Large-scale time series microscopy of neovessel growth during angiogenesis.

During angiogenesis, growing neovessels must effectively navigate through the tissue space as they elongate and subsequently integrate into a microvascular network. While time series microscopy has provided insight into the cell activities within single growing neovessel sprouts, less is known concerning neovascular dynamics within a large angiogenic tissue bed. Here, we developed a time-lapse imaging technique that allowed visualization and quantification of sprouting neovessels as they form and grow away from adult parent microvessels in three dimensions over cubic millimeters of matrix volume during the course of up to 5 days on the microscope. Using a new image acquisition procedure and novel morphometric analysis tools, we quantified the elongation dynamics of growing neovessels and found an episodic growth pattern accompanied by fluctuations in neovessel diameter. Average elongation rate was 5 µm/h for individual vessels, but we also observed considerable dynamic variability in growth character including retraction and complete regression of entire neovessels. We observed neovessel-to-neovessel directed growth over tens to hundreds of microns preceding tip-to-tip inosculation. As we have previously described via static 3D imaging at discrete time points, we identified different collagen fibril structures associated with the growing neovessel tip and stalk, and observed the coordinated alignment of growing neovessels in a deforming matrix. Overall analysis of the entire image volumes demonstrated that although individual neovessels exhibited episodic growth and regression, there was a monotonic increase in parameters associated with the entire vascular bed such as total network length and number of branch points. This new time-lapse imaging approach corroborated morphometric changes in individual neovessels described by us and others, as well as captured dynamic neovessel behaviors unique to days-long angiogenesis within the forming neovascular network.

Mechanical interaction of angiogenic microvessels with the extracellular matrix.

Angiogenesis is the process by which new blood vessels sprout from existing blood vessels, enabling new vascular elements to be added to an existing vasculature. This review discusses our investigations into the role of cell-matrix mechanics in the mechanical regulation of angiogenesis. The experimental aspects of the research are based on in vitro experiments using an organ culture model of sprouting angiogenesis with the goal of developing new treatments and techniques to either promote or inhibit angiogenic outgrowth, depending on the application. Computational simulations were performed to simulate angiogenic growth coupled to matrix deformation, and live two-photon microscopy was used to obtain insight into the dynamic mechanical interaction between angiogenic neovessels and the extracellular matrix. In these studies, we characterized how angiogenic neovessels remodel the extracellular matrix (ECM) and how properties of the matrix such as density and boundary conditions influence vascular growth and alignment. Angiogenic neovessels extensively deform and remodel the matrix through a combination of applied traction, proteolytic activity, and generation of new cell-matrix adhesions. The angiogenic phenotype within endothelial cells is promoted by ECM deformation and remodeling. Sensitivity analysis using our finite element model of angiogenesis suggests that cell-generated traction during growth is the most important parameter controlling the deformation of the matrix and, therefore, angiogenic growth and remodeling. Live two-photon imaging has also revealed numerous neovessel behaviors during angiogenesis that are poorly understood such as episodic growth/regression, neovessel colocation, and anastomosis. Our research demonstrates that the topology of a resulting vascular network can be manipulated directly by modifying the mechanical interaction between angiogenic neovessels and the matrix.

Mitochondria as a primary determinant of angiogenic modality in pulmonary arterial hypertension.

Impaired pulmonary angiogenesis plays a pivotal role in the progression of pulmonary arterial hypertension (PAH) and patient mortality, yet the molecular mechanisms driving this process remain enigmatic. Our study uncovered a striking connection between mitochondrial dysfunction (MD), caused by a humanized mutation in the NFU1 gene, and severely disrupted pulmonary angiogenesis in adult lungs. Restoring the bioavailability of the NFU1 downstream target, lipoic acid (LA), alleviated MD and angiogenic deficiency and rescued the progressive PAH phenotype in the NFU1G206C model. Notably, significant NFU1 expression and signaling insufficiencies were also identified in idiopathic PAH (iPAH) patients' lungs, emphasizing this study's relevance beyond NFU1 mutation cases. The remarkable improvement in mitochondrial function of PAH patient-derived pulmonary artery endothelial cells (PAECs) following LA supplementation introduces LA as a potential therapeutic approach. In conclusion, this study unveils a novel role for MD in dysregulated pulmonary angiogenesis and PAH manifestation, emphasizing the need to correct MD in PAH patients with unrecognized NFU1/LA deficiency.

