Collective cancer invasion forms an integrin-dependent radioresistant niche.

Cancer fatalities result from metastatic dissemination and therapy resistance, both processes that depend on signals from the tumor microenvironment. To identify how invasion and resistance programs cooperate, we used intravital microscopy of orthotopic sarcoma and melanoma xenografts. We demonstrate that these tumors invade collectively and that, specifically, cells within the invasion zone acquire increased resistance to radiotherapy, rapidly normalize DNA damage, and preferentially survive. Using a candidate-based approach to identify effectors of invasion-associated resistance, we targeted ß1 and αVß3/ß5 integrins, essential extracellular matrix receptors in mesenchymal tumors, which mediate cancer progression and resistance. Combining radiotherapy with ß1 or αV integrin monotargeting in invading tumors led to relapse and metastasis in 40-60% of the cohort, in line with recently failed clinical trials individually targeting integrins. However, when combined, anti-ß1/αV integrin dual targeting achieved relapse-free radiosensitization and prevented metastatic escape. Collectively, invading cancer cells thus withstand radiotherapy and DNA damage by ß1/αVß3/ß5 integrin cross-talk, but efficient radiosensitization can be achieved by multiple integrin targeting.

Elemental F2 with transannular dienes: regioselectivities and mechanisms.

Three reaction paths, namely, molecule-induced homolytic, free radical, and electrophilic, were modeled computationally at the MP2 level of ab initio theory and studied experimentally for the reaction of F2 with the terminal dienes of bicyclo[3.3.1]nonane series. The addition of fluorine is accompanied by transannular cyclization to the adamantane derivatives in which strong evidence for the electrophilic mechanism both in nucleophilic (acetonitrile) and non-nucleophilic (CFCl3 , CHCl3 ) solvents were found. The presence of KF in CFCl3 and CHCl3 facilitates the addition and substantially reduces the formation of tar products.

An experimental protocol for in vivo imaging of neuronal structural plasticity with 2-photon microscopy in mice.

INTRODUCTION: Structural plasticity with synapse formation and elimination is a key component of memory capacity and may be critical for functional recovery after brain injury. Here we describe in detail two surgical techniques to create a cranial window in mice and show crucial points in the procedure for long-term repeated in vivo imaging of synaptic structural plasticity in the mouse neocortex. METHODS: Transgenic Thy1-YFP(H) mice expressing yellow-fluorescent protein (YFP) in layer-5 pyramidal neurons were prepared under anesthesia for in vivo imaging of dendritic spines in the parietal cortex either with an open-skull glass or thinned skull window. After a recovery period of 14 days, imaging sessions of 45-60 min in duration were started under fluothane anesthesia. To reduce respiration-induced movement artifacts, the skull was glued to a stainless steel plate fixed to metal base. The animals were set under a two-photon microscope with multifocal scanhead splitter (TriMScope, LaVision BioTec) and the Ti-sapphire laser was tuned to the optimal excitation wavelength for YFP (890 nm). Images were acquired by using a 20×, 0.95 NA, water-immersion objective (Olympus) in imaging depth of 100-200 µm from the pial surface. Two-dimensional projections of three-dimensional image stacks containing dendritic segments of interest were saved for further analysis. At the end of the last imaging session, the mice were decapitated and the brains removed for histological analysis. RESULTS: Repeated in vivo imaging of dendritic spines of the layer-5 pyramidal neurons was successful using both open-skull glass and thinned skull windows. Both window techniques were associated with low phototoxicity after repeated sessions of imaging. CONCLUSIONS: Repeated imaging of dendritic spines in vivo allows monitoring of long-term structural dynamics of synapses. When carefully controlled for influence of repeated anesthesia and phototoxicity, the method will be suitable to study changes in synaptic structural plasticity after brain injury.

Influence of corneal collagen crosslinking with riboflavin and ultraviolet-a irradiation on excimer laser surgery.

PURPOSE: Riboflavin/ultraviolet A (UVA) cross-linking (CXL) of corneal collagen is a novel method of stabilizing corneal mechanical properties and preventing progression of keratectasias. This study was conducted to investigate whether CXL influences ablation rate, flap thickness, and refractive results of excimer laser procedures ex vivo. METHODS: Corneal epithelium was removed from enucleated porcine eyes, and CXL was performed with riboflavin 0.1% and UVA radiation (365 nm, 3 mW/cm(2)) for 30 minutes. Control eyes received epithelial abrasion only. Diffusion of riboflavin through the cornea was assessed by using infrared-excited, two-photon microscopy of riboflavin autofluorescence, combined with second-harmonic generation of fibrillar collagen. During phototherapeutic keratectomy, corneal thickness was measured by optical coherence pachymetry. During LASIK for myopia, the flap thickness of microkeratome cuts was measured and the induced refractive change assessed by Placido topography. Data were analyzed by Shapiro-Wilk test and Student's t-test. RESULTS: Multiphoton imaging showed a rapid (30-minute) and even distribution of riboflavin throughout the corneal stroma. No difference in ablation rate was measured in treated and untreated corneas (P = 0.90). Mean flap thickness was increased by 44% in cross-linked corneas (P < 0.01). After LASIK for myopia of 4 to 25 D, the mean corneal refractive change was reduced in CXL-treated eyes by 20.1% (P < 0.05). This effect was less pronounced in thinner flaps. CONCLUSIONS: CXL reduces the amount of refractive change after LASIK for myopia. Although the laser ablation rate is unaffected, CXL results in an increased flap thickness. This study suggests the need for adjustment of microkeratome and laser parameters for LASIK after CXL and indirectly endorses the theory of a direct stiffening effect of CXL.

Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging.

Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and fluorescent proteins, doubles imaging depth, improves second harmonic generation of tissue structures, and strongly reduces phototoxicity and photobleaching, compared with conventional MPM. Furthermore, it still provides subcellular resolution at depths of several hundred micrometers and thus will enhance long-term live cell and deep tissue microscopy.

Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.

The metastatic invasion of cancer cells from the primary lesion into the adjacent stroma is a key step in cancer progression, and is associated with poor outcome. The principles of cancer invasion have been experimentally addressed in various in vitro models; however, key steps and mechanisms in vivo remain unclear. Here, we establish a modified skin-fold chamber model for orthotopic implantation, growth and invasion of human HT-1080 fibrosarcoma cells, dynamically reconstructed by epifluorescence and multiphoton microscopy. This strategy allows repeated imaging of tumor growth, tumor-induced angiogenesis and invasion, as either individual cells, or collective strands and cell masses that move along collagen-rich extracellular matrix and coopt host tissue including striated muscle strands and lymph vessels. This modified window model will be suited to address mechanisms of cancer invasion and metastasis, and related experimental therapy.