Michael Bouvet

Development of Real-time Subcellular Dynamic Multicolor Imaging of Cancer-Cell Trafficking in Live Mice with a Variable-Magnification Whole-Mouse Imaging System

With the use of dual-color fluorescent cells and a highly sensitive whole-mouse imaging system with both macro-optics and micro-optics, we report here the development of subcellular real-time imaging of cancer cell trafficking in live mice. To observe cytoplasmic and nuclear dynamics in the living mouse, tumor cells were labeled in the nucleus with green fluorescent protein and with red fluorescent protein in the cytoplasm. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 whole-mouse imaging system with a sensitive CCD camera and five objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. With the dual-color cancer cells and the highly sensitive whole-mouse imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time. This imaging technology will enable further understanding of the critical steps of metastasis and provide visible targets for antimetastasis drug development.

Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model

Mouse tumor models have undergone profound improvements in the fidelity of emulating human disease. Replacing ectopic s.c. implantation with organ-specific orthotopic implantation reproduces human tumor growth and metastasis. Strong fluorescent labeling with green fluorescent protein along with inexpensive video detectors, positioned externally to the mouse, allows the monitoring of details of tumor growth, angiogenesis, and metastatic spread. However, the sensitivity of external imaging is limited by light scattering in intervening tissue, most especially in skin. Opening a reversible skin-flap in the light path markedly reduces signal attenuation, increasing detection sensitivity many-fold. The observable depth of tissue is thereby greatly increased and many tumors that were previously hidden are now clearly observable. This report presents tumor images and related quantitative growth data previously impossible to obtain. Single tumor cells, expressing green fluorescent protein, were seeded on the brain image through a scalp skin-flap. Lung tumor microfoci representing a few cells are viewed through a skin-flap over the chest wall, while contralateral micrometastases were imaged through the corresponding skin-flap. Pancreatic tumors and their angiogenic microvessels were imaged by means of a peritoneal wall skin-flap. A skin-flap over the liver allowed imaging of physiologically relevant micrometastases originating in an orthotopically implanted tumor. Single tumor cells on the liver arising from intraportal injection also were detectable. Possible future technical developments are suggested by the image, through a lower-abdominal skin-flap, of an invasive prostate tumor expressing both red and green fluorescent proteins in separate colonies.

Nestin-Linked Green Fluorescent Protein Transgenic Nude Mouse for Imaging Human Tumor Angiogenesis

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein-expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.

A novel red fluorescent protein orthotopic pancreatic cancer model for the preclinical evaluation of chemotherapeutics

BACKGROUND Realistic models of pancreatic cancer are necessary to develop effective drugs for the disease. More aggressive tumor models enhanced by brighter fluorescent biomarkers to follow the disease in real time would enhance the ability to predict accurately the effect of novel therapeutics on this particularly malignant human cancer. MATERIALS AND METHODS A novel, highly fluorescent, red fluorescent protein (RFP)-expressing pancreatic cancer model was orthotopically established in nude mice. The MIA-PaCa-2 human pancreatic cancer cell line was transduced with RFP and grown subcutaneously. Fluorescent tumor fragments were then surgically transplanted onto the nude mouse pancreas. Groups treated with intraperitoneal gemcitabine or intravenous irinotecan were sequentially imaged to compare, in real time, the antimetastatic and antitumor effects of these agents compared with untreated controls. RESULTS Rapid tumor growth and widespread metastases developed in untreated mice within 2 weeks, leading to a median survival of 21 days. In contrast, significant tumor growth suppression and consequent increase in survival (32.5 days, P = 0.009) were achieved with CPT-11. Gemcitabine highly improved survival (72 days, P = 0.004) by inducing transient tumor regression over the first 3 weeks. However, at this time, growth and dissemination occurred despite continued treatment, suggesting the development of tumor resistance. The antimetastatic efficacy of each drug was followed noninvasively in real time by imaging the RFP-expressing tumor and metastases, and was confirmed by fluorescent open imaging of autopsy specimens. CONCLUSIONS This highly metastatic model reliably simulates the aggressive course of human pancreatic cancer. Noninvasive, sequential imaging permits quantification of tumor growth and dissemination and, thereby, real time evaluation of therapeutic efficacy. These features make this model an ideal, preclinical system with which to study novel therapeutics for pancreatic cancer.

Real-time in vivo dual-color imaging of intracapillary cancer cell and nucleus deformation and migration.

The mechanism of cancer cell deformation and migration in narrow vessels is incompletely understood. In order to visualize the cytoplasmic and nuclear dynamics of cells migrating in capillaries, red fluorescent protein was expressed in the cytoplasm, and green fluorescent protein, linked to histone H2B, was expressed in the nucleus of cancer cells. Immediately after the cells were injected in the heart of nude mice, a skin flap on the abdomen was made. With a color CCD camera, we could observe highly elongated cancer cells and nuclei in capillaries in the skin flap in living mice. The migration velocities of the cancer cells in the capillaries were measured by capturing images of the dual-color fluorescent cells over time. The cells and nuclei in the capillaries elongated to fit the width of these vessels. The average length of the major axis of the cancer cells in the capillaries increased to approximately four times their normal length. The nuclei increased their length 1.6 times in the capillaries. Cancer cells in capillaries over 8 microm in diameter could migrate up to 48.3 microm/hour. The data suggests that the minimum diameter of capillaries where cancer cells are able to migrate is approximately 8 microm. The use of the dual-color cancer cells differentially labeled in the cytoplasm and nucleus and associated fluorescent imaging provide a powerful tool to understand the mechanism of cancer cell migration and deformation in small vessels.

Metastases to the Thyroid: A Review of the Literature from the Last Decade

BACKGROUND Although clinically evident metastases of nonthyroid malignancies (NTMs) to the thyroid gland are uncommon, it is important to suspect them in patients who present with a new thyroid mass and a history, however far back, of prior malignancy. In fact, metastases from NTMs to the thyroid gland have been reported in 1.4%-3% of all patients who have surgery for suspected cancer in the thyroid gland. Here we review the literature over the last decade regarding this topic. SUMMARY Based on recent literature, the most common NTMs that metastasize to the thyroid gland are renal cell (48.1%), colorectal (10.4%), lung (8.3%), and breast carcinoma (7.8%), and sarcoma (4.0%). Metastases of NTMs to the thyroid are more common in women than men (female to male ratio=1.4 to 1) and in nodular thyroid glands (44.2%). The mean and median intervals between diagnosing NTMs and their