Jennifer A. Lee

Length of mitotic arrest induced by microtubule-stabilizing drugs determines cell death after mitotic exit

Cell death induced by agents that disrupt microtubules can kill cells by inducing a prolonged mitotic block. This mitotic block is dependent on the spindle assembly checkpoint, a surveillance system that ensures the bipolar attachment of chromosomes to the mitotic spindle before the onset of anaphase. Under some conditions, the spindle assembly checkpoint can become weakened, allowing cells to exit mitosis despite the presence of chromosomes that are not properly attached to the mitotic spindle. Here, we use an Aurora kinase inhibitor to drive mitotic exit and test the effect of mitotic arrest length on death in the subsequent interphase. Cells that are blocked in mitosis for >15 h die shortly after exiting from mitosis, whereas cells that exit after being blocked for <15 h show variable fates, with some living for days after exiting mitosis. Cells blocked in mitosis by either Taxol or epothilone B are acutely sensitive to the death ligand tumor necrosis factor-related apoptosis-inducing ligand, suggesting that prolonged mitosis allows the gradual accumulation of internal death signals, rendering cells hypersensitive to additional prodeath cues. Death under these conditions is initiated while cyclin B1 is still present, indicating that cells are in mitosis. Our experiments suggest that there is a point of no return during prolonged mitotic block after which mitotic exit can no longer block death. [Mol Cancer Ther 2009;8(6):1646–54]

Adaptive control of laser modal properties

An adaptive optical system for precise control of a laser beams mode structure has been developed. The system uses a dynamic lens based on controlled optical path deformation in a dichroic optical element that is heated with an auxiliary laser. Our method is essentially aberration free, has high dynamic range, and can be implemented with high average power laser beams where other adaptive optics methods fail. A quantitative model agrees well with our experimental data and demonstrates the potential of our method as a mode-matching and beam-shaping element for future large-scale gravitational wave detectors.

In vivo spectral and fluorescence microscopy comparison of microvascular function after treatment with OXi4503, Sunitinib and their combination in Caki-2 tumors

Vascular targeting agents on their own have been shown to be insufficient for complete treatment of solid tumors, emphasizing the importance of studying the vascular effects of these drugs for their use with conventional therapies in the clinic. First-pass fluorescence imaging combined with hyperspectral imaging of hemoglobin saturation of microvessels in the murine dorsal window chamber model provides an easily implementable, low cost method to analyze tumor vascular response to these agents in real-time. In this study, the authors utilized these methods to spectroscopically demonstrate distinct vessel structure, blood flow and oxygenation changes in human Caki-2 renal cell carcinoma following treatment with OXi4503 alone, Sunitinib alone and both drugs together. We showed that treatment with OXi4503 plus Sunitinib destroyed existing tumor microvessels, inhibited blood vessel recovery and impaired Caki-2 tumor growth significantly more than either treatment alone.

Combination of spectral and fluorescence imaging microscopy for wide-field in vivo analysis of microvessel blood supply and oxygenation

Hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence imaging of blood transit time were combined to analyze the oxygenation of and blood flow through microvessel networks. The combination imaging technique was demonstrated in a mouse dorsal window chamber model of a growing Caki-2 human renal cell carcinoma over time. Data from Hb saturation and blood supply time maps show the formation of arteriovenous malformations and shunting of blood directly from arteries to the tumor core and into veins in the periphery of the tumor. Images and data analysis show these malformations result in an oxygenated environment ideal for a tumor to proliferate.

Comparative evaluation of differential laser-induced perturbation spectroscopy as a technique to discriminate emerging skin pathology

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology. Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

In vivo microscopy of microvessel oxygenation and network connections.

Abnormal or compromised microvascular function is a key component of various diseases. In vivo microscopy of microvessel function in preclinical models can be useful for the study of a disease state and effects of new treatments. Wide-field imaging of microvascular oxygenation via hemoglobin (Hb) saturation measurements has been applied in various applications alone and in combination with other measures of microvessel function, such as blood flow. However, most current combined imaging methods of microvessel function do not provide direct information on microvessel network connectivity or changes in connections and blood flow pathways. First-pass fluorescence (FPF) imaging of a systemically administered fluorescent contrast agent can be used to directly image blood flow pathways and connections relative to a local supplying arteriole in a quantitative manner through measurement of blood supply time (BST). Here, we demonstrate the utility of information produced by the combination of Hb saturation measurements via spectral imaging with BST measurements via FPF imaging for correlation of microvessel oxygenation with blood flow pathways and connections throughout a local network. Specifically, we show network pathway effects on oxygen transport in normal microvessels, dynamic changes associated with wound healing, and pathological effects of abnormal angiogenesis in tumor growth and development.

Abstract B24: Impact of aerobic exercise on tumor oxygenation and perfusion in breast cancer

The purpose of this study is to evaluate the effects of low to moderate aerobic exercise on tumor vasculature, perfusion and oxygenation in breast cancer. Poor perfusion and hypoxia are common features of solid tumors that are a result of abnormal tumor angiogenesis. Both chemo- and radiation therapies are negatively affected by tumor hypoxia, with the presence of hypoxia associated with treatment failure and poor outcomes in cancer patients. While strategies to enhance tumor oxygenation and perfusion have been developed, their success in the clinic has been limited. A potential method to increase oxygenation and decrease hypoxia that is safe and well-tolerated is aerobic exercise. The benefits of exercise as a palliative measure are well documented, however the effects of exercise on tumor blood flow and oxygenation have not been extensively characterized. Histological analysis of vessel density (MECA-32), vessel perfusion (Hoechst-33342) and hypoxia (EF5) were quantified in tumor bearing mice subjected to low to moderate intensity treadmill running and compared to sedentary control mice. In a separate model, a murine dorsal skinfold window chamber model and in vivo microscopy of microvessel function was utilized to characterize changes in tumor vascular structure and function in real time. In these studies, the combination of hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence (FPF) imaging of blood transit time were used. Hb saturation imaging reveals the oxygenation of blood within microvessels while FPF imaging gives information regarding blood flow and network connections by recording a fluorescent contrast agent as it is injected into circulation. Spectroscopic analysis was performed in female nude mice with surgically installed titanium window chambers. Mammary tumors were initiated in the windows and once tumors reached a specified volume, animals were subjected to daily treadmill running for up to a week. As a sedentary control, mice were placed on a stationary treadmill for equivalent periods of time. Combination imaging was performed daily to monitor microvessel oxygenation and blood flow. Preliminary results indicate that following one bout of exercise, tumor perfusion is increased, however there was no significant correlation between exercise and tumor microvessel oxygenation. For histological analysis, mammary tumors were initiated in the fat pad of female nude mice. Once tumors nodules were established, animals were subjected to either a single bout or daily bouts of exercise for 1 to 5 weeks. Sedentary controls were exposed to a stationary treadmill for equivalent periods of time. Following exercise protocols, tumors were harvested and sectioned for immunohistochemical analysis. Staining analysis revealed an improvement in tumor oxygenation and an increase in the number of perfused vessels in tumors of exercising animals in comparison to sedentary controls. Our results demonstrate aerobic exercise can increase tumor oxygenation and perfusion in breast cancer, suggesting that exercise can potentially be used as an adjunctive therapy to overcome tumor hypoxia and enhance the efficacy of chemo- and radiation therapy. Citation Format: Jennifer A. Lee, Jennifer Wiggins, Dietmar W. Siemann. Impact of aerobic exercise on tumor oxygenation and perfusion in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B24.

Abstract 5211: In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer

Abnormal tumor angiogenesis leads to inefficient blood flow and inhibited delivery of oxygen, resulting in a hypoxic tumor microenvironment. In radiation therapy the presence of oxygen is necessary to impart lasting damage to cancer cells, therefore the modulation of tumor hypoxia has been an important focus to improve radiation therapy outcome. While strategies such as hyperbaric chambers and hypoxic cell radiosensitizers or cytotoxins have shown merit, their success in the clinic has been limited. A potential method to increase oxygenation and decrease hypoxia in a tumor mass that is safe and well-tolerated is aerobic exercise. For cancer patients, the benefits of exercise in a palliative setting are well known, however the effects of exercise on tumor microvasculature and blood flow have not been extensively characterized. In vivo microscopy of microvessel function in preclinical models is a useful tool for the study of tumor vascular structure and function. The combination of hyperspectral imaging of hemoglobin (Hb) saturation and first-pass fluorescence (FPF) imaging of blood transit time in the murine dorsal skinfold window chamber has been previously used to characterize tumor angiogenesis, wound healing and the response of tumor microvasculature to vascular targeting agents. Hb saturation imaging reveals the oxygenation of blood within microvessels while FPF imaging gives information regarding blood flow and network connections by recording a fluorescent contrast agent as it is injected into circulation. In the present study, this combination imaging technique is used to evaluate the impact of treadmill running on tumor blood flow and microvessel oxygenation in real time. 4T1 mammary tumors were initiated in nude mice bearing titanium window chambers. Once tumors reached a volume of ∼5mm3, mice were subjected to daily treadmill running for 5-7 consecutive days (30 min, 15-18 m/min, 10° incline). As a sedentary control, mice were placed on a stationary treadmill for equivalent periods of time. Combination imaging was performed daily to monitor microvessel oxygenation and blood flow. On the first day of exercise, imaging was performed both before and after animals were exercised. Preliminary results indicate that following one bout of exercise, tumor perfusion is increased, however there was no significant correlation between exercise and tumor microvessel oxygenation. Citation Format: Jennifer A. Lee, Jennifer M. Wiggins, Lori P. Rice, Dietmar W. Siemann. In vivo fluorescence and spectral microscopy of the effects of aerobic exercise on tumor oxygenation and perfusion in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2015-5211

