Lezlee Coghlan

Plasmon resonance coupling of metal nanoparticles for molecular imaging of carcinogenesis in vivo

An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-κB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IκB kinase (IKK)α, a required intermediate in the noncanonical NF-κB signaling pathway. Introduction of an IKKα transgene driven by a K5 promoter restores the K5+K14+ mTEC subset in IKKα−/− mice. Unexpectedly, the K5-IKKα transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKα transgene ameliorates autoimmune symptoms in IKKα−/− mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-κB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

Transgenic Expression of Cyclin D1 in Thymic Epithelial Precursors Promotes Epithelial and T Cell Development

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5− TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3ε transgenic and hCD3ε/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: Results from an animal model

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

Changes in protein expression during multistage mouse skin carcinogenesis

To directly compare the expression patterns of different proteins known to be altered during mouse skin carcinogenesis, serial sections of normal and hyperplastic skin and tumors from various stages of 7,12‐dimethylbenz[a]anthracene–initiated, 12‐O‐tetradecanoylphorbol‐13‐acetate–promoted female SENCAR mice were examined by immunohistochemistry. In untreated, normal mouse skin, keratin 1 (K1) and transforming growth factor‐β1 (TGFβ1) were strongly expressed in the suprabasal layers, whereas integrin α6β4 was expressed only in basal cells and only moderate staining for transforming growth factor‐α (TGFα) was seen. In hyperplastic skin, TGFα expression became stronger, whereas expression of another epidermal growth factor (EGF) receptor ligand, heparin‐binding EGF‐like growth factor (HB‐EGF), was strongly induced in all epidermal layers from no expression in normal skin. Likewise, the gap‐junctional protein connexin 26 (Cx26) became highly expressed in the differentiated granular layers of hyperplastic skin relative to undetectable expression in normal skin. Expression of cyclin D1 in the proliferative cell compartment was seen in all benign and malignant tumors but not in hyperplastic skin. Beginning with very early papillomas (after 10 wk of promotion), expression of α6β4 in suprabasal cells and small, focal staining for keratin 13 (K13) were seen in some tumors. Later (after 20–30 wk), focal areas of γ‐glutamyl transpeptidase (GGT) activity appeared in a few papillomas, whereas TGFβ1 expression began to decrease. Cx26 and TGFα staining became patchier in some late‐stage papillomas (30–40 wk), whereas suprabasal α6β4, K13, and GGT expression progressively increased and K1 expression decreased. Finally, in squamous cell carcinomas (SCCs), there was an almost complete loss of K1 and a further decline in TGFα, HB‐EGF, TGFβ1, and Cx26 expression. On the other hand, almost all SCCs showed suprabasal staining for α6β4 and widespread cyclin D1 and K13 expression, whereas only about half showed positive focal staining for GGT activity. Mol. Carcinog. 20:125–136, 1997.

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents.

Feasibility and preliminary accuracy of high-resolution imaging of the liver and pancreas using FNA compatible microendoscopy (with video).

BACKGROUND EUS-guided FNA is one of the few techniques that can obtain cells and tissue from the liver and pancreas. However, the technique remains vulnerable to poor specimen quality and sampling error. OBJECTIVE To evaluate the ability of a high-resolution microendoscope (HRME) to visualize the cellular and architectural features of normal and malignant liver and pancreatic tissue ex vivo, to assess the ability of endosonographers to identify normal and neoplastic tissue by using HRME images, and to demonstrate preliminary technical feasibility of in vivo HRME imaging via EUS fine-needle puncture (FNP). DESIGN Ex vivo pilot feasibility study in human tissue; in vivo swine model. SETTING Two academic medical centers. PATIENTS Co-registered HRME images and biopsies were obtained from surgically resected hepatic and pancreatic tissues from 44 patients. INTERVENTION Images were divided into training (12 images) and test (80 images) sets containing a range of normal and pathologic conditions for each organ. After viewing the training sets, 9 endosonographers attempted to distinguish malignant tissue from normal or benign lesions in the test sets, each of which contained 40 unique images with individual diagnoses from pathology. MAIN OUTCOME MEASUREMENTS Image acquisition feasibility, ex vivo and in vivo. Ability of endosonographers to recognize features of normal/benign or malignant tissue from the liver and pancreas. RESULTS Overall, the 9 endosonographers achieved median accuracy figures of 85% in the liver and 90% in the pancreas. The endosonographers with prior experience in reading HRME images achieved accuracy rates between 90% and 95%. Technical feasibility of HRME imaging through a 19-gauge EUS-FNP needle was demonstrated in an in vivo swine model. LIMITATIONS Ex vivo study. CONCLUSION High-resolution microendoscopy allows real-time imaging of cellular-level morphology and tissue architecture in the liver and pancreas. The technique appears to have a short learning curve, after which endosonographers achieved high accuracy rates in distinguishing malignant tissue from normal and benign pathology in both organs. Translating this imaging platform to the in vivo setting appears technically feasible.

Multispectral digital microscopy for in vivo monitoring of oral neoplasia in the hamster cheek pouch model of carcinogenesis

In this study we use a multi-spectral digital microscope (MDM) to measure multi-spectral auto-fluorescence and reflectance images of the hamster cheek pouch model of DMBA (dimethylbenz[alpha]anthracene)- induced oral carcinogenesis. The multi-spectral images are analyzed both in the RGB (red, green, blue) color space as well as in the YCbCr (luminance, chromatic minus blue, chromatic minus red) color space. Mean image intensity, standard deviation, skewness, and kurtosis are selected as features to design a classification algorithm to discriminate normal mucosa from neoplastic tissue. The best diagnostic performance is achieved using features extracted from the YCbCr space, indicating the importance of chromatic information for classification. A sensitivity of 96% and a specificity of 84% were achieved in separating normal from abnormal cheek pouch lesions. The results of this study suggest that a simple and inexpensive MDM has the potential to provide a cost-effective and accurate alternative to standard white light endoscopy.

Triplex-forming oligonucleotides targeting c-MYC potentiate the anti-tumor activity of gemcitabine in a mouse model of human cancer

Antimetabolite chemotherapy remains an essential cancer treatment modality, but often produces only marginal benefit due to the lack of tumor specificity, the development of drug resistance, and the refractoriness of slowly proliferating cells in solid tumors. Here, we report a novel strategy to circumvent the proliferation‐dependence of traditional antimetabolite‐based therapies. Triplex‐forming oligonucleotides (TFOs) were used to target site‐specific DNA damage to the human c‐MYC oncogene, thereby inducing replication‐independent, unscheduled DNA repair synthesis (UDS) preferentially in the TFO‐targeted region. The TFO‐directed UDS facilitated incorporation of the antimetabolite, gemcitabine (GEM), into the damaged oncogene, thereby potentiating the anti‐tumor activity of GEM. Mice bearing COLO 320DM human colon cancer xenografts (containing amplified c‐MYC) were treated with a TFO targeted to c‐MYC in combination with GEM. Tumor growth inhibition produced by the combination was significantly greater than with either TFO or GEM alone. Specific TFO binding to the genomic c‐MYC gene was demonstrated, and TFO‐induced DNA damage was confirmed by NBS1 accumulation, supporting a mechanism of enhanced efficacy of GEM via TFO‐targeted DNA damage‐induced UDS. Thus, coupling antimetabolite chemotherapeutics with a strategy that facilitates selective targeting of cells containing amplification of cancer‐relevant genes can improve their activity against solid tumors, while possibly minimizing host toxicity.