting the growth and metastasis of pancreatic cancer cells by means of paracrine effects and extracellular matrix protein deposition. PSCs remain in a quiescent/dormant state until they are 'activated' by various environmental cues. While the mechanisms of PSC activation are increasingly being described in literature, the influence of matrix stiffness on PSC activation is largely unexplored. To test the hypothesis that matrix stiffness affects myofibroblastic activation of PSCs, we have prepared cell-laden hydrogels capable of being dynamically stiffened through an enzymatic reaction. The stiffening of the microenvironment was created by using a peptide linker with additional tyrosine residues, which were susceptible to tyrosinase-mediated crosslinking. Tyrosinase catalyzes the oxidation of tyrosine into dihydroxyphenylalanine (DOPA), DOPA quinone, and finally into DOPA dimer. The formation of DOPA dimer led to additional crosslinks and thus stiffening the cell-laden hydrogel. In addition to systematically studying the various parameters relevant to the enzymatic reaction and hydrogel stiffening, we also designed experiments to probe the influence of dynamic matrix stiffening on cell fate. Protease-sensitive peptides were used to crosslink hydrogels, whereas integrin-binding ligands (e.g., RGD motif) were immobilized in the network to afford cell-matrix interaction. PSC-laden hydrogels were placed in media containing tyrosinase for 6h to achieve in situ gel stiffening. We found that PSCs encapsulated and cultured in a stiffened matrix expressed higher levels of αSMA and hypoxia-inducible factor 1α (HIF-1α), suggestive of a myofibroblastic phenotype. This hydrogel platform offers a facile means of in situ stiffening of cell-laden matrices and should be valuable for probing cell fate process dictated by dynamic matrix stiffness. STATEMENT OF SIGNIFICANCE: Hydrogels with spatial-temporal controls over crosslinking kinetics (i.e., dynamic hydrogel) are increasingly being developed for studying mechanobiology in 3D. The general principle of designing dynamic hydrogel is to perform cell encapsulation within a hydrogel network that allows for postgelation modification in gel crosslinking density. The enzyme-mediated in situ gel stiffening is innovative because of the specificity and efficiency of enzymatic reaction. Although tyrosinase has been used for hydrogel crosslinking and in situ cell encapsulation, to the best of our knowledge tyrosinase-mediated DOPA formation has not been explored for in situ stiffening of cell-laden hydrogels. Furthermore, the current work provides a gradual matrix stiffening strategy that may more closely mimic the process of tumor development

Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer

Background: Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). Methods and Findings: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. Conclusions: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations

Pathema: a clade-specific bioinformatics resource center for pathogen research

Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A–C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms

Identification of six new susceptibility loci for invasive epithelial ovarian cancer.

Genome-wide association studies (GWAS) have identified 12 epithelial ovarian cancer (EOC) susceptibility alleles. The pattern of association at these loci is consistent in BRCA1 and BRCA2 mutation carriers who are at high risk of EOC. After imputation to 1000 Genomes Project data, we assessed associations of 11 million genetic variants with EOC risk from 15,437 cases unselected for family history and 30,845 controls and from 15,252 BRCA1 mutation carriers and 8,211 BRCA2 mutation carriers (3,096 with ovarian cancer), and we combined the results in a meta-analysis. This new study design yielded increased statistical power, leading to the discovery of six new EOC susceptibility loci. Variants at 1p36 (nearest gene, WNT4), 4q26 (SYNPO2), 9q34.2 (ABO) and 17q11.2 (ATAD5) were associated with EOC risk, and at 1p34.3 (RSPO1) and 6p22.1 (GPX6) variants were specifically associated with the serous EOC subtype, all with P < 5 × 10(-8). Incorporating these variants into risk assessment tools will improve clinical risk predictions for BRCA1 and BRCA2 mutation carriers.COGS project is funded through a European Commission's Seventh Framework Programme grant (agreement number 223175 ] HEALTH ]F2 ]2009 ]223175). The CIMBA data management and data analysis were supported by Cancer Research.UK grants 12292/A11174 and C1287/A10118. The Ovarian Cancer Association Consortium is supported by a grant from the Ovarian Cancer Research Fund thanks to donations by the family and friends of Kathryn Sladek Smith (PPD/RPCI.07). The scientific development and funding for this project were in part supported by the US National Cancer Institute GAME ]ON Post ]GWAS Initiative (U19 ]CA148112). This study made use of data generated by the Wellcome Trust Case Control consortium. Funding for the project was provided by the Wellcome Trust under award 076113. The results published here are in part based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancer Institute and National Human Genome Research Institute (dbGap accession number phs000178.v8.p7). The cBio portal is developed and maintained by the Computational Biology Center at Memorial Sloan ] Kettering Cancer Center. SH is supported by an NHMRC Program Grant to GCT. Details of the funding of individual investigators and studies are provided in the Supplementary Note. This study made use of data generated by the Wellcome Trust Case Control consortium, funding for which was provided by the Wellcome Trust under award 076113. The results published here are, in part, based upon data generated by The Cancer Genome Atlas Pilot Project established by the National Cancerhttp://dx.doi.org/10.1038/ng.3185This is the Author Accepted Manuscript of 'Identification of six new susceptibility loci for invasive epithelial ovarian cancer' which was published in Nature Genetics 47, 164–171 (2015) © Nature Publishing Group - content may only be used for academic research

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10-20), ER-negative BC (P=1.1 × 10-13), BRCA1-associated BC (P=7.7 × 10-16) and triple negative BC (P-diff=2 × 10-5). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10-3) and ABHD8 (P<2 × 10-3). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3′-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk