Photothermal modulation of human stem cells using light-responsive 2D nanomaterials.

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS2 to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS2 and NIR stimulation of MoS2 with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and wound healing. The combination of MoS2 and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.

Analysis of the wnt1 regulatory chromosomal landscape.

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.

Nck deficiency is associated with delayed breast carcinoma progression and reduced metastasis.

Although it is known that noncatalytic region of tyrosine kinase (Nck) regulates cell adhesion and migration by bridging tyrosine phosphorylation with cytoskeletal remodeling, the role of Nck in tumorigenesis and metastasis has remained undetermined. Here we report that Nck is required for the growth and vascularization of primary tumors and lung metastases in a breast cancer xenograft model as well as extravasation following injection of carcinoma cells into the tail vein. We provide evidence that Nck directs the polarization of cell-matrix interactions for efficient migration in three-dimensional microenvironments. We show that Nck advances breast carcinoma cell invasion by regulating actin dynamics at invadopodia and enhancing focalized extracellular matrix proteolysis by directing the delivery and accumulation of MMP14 at the cell surface. We find that Nck-dependent cytoskeletal changes are mechanistically linked to enhanced RhoA but restricted spatiotemporal activation of Cdc42. Using a combination of protein silencing and forced expression of wild-type/constitutively active variants, we provide evidence that Nck is an upstream regulator of RhoA-dependent, MMP14-mediated breast carcinoma cell invasion. By identifying Nck as an important driver of breast carcinoma progression and metastasis, these results lay the groundwork for future studies assessing the therapeutic potential of targeting Nck in aggressive cancers.

Revisiting optical clearing with dimethyl sulfoxide (DMSO).

Functional optical characterization of disease progression and response to therapy suffers from loss of spatial resolution and imaging depth due to scattering. Here we report on the ability of dimethyl sulfoxide (DMSO) alone to reduce the optical scattering of skin. We observed a threefold reduction in the scattering of skin with topical DMSO application. With an in vivo window chamber model, we observed a threefold increase in light transmittance through the preparation and enhanced visualization of subsurface microvasculature. Collectively, our data demonstrate the potential of DMSO alone to mitigate effects of scattering, which we expect will improve molecular imaging studies.

Correlation between collagen solubility and skin optical clearing using sugars.

BACKGROUND AND OBJECTIVE: Light scattering from collagen within skin limits light-based therapeutics while increasing the risk of epidermal thermal injury. Specific chemicals show the ability to reduce light scattering by reversibly altering the optical properties of skin. This study examines the correlation between collagen solubility and the optical clearing potential (OCP) of sugars and sugar-alcohols using in vitro rodent skin. MATERIALS AND METHODS: Collagen solubility in dextrose, fructose, sucrose, and sorbitol was measured using near-UV spectroscopy. Light transmittance, reflectance, and rodent skin thickness were measured (giving skin reduced scattering coefficient) before and after exposure of the dermal surface to sugars and sugar-alcohols. OCP was calculated as the ratio of reduced scattering coefficients before and after exposures. RESULTS: Dextrose, fructose, sucrose, and sorbitol had at least twice the collagen solubility and twice the OCP as compared to glycerol. In general, collagen solubility correlated with each agent's ability to optically clear rodent skin. CONCLUSION: These results demonstrate that sugar and sugar-alcohol interaction with collagen are a primary event in tissue optical clearing.

Photodynamic therapy on keloid fibroblasts in tissue-engineered keratinocyte-fibroblast co-culture.

BACKGROUND AND OBJECTIVES: Keloids are disfiguring, proliferative scars that are a pathologic response to cutaneous injury. An organotypic tissue culture system (the Raft model 1-10) was used to investigate the feasibility of using photodynamic therapy (PDT) as an adjunctive therapy to treat keloids following surgical excision. The Raft co-culture system mimics skin by layering keratinocytes on top of fibroblasts embedded in a collagen matrix. PDT uses drugs that produce singlet oxygen in situ when irradiated by light, and may lead to a number of effects in living tissues varying from the modulation of growth to apoptosis. PDT is already used to treat several benign and malignant diseases in organs such as the skin, retina, and esophagus. STUDY DESIGN/MATERIALS AND METHODS: Normal adult, neonatal, and keloid fibroblasts and keratinocytes were isolated from skin obtained from patients undergoing elective procedures and used to construct the Rafts. Mature Rafts (after 4 days) were incubated with 5-amino levulinic acid (5-ALA), a photosensitizer, for 3 hours and were laser-irradiated (635 nm) for total energy delivery of 5 J/cm2, 10 J/cm2, or 20 J/cm2. Rafts were examined 24 hours and 14 days later. Cell viability was determined using confocal imaging combined with live-dead fluorescent dyes. Multi-photon microscope (MPM) imaged collagen structure and density. As Rafts contract over time, surface area was measured using optical micrometry daily. RESULTS: At 10 and 20 J/cm2, near-total cell death was observed in all constructs, while at 5 J/cm2 cell viability was comparable to controls. Cell viability in keloid and neonatal Rafts was greater than that observed in normal adult Rafts. Treated Rafts contracted less over the 14-day period compared to controls. Contraction and collagen density were greatest in keloid and neonatal Rafts. CONCLUSIONS: A PDT dosimetry range was established, which reduces tissue contraction and collagen density while minimizing injury to fibroblasts.

Nonlinear optical microscopy of articular cartilage.

OBJECTIVE: To assess the ability of nonlinear optical microscopy (NLOM) to image ex vivo healthy and degenerative bovine articular cartilage. METHOD: Fresh bovine femoral-tibial joints were obtained from an abattoir. Articular cartilage specimens were harvested from the tibial plateau. Normal and degenerative specimens were imaged by NLOM and subsequently fixed and processed for histological examination. RESULTS: NLOM provided high resolution images of articular cartilage at varying depths with high sensitivity to tissue morphology and high specificity to tissue components without fixing, sectioning or staining. Spectroscopic segmentation of nonlinear optical signals isolated the collagen matrix from the chondron (chondrocyte and non-collagen pericellular matrix). Images from the superficial zone were consistent with the presence of a matrix composed of both elastin-like and collagen fibers distributed in a depth-dependent morphological arrangement, whereas only collagen was demonstrated in the middle and deep zones. Alterations of collagen matrix associated with advanced degenerative joint disease (fibrocartilage) were observed with NLOM. Individual chondrocytes were imaged and demonstrated intracellular fluorescence consistent with the presence of products of intracellular biochemical processes. CONCLUSION: Thin images of living articular cartilage using NLOM may be obtained with (sub-)cellular resolution at varying depths without fixing, sectioning or staining. Extracellular matrical collagen and chondron may be imaged separately in native tissue using spectrally distinct, endogenous, nonlinear optical signals. NLOM was sensitive to macromolecular composition and pathologic changes in articular cartilage matrix. Advances in instrumentation may lead to the application of NLOM to study articular cartilage in vivo.

Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy.

BACKGROUND: Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image. OBJECTIVE: To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time. METHODS: Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days. RESULTS: The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts. CONCLUSIONS: The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.

Enhanced cytotoxic effects of 5-aminolevulinic acid-mediated photodynamic therapy by concurrent hyperthermia in glioma spheroids.

During photodynamic therapy (PDT) both normal and pathological brain tissue, in close proximity to the light source, can experience significant temperature increases. The purpose of this study was to investigate the anti-tumor effects of concurrent 5-aminolevulinic acid (ALA)-mediated PDT and hyperthermia (HT) in human and rat glioma spheroids. Human or rat glioma spheroids were subjected to PDT, HT, or a combination of the two treatments. Therapies were given concurrently to simulate the conditions that will occur during patient PDT. Predictions of diffusion theory suggest that brain tissue immediately adjacent to a spherical light applicator may experience temperature increases approaching 8 degrees C for laser input powers of 2 W. In the in vitro model employed here, HT had no effect on spheroid survival at temperatures below 49 degrees C, while sub-threshold fluence PDT results in only modest decrease in survival. HT (40-46 degrees C) and PDT interact in a synergistic manner if the two treatments are given concurrently. The degree of synergism increases with increasing temperature and light fluence. Apoptosis is the primary mode of cell death following both low-fluence rate PDT and combined HT + PDT.

Effects of combined photodynamic therapy and ionizing radiation on human glioma spheroids.

The effects of combined photodynamic therapy (PDT) and ionizing radiation are studied in a human glioma spheroid model. The degree of interaction between the two modalities depends in a complex manner on factors such as PDT irradiation fluence, fluence rate and dose of ionizing radiation. It is shown that gamma radiation and PDT interact in a synergistic manner only if both light fluence and gamma radiation dose exceed approximately 25 J cm(-2) and 8 Gy, respectively. Synergistic interactions are observed only for the lower fluence rate (25 mW cm(-2)) investigated. The degree of interaction appears to be independent of both sequence and the PDT or ionizing radiation time intervals investigated (1 and 24 h). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays show that low-fluence rate PDT is very efficient at inducing apoptotic cell death, whereas neither high-fluence rate PDT nor ionizing radiation produces significant apoptosis. Although the mechanisms remain to be elucidated, the data imply that the observed synergism is likely not due to gamma-induced cell cycle arrest or to PDT-induced inhibition of DNA repair.