Chung Ho Sun

An integrated computational/experimental model of tumor invasion

The intracellular and extracellular dynamics that govern tumor growth and invasiveness in vivo remain poorly understood. Cell genotype and phenotype, and nutrient, oxygen, and growth factor concentrations are key variables. In previous work, using a reaction-diffusion mathematical model based on variables that directly describe tumor cell cycle and biology, we formulated the hypothesis that tumor morphology is determined by the competition between heterogeneous cell proliferation caused by spatial diffusion gradients, e.g., of cell nutrients, driving shape instability and invasive tumor morphologies, and stabilizing mechanical forces, e.g., cell-to-cell and cell-to-matrix adhesion. To test this hypothesis, we here obtain variable-based statistics for input to the mathematical model from in vitro human and rat glioblastoma cultures. A linear stability analysis of the model predicts that glioma spheroid morphology is marginally stable. In agreement with this prediction, for a range of variable values, unbounded growth of the tumor mass and invasion of the environment are observed in vitro. The mechanism of invasion is recursive subspheroid component development at the tumor viable rim and separation from the parent spheroid. Results of computer simulations of the mathematical model closely resemble the morphologies and spatial arrangement of tumor cells from the in vitro model. We propose that tumor morphogenesis in vivo may be a function of marginally stable environmental conditions caused by spatial variations in cell nutrients, oxygen, and growth factors, and that controlling these conditions by decreasing spatial gradients could benefit treatment outcomes, whereas current treatment, and especially antiangiogenic therapy, may trigger spatial heterogeneity (e.g., local hypoxia), thus causing invasive instability.

Photothermal treatment of glioma; an in vitro study of macrophage-mediated delivery of gold nanoshells

One of the major factors that limits the treatment effectiveness for gliomas is the presence of the blood–brain barrier (BBB) which protects infiltrating glioma cells from the effects of anti-cancer agents. Circulating monocytes/macrophages (Ma) have a natural ability to traverse the intact and compromised BBB and loaded with anti cancer agents could be used as vectors to target tumors and surrounding tumor infiltrated tissue. Nanoshells (NS) are composed of a dielectric core (silica) coated with an ultrathin gold layer which converts absorbed near-infrared light (NIR) to heat with an extremely high efficacy and stability. We have investigated the effects of exposure to laser NIR on multicell human glioma spheroids infiltrated with empty (containing no nanoshells) or nanoshell loaded macrophages. Our results demonstrated that; (1) macrophages could efficiently take up bare or coated (PEGylated) gold NS: (2) NS loaded macrophages infiltrated into glioma spheroids to the same or, in some cases, to a greater degree than empty Ma; (3) NIR laser irradiation of spheroids incorporating NS loaded macrophages resulted in complete growth inhibition in an irradiance dependent manner, and (4) spheroids infiltrated with empty macrophages had growth curves identical to untreated control cultures. The results of this study provide proof of concept for the use of macrophages as a delivery vector of NS into gliomas for photothermal ablation and open the possibility of developing such regimens for patient treatment.

Photodynamic Therapy of Human Glioma Spheroids Using 5-Aminolevulinic Acid¶

Abstract The response of human glioma spheroids to 5-aminolevulinic acid (ALA)–mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (≤50 J cm−2), lower fluence rates are more effective. At a fluence of 50 J cm−2, near-total spheroid kill is observed at fluence rates of as low as 10 mW cm−2. The fluence rate effect is not as pronounced at higher fluences (>50 J cm−2), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.

A Role for Fibroblasts in Mediating the Effects of Tobacco-Induced Epithelial Cell Growth and Invasion

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts–exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts–exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment. (Mol Cancer Res 2008;6(7):1085–98)

Nondestructive Imaging of Live Human Keloid and Facial Tissue Using Multiphoton Microscopy

OBJECTIVES To use multiphoton microscopy to image collagen fibers and matrix structure in nonfixed human keloid tissue and normal human facial skin obtained following surgery and to compare the findings to existing knowledge of normal skin and keloid morphology to determine if this technology is a suitable adjunct for conventional histology. METHODS Epidermis was removed to expose the fibroblast-rich dermal layer that was then imaged using a multiphoton confocal microscope (Zeiss-Meta 510; Carl Zeiss, Jena, Germany). An 800-nm tunable titanium/sapphire femtosecond laser (Mai-Tai; Newport Co Spectra-Physics, Mountain View, California) was used to excite the tissue; second harmonic generation between 397 and 408 nm and autofluorescent signals were collected. Images were obtained using a Plan-Neofluar x40 oil immersion objective lens and a Plan-Apochromat x63 oil immersion lens. RESULTS Compared with normal skin, keloids showed disorganized collagen fibers arranged in complex swirls and bundles 20 to 30 microm in diameter. Normal tissue showed collagen fibers as distinct, straight strands less than 10 microm in diameter. Differences between normal and keloid tissue were subtle but apparent. CONCLUSIONS The value of imaging living tissue is a significant benefit. Because keloids and hypertrophic scars result from altered collagen metabolism, the development of clinical multiphoton microscopy systems may allow examination of wound healing dynamics in vivo and potentially provides a means to monitor therapy without the need for biopsy or the risk of injury to tissue.

Bypassing the blood brain barrier: delivery of therapeutic agents by macrophages

Introduction: Failure to eradicate infiltrating glioma cells using conventional treatment regimens results in tumor recurrence and is responsible for the dismal prognosis of patients with glioblastoma multiforme (GBM). This is due to the fact that these migratory cells are protected by the blood-brain barrier (BBB) and the blood brain tumor barrier (BBTB) which prevents the delivery of most anti-cancer agents. We have evaluated the ability of monocytes/macrophages (Mo/Ma) to cross the BBB in rats. This will permit access of anti-cancer agents such as nanoparticles to effectively target the infiltrating tumor cells, and potentially improve the treatment effectiveness for malignant gliomas. Materials and Methods: The infiltration of Mo/Ma into brain tumor spheroids in vitro was determined using fluorescent stained Mo/Ma. Tumors were also established in the brains of inbred rats and ALA-PDT was given 18 days following tumor induction. The degredation of the BBTB and quantification of the number of infiltrating Mo/Ma was examined on histological sections from removed brains. Results & Conclusion: PDT was highly effective in locally opening the BBTB and inducing macrophage migration into the irradiated portions of brain tumors.

Two-photon excited imaging of photosensitizers in tissues

Two-photon microscopy (TPM) is a non-invasive biological imaging technique that can be used to selectively image cellular activity and photosensitizer (PS) localization within highly scattering epithelial tissues at depths of approximately 200 micrometer with submicron resolution. The principal objective of this study was to develop a model system for understanding the impact of photodynamic therapy on cellular and extracellular matrix remodeling in biological tissues. An artificial tissue model (RAFT) composed of collagen, embedded fibroblasts, and macrophage cells has been developed for this purpose. TPM is utilized to monitor extracellular matrix remodeling following PDT by imaging collagen/elastin autofluorescence. Selective uptake of photosensitizers by specific cellular components in the matrix can also be visualized by TPM.

Photothermal ablation of malignant brain tumors by nanoparticle loaded macrophages

Nanoshells are a new class of optically tunable nanoparticles composed of a dielectric core (silica) coated with an ultrathin metallic layer (gold). Since nanoshells are roughly one million times more efficient at converting NIR light into heat than conventional dyes when exposed to NIR light, they can generate sufficient heat to induce cell death. Macrophages are frequently found in and around glioblastomas in both experimental animals and patient biopsies. Inflammatory cells loaded with nanoparticles could therefore be used to target tumors.

Photochemical internalization (PCI) enhanced nonviral transfection of tumor suppressor and pro-drug activating genes; A potential treatment modality for gliomas

The overall objective of the research is to investigate the utility of photochemical internalization for the enhanced nonviral transfection of genes into cells. We have examined, in detail, the evaluation of photochemical internalization (PCI) as a method for the non-viral introduction of the tumor suppressor gene PTEN and the PCI mediated transfection of the cytosine deaminase (CD) pro drug activating gene into glioma cell monolayers and multi-cell tumor spheroids. Expression of the CD gene within the target cell produces an enzyme that converts the nontoxic prodrug, 5-fluorocytosine (5-FC), to the toxic metabolite, 5-fluorouracil (5-FU).

Multi-photon microscopy of tobacco-exposed organotypic skin models

Cigarette smoking is the most preventable cause of death in the United States. Researchers have extensively studied smoking in regards to its association with cancer, cardiovascular, and pulmonary disease. In contrast, the impact of cigarette smoking on skin has received much less attention. To provide a better understanding of the effect of cigarette smoking on the human dermal layer, this study used multi-photon microscopy (MPM) to examine collagen in organotypic skin models exposed to cigarette smoke condensate (CSC). Adult and neonatal organotypic tissue-engineered artificial skin models (RAFTs) were constructed and exposed to varying concentrations of CSC. Imaging of the RAFTs was performed using MPM and second-harmonic generation signals (SHG), which allowed for collagen structure to be viewed and analyzed as well as for collagen density to be assessed from derived depth-dependent decay (DDD) values. RAFT contraction as related to exposure concentration was monitored as well. Results indicated a dose dependent between contraction rates and CSC concentration. Collagen structure showed more preservation of its original structure at a greater depth in RAFTs with higher concentrations of CSC. No clear trends could be drawn from analysis of derived DDD values.