Intravital confocal and two-photon imaging of dual-color cells and extracellular matrix mimics.

We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

Ultra-slim plastic endomicroscope objective for non-linear microscopy.

Non-linear microscopy has the potential to provide clinically useful information on the structure of biological tissue in vivo via an endomicroscope. The ability to use plastic as the optical material in a multiphoton objective was evaluated based on several criteria including autofluorescence, injection molding induced birefringence, and pulse broadening due to group velocity dispersion. An all-plastic, refractive ultra-slim endoscope objective was built with design specifications of NA=0.4, FOV=250 µm, 1.27 mm outer diameter, and 0.8 mm clear aperture. Initial images of second-harmonic generation signal (illumination at 780 nm) in collagen fibers and two-photon excited fluorescence (illumination at 920 nm) of Convallaria rhizome are reported.

Acid treatment of melanoma cells selects for invasive phenotypes.

Solid tumors become acidic due to hypoxia and upregulated glycolysis. We have hypothesized that this acidosis leads to more aggressive invasive behavior during carcinogenesis (Nature Reviews Cancer 4:891-899, 2004). Previous work on this subject has shown mixed results. While some have observed an induction of metastasis and invasion with acid treatments, others have not. To investigate this, human melanoma cells were acclimated to low pH growth conditions. Significant cell mortality occurred during acclimation, suggesting that acidosis selected for resistant phenotypes. Cells maintained under acidic conditions exhibited a greater range of motility, a reduced capacity to form flank tumors in SCID mice and did not invade more rapidly in vitro, compared to non-selected control cells. However, re-acclimation of these selected cells to physiological pH gave rise to stable populations with significantly higher in vitro invasion. These re-acclimated cells maintained higher invasion and higher motility for multiple generations. Transcriptomic analyses of these three phenotypes revealed significant differences, including upregulation of relevant pathways important for tissue remodeling, cell cycle control and proliferation. These results reinforce the hypothesis that acidosis promotes selection of stable, more invasive phenotypes, rather than inductive changes, which would be reversible.

Multispectral fluorescence imaging of human ovarian and fallopian tube tissue for early-stage cancer detection.

With early detection, 5-year survival rates for ovarian cancer exceed 90%, yet no effective early screening method exists. Emerging consensus suggests over 50% of the most lethal form of the disease originates in the fallopian tube. Twenty-eight women undergoing oophorectomy or debulking surgery provided informed consent for the use of surgical discard tissue samples for multispectral fluorescence imaging. Using multiple ultraviolet and visible excitation wavelengths and emissions bands, 12 fluorescence and 6 reflectance images of 47 ovarian and 31 fallopian tube tissue samples were recorded. After imaging, each sample was fixed, sectioned, and stained for pathological evaluation. Univariate logistic regression showed cancerous tissue samples had significantly lower intensity than noncancerous tissue for 17 image types. The predictive power of multiple image types was evaluated using multivariate logistic regression (MLR) and quadratic discriminant analysis (QDA). Two MLR models each using two image types had receiver operating characteristic curves with area under the curve exceeding 0.9. QDA determined 56 image type combinations with perfect resubstituting using as few as five image types. Adaption of the system for future in vivo fallopian tube and ovary endoscopic imaging is possible, which may enable sensitive detection of ovarian cancer with no exogenous contrast agents.

Hypoxia-inducible factor-1alpha and the glycolytic phenotype in tumors.

Metastatic tumors generally exhibit aerobic glycolysis (the Warburg effect). The advent of [18F]fluorodeoxyglucose positron emission tomography imaging, coupled with recent findings linking hypoxia-inducible factor (HIF-1alpha) overexpression to aggressive cancers, has rekindled an interest in this aspect of tumor metabolism. These studies explore the role of HIF-1alpha in human breast cancer lines and its relationship to glycolytic regulation. Here we demonstrate that, under normal oxygen conditions, nonmetastatic cells consume less glucose and express low HIF-1alpha, whereas metastatic cells constitutively express high glycolysis and HIF-1alpha, suggesting that dysregulation of HIF-1alpha may induce the Warburg effect. This hypothesis was tested by renormalizing HIF-1alpha levels in renal carcinoma cells, leading to inhibition of aerobic glycolysis.

Tumor acidity, ion trapping and chemotherapeutics. I. Acid pH affects the distribution of chemotherapeutic agents in vitro.

Resistance to anti-cancer chemotherapies often leads to regional failure, and can be caused by biochemical and/or physiological mechanisms. Biochemical mechanisms include the overexpression of resistance-conferring proteins. In contrast, physiological resistance involves the tumor microenvironment, and can be caused by poor perfusion, hypoxia and/or acidity. This communication investigates the role of tumor acidity in resistance to a panel of chemotherapeutic agents commonly used against breast cancer, such as anthracyclines, taxanes, anti-metabolites and alkylating agents. The effects of pH on the cytotoxicity of these agents were determined, and ion trapping was confirmed by monitoring the effect of pH on the cellular uptake of radiolabeled anthracyclines. Furthermore, pH-dependent cytotoxicity and uptake were compared between parental drug sensitive MCF-7 cells and variants overexpressing p-glycoprotein (MDR-1) and Breast Cancer Resistance Protein. These data indicate that the magnitude of physiological resistance from pH-dependent ion trapping is comparable to biochemical resistance caused by overexpression of drug efflux pumps. Hence, microenvironment-based ion trapping is a significant barrier to anthracycline-based chemotherapy and can itself be a therapeutic target to enhance the efficacy of existing chemotherapies.

The efficacy of image correlation spectroscopy for characterization of the extracellular matrix.

Image correlation spectroscopy (ICS) is known to be a useful tool for the evaluation of fiber width in the extracellular matrix. Here we evaluate a more general from of ICS fit parameters for fiber networks and arrive at a means of quantifying the fiber density, pore size and length which facilitates the characterization of the extracellular matrix. A simulation package was made to create images with different structural properties of fiber networks such as fiber orientation, width, fiber density and length. A pore finding algorithm was developed which determines the distribution of circular voids in the image. Collagen I hydrogels were prepared under different polymerization conditions for validation of our pore size algorithm with microscopy data. ICS parameters included amplitude, standard deviation and ellipticity and are shown to predict the structural properties of fiber networks in a quantitative manner. While the fiber width is related to the ICS sigma; the fiber density relates to the pore size distribution which correlates with the ICS amplitude in thresholded images. Fiber length is related to ICS ellipticity if the fibers have a preferred orientation. Findings from ICS and pore distribution algorithms were verified for both simulated and microscopy data. Based on these findings, we conclude that ICS can be used in the assessment of the extracellular matrix and the prediction of fiber orientation, width, density, length and matrix pore size.

Solid-phase synthetic strategy and bioevaluation of a labeled delta-opioid receptor ligand Dmt-Tic-Lys for in vivo imaging.

A general solid-phase synthetic strategy is developed to prepare fluorescent and/or lanthanide-labeled derivatives of the delta-opioid receptor (deltaOR) ligand H-Dmt-Tic-Lys(R)-OH. The high delta-OR affinity (K(i) = 3 nM) and desirable in vivo characteristics of the Cy5 derivative 1 suggest its usefulness for structure-function studies and receptor localization and as a high-contrast noninvasive molecular marker for live imaging ex vivo or in vivo.

Enhanced targeting with heterobivalent ligands.

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human delta-opioid receptor (deltaOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-alpha-MSH, and Eu-CCK8 for the deltaOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a deltaOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers.

Characterization of breast cancers and therapy response by MRS and quantitative gene expression profiling in the choline pathway.

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non-invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image-guided 31P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high-resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA-mb-231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3-phosphate, are all raised in breast cancer lines relative to an immortalized non-malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase-mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A2-mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model.

Bicarbonate increases tumor pH and inhibits spontaneous metastases.

The external pH of solid tumors is acidic as a consequence of increased metabolism of glucose and poor perfusion. Acid pH has been shown to stimulate tumor cell invasion and metastasis in vitro and in cells before tail vein injection in vivo. The present study investigates whether inhibition of this tumor acidity will reduce the incidence of in vivo metastases. Here, we show that oral NaHCO(3) selectively increased the pH of tumors and reduced the formation of spontaneous metastases in mouse models of metastatic breast cancer. This treatment regimen was shown to significantly increase the extracellular pH, but not the intracellular pH, of tumors by (31)P magnetic resonance spectroscopy and the export of acid from growing tumors by fluorescence microscopy of tumors grown in window chambers. NaHCO(3) therapy also reduced the rate of lymph node involvement, yet did not affect the levels of circulating tumor cells, suggesting that reduced organ metastases were not due to increased intravasation. In contrast, NaHCO(3) therapy significantly reduced the formation of hepatic metastases following intrasplenic injection, suggesting that it did inhibit extravasation and colonization. In tail vein injections of alternative cancer models, bicarbonate had mixed results, inhibiting the formation of metastases from PC3M prostate cancer cells, but not those of B16 melanoma. Although the mechanism of this therapy is not known with certainty, low pH was shown to increase the release of active cathepsin B, an important matrix remodeling protease.

Regulation of the Warburg effect in early-passage breast cancer cells.

Malignancy in cancer is associated with aerobic glycolysis (Warburg effect) evidenced by increased trapping of [(18)F]deoxyglucose (FdG) in patients imaged by positron emission tomography (PET). [(18)F]deoxyglucose uptake correlates with glucose transporter (GLUT-1) expression, which can be regulated by hypoxia-inducible factor 1 alpha (HIF-1alpha). We have previously reported in established breast lines that HIF-1alpha levels in the presence of oxygen leads to the Warburg effect. However, glycolysis and GLUT-1 can also be induced independent of HIF-1alpha by other factors, such as c-Myc and phosphorylated Akt (pAkt). This study investigates HIF-1alpha, c-Myc, pAkt, and aerobic glycolysis in low-passage breast cancer cells under the assumption that these represent the in vivo condition better than established lines. Similar to in vivo FdG-PET or primary breast cancers, rates of glycolysis were diverse, being higher in cells expressing both c-Myc and HIF-1alpha and lower in cell lines low or negative in both transcription factors. No correlations were observed between glycolytic rates and pAkt levels. Two of 12 cell lines formed xenografts in mice. Both were positive for HIF-1alpha and phosphorylated c-Myc, and only one was positive for pAkt. Glycolysis was affected by pharmacological regulation of c-Myc and HIF-1alpha. These findings suggest that c-Myc and/or HIF-1alpha activities are both involved in the regulation of glycolysis in breast cancers.

Thermostability of firefly luciferases affects efficiency of detection by in vivo bioluminescence.

Luciferase from the North American firefly (Photinis pyralis) is a useful reporter gene in vivo, allowing noninvasive imaging of tumor growth, metastasis, gene transfer, drug treatment, and gene expression. Luciferase is heat labile with an in vitro halflife of approximately 3 min at 37 degrees C. We have characterized wild type and six thermostabilized mutant luciferases. In vitro, mutants showed half-lives between 2- and 25-fold higher than wild type. Luciferase transfected mammalian cells were used to determine in vivo half-lives following cycloheximide inhibition of de novo protein synthesis. This showed increased in vivo thermostability in both wild-type and mutant luciferases. This may be due to a variety of factors, including chaperone activity, as steady-state luciferase levels were reduced by geldanamycin, an Hsp90 inhibitor. Mice inoculated with tumor cells stably transfected with mutant or wild-type luciferases were imaged. Increased light production and sensitivity were observed in the tumors bearing thermostable luciferase. Thermostable proteins increase imaging sensitivity. Presumably, as more active protein accumulates, detection is possible from a smaller number of mutant transfected cells compared to wild-type transfected cells.