Correlation of manual semi-quantitative and automated quantitative Ki-67 proliferative index with OncotypeDXTM recurrence score in invasive breast carcinoma.

BACKGROUND: Ki-67 immunohistochemistry (IHC) staining is a widely used cancer proliferation assay; however, its limitations could be improved with automated scoring. The OncotypeDXTM Recurrence Score (ORS), which primarily evaluates cancer proliferation genes, is a prognostic indicator for breast cancer chemotherapy response; however, it is more expensive and slower than Ki-67. OBJECTIVE: To compare manual Ki-67 (mKi-67) with automated Ki-67 (aKi-67) algorithm results based on manually selected Ki-67 "hot spots" in breast cancer, and correlate both with ORS. METHODS: 105 invasive breast carcinoma cases from 100 patients at our institution (2011-2013) with available ORS were evaluated. Concordance was assessed via Cohen's Kappa (κ). RESULTS: 57/105 cases showed agreement between mKi-67 and aKi-67 (κ 0.31, 95% CI 0.18-0.45), with 41 cases overestimated by aKi-67. Concordance was higher when estimated on the same image (κ 0.53, 95% CI 0.37-0.69). Concordance between mKi-67 score and ORS was fair (κ 0.27, 95% CI 0.11-0.42), and concordance between aKi-67 and ORS was poor (κ 0.10, 95% CI -0.03-0.23). CONCLUSIONS: These results highlight the limits of Ki-67 algorithms that use manual "hot spot" selection. Due to suboptimal concordance, Ki-67 is likely most useful as a complement to, rather than a surrogate for ORS, regardless of scoring method.

Nanomaterials for Cardiac Tissue Engineering.

End stage heart failure is a major cause of death in the US. At present, organ transplant and left-ventricular assist devices remain the only viable treatments for these patients. Cardiac tissue engineering presents the possibility of a new option. Nanomaterials such as gold nanorods (AuNRs) and carbon nanotubes (CNTs) present unique properties that are beneficial for cardiac tissue engineering approaches. In particular, these nanomaterials can modulate electrical conductivity, hardness, and roughness of bulk materials to improve tissue functionality. Moreover, they can deliver bioactive cargo to affect cell phenotypes. This review covers recent advances in the use of nanomaterials for cardiac tissue engineering.

Attenuation of Abnormal Scarring Using Spherical Nucleic Acids Targeting Transforming Growth Factor Beta 1.

Abnormal scarring is a consequence of dysregulation in the wound healing process, with limited options for effective and noninvasive therapies. Given the ability of spherical nucleic acids (SNAs) to penetrate skin and regulate gene expression within, we investigated whether gold-core SNAs (AuSNAs) and liposome-core SNAs (LSNAs) bearing antisense oligonucleotides targeting transforming growth factor beta 1 (TGF-ß1) can function as a topical therapy for scarring. Importantly, both SNA constructs appreciably downregulated TGF-ß1 protein expression in primary hypertrophic and keloid scar fibroblasts in vitro. In vivo, topically applied AuSNAs and LSNAs downregulated TGF-ß1 protein expression levels and improved scar histology as determined by the scar elevation index. These data underscore the potential of SNAs as a localized, self-manageable treatment for skin-related diseases and disorders that are driven by increased gene expression.

Cross-Linked Micellar Spherical Nucleic Acids from Thermoresponsive Templates.

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs.

Drug-Loaded Polymeric Spherical Nucleic Acids: Enhancing Colloidal Stability and Cellular Uptake of Polymeric Nanoparticles through DNA Surface-Functionalization.

Small-sized (∼65 nm) doxorubicin (Dox)-loaded polymeric nanoparticles (PNPs) were modified with oligonucleotides to form colloidally stable Dox-loaded polymeric spherical nucleic acid (Dox-PSNA) nanostructures in biological media. The nucleic acid shell facilitates the cellular uptake of Dox-PSNA, which results in in vitro cytotoxicity against SKOV3 cancer cells.

The Sequence-Specific Cellular Uptake of Spherical Nucleic Acid Nanoparticle Conjugates.

The sequence-dependent cellular uptake of spherical nucleic acid nanoparticle conjugates (SNAs) is investigated. This process occurs by interaction with class A scavenger receptors (SR-A) and caveolae-mediated endocytosis. It is known that linear poly(guanine) (poly G) is a natural ligand for SR-A, and it has been proposed that interaction of poly G with SR-A is dependent on the formation of G-quadruplexes. Since G-rich oligonucleotides are known to interact strongly with SR-A, it is hypothesized that SNAs with higher G contents would be able to enter cells in larger amounts than SNAs composed of other nucleotides, and as such, cellular internalization of SNAs is measured as a function of constituent oligonucleotide sequence. Indeed, SNAs with enriched G content show the highest cellular uptake. Using this hypothesis, a small molecule (camptothecin) is chemically conjugated with SNAs to create drug-SNA conjugates and it is observed that poly G SNAs deliver the most camptothecin to cells and have the highest cytotoxicity in cancer cells. Our data elucidate important design considerations for enhancing the intracellular delivery of spherical nucleic acids.

Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation.

Herein, we report the synthesis of DNA-functionalized infinite-coordination-polymer (ICP) nanoparticles as biocompatible gene-regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe(III) to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe(III)-HOPO ICP particles by copper-free click chemistry afforded colloidally stable nucleic-acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Liposomal spherical nucleic acids.

A novel class of metal-free spherical nucleic acid nanostructures was synthesized from readily available starting components. These particles consist of 30 nm liposomal cores, composed of an FDA-approved 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid monomer. The surface of the liposomes was functionalized with DNA strands modified with a tocopherol tail that intercalates into the phospholipid layer of the liposomal core via hydrophobic interactions. The spherical nucleic acid architecture not only stabilizes these constructs but also facilitates cellular internalization and gene regulation in SKOV-3 cells.

Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates.

Intracellular delivery of nucleic acids as gene regulation agents typically requires the use of cationic carriers or viral vectors, yet issues related to cellular toxicity or immune responses hamper their attractiveness as therapeutic candidates. The discovery that spherical nucleic acids (SNAs), polyanionic structures comprised of densely packed, highly oriented oligonucleotides covalently attached to the surface of nanoparticles, can effectively enter more than 50 different cell types presents a potential strategy for overcoming the limitations of conventional transfection agents. Unfortunately, little is known about the mechanism of endocytosis of SNAs, including the pathway of entry and specific proteins involved. Here, we demonstrate that the rapid cellular uptake kinetics and intracellular transport of SNAs stem from the arrangement of oligonucleotides into a 3D architecture, which supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. These results reinforce the notion that SNAs can serve as therapeutic payloads and targeting structures to engage biological pathways not readily accessible with linear oligonucleotides.