In vivo spectral and fluorescence imaging microscopy of tumor microvessel blood supply and oxygenation changes following vascular targeting agent treatment

The formation of new microvasculature is essential for a tumor mass to grow. Vascular targeting agents (VTAs), including anti-angiogenic drugs and vascular disrupting agents, aim to either inhibit new vasculature growth or destroy existing vasculature, respectively. Because the mechanisms for anti-angiogenic drugs and vascular disrupting agents are complementary, analysis of these drugs used together is under investigation for the enhanced treatment of tumors in comparison to each treatment alone. The preclinical evaluation of the effects of VTAs on tumor growth in small animal models is vital for the development of effective drugs for clinical use. In vivo hyperspectral imaging microscopy of hemoglobin saturation has been used previously to investigate the efficacy of VTAs through analysis of tumor microvessel oxygenation after drug administration. Combining this imaging modality with first-pass fluorescence angiographic imaging can give additional important information about the vessel morphology and blood flow changes that occur after VTA treatment, thus elucidating the relationship between microvessel structure changes and oxygenation. In this study, we report the combined use of hyperspectral and first pass fluorescence angiographic imaging to examine the relationship between vessel morphology and oxygenation of human prostate cancer tumors in mice following treatment with vascular disrupting agents, OXi4503, and anti-VEGF angiogenesis inhibitor, cediranib. Imaging of the tumors is completed before treatment as well as in the days following treatment.

Abstract B56: A phase 1, open-label, dose escalation, nonrandomized study to assess the maximum tolerated dose, dose limiting toxicity, and pharmacokinetics of OPB-31121 in subjects with advanced solid tumors.

Background: OPB-31121 is a novel compound exhibiting potent growth inhibition of cancer cell lines in vitro and xenografts in vivo. The exact mechanism of action of OPB-31121 has not been fully characterized, but studies indicate that a major effect is inhibition of STAT3 phosphorylation. STAT3 is frequently activated in a variety of solid and hematologic malignancies and may present an important target for antitumor therapy. Methods: Open-label, non-randomized, multi-center study in subjects with advanced solid tumors, using a 3+3 dose escalation design. OPB-31121 was administered orally for 21 days followed by 7 days rest per cycle (28-day cycle). The starting dose was 50 mg BID with escalations planned until the dose-limiting toxicity (DLT) was reached. The primary endpoint was determination of maximum tolerated dose (MTD). Additional endpoints included safety, pharmacokinetics, and anti-tumor effect of OPB-31121. Results: 30 subjects received treatment with OPB-31121. Most common tumor types were colorectal cancer (15), breast (3) and thyroid (2). Mean age was 55.9 (range 35–80) years and 17 of the patients were female. Most common adverse events (AEs) potentially attributed to treatment were gastrointestinal: nausea (80%), vomiting (73%), diarrhea (63%), anorexia (20%), and constipation (17%). Most AEs were CTCAE grades 1–2 and manageable with supportive treatment. Three DLTs were observed: one at 300 mg BID (grade 3 lactic acidosis), and two at 350 mg BID (a grade 3 diarrhea and a grade 3 vomiting); the MTD was 300 mg BID. All patients recovered from DLTs after discontinuing the drug. Six additional (9 total) subjects discontinued during the first cycle. Eight subjects completed only one cycle and 13 completed two cycles. No objective responses were observed. Disease progression was observed in all evaluable patients at first restaging. Pharmacokinetic measurements showed low and transient plasma levels of OPB-31121. Inter-patient variability was high. Exposure was low - area-under-the-curve (AUC) values were 2–3 orders of magnitude lower than those measured at active doses in mouse models. Analysis of metabolites in plasma samples indicates extensive CYP3A4 metabolism and suggests a large first-pass effect in humans, which had not been observed in rodents. Conclusions: OPB-31121 does not show potential as a therapeutic option for most malignancies. The extensive first-pass metabolism and dose-limiting gastrointestinal AEs limit the level of systemic exposure that can be achieved with this agent. Further studies may be warranted in cancers of organs where local concentrations of the compound may be higher (e.g., liver) and an exploratory study in hepatocellular carcinoma is ongoing in Asia. Because STAT3 remains an attractive antitumor target, chemically related compounds with similar pharmacologic activities are currently being evaluated preclinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B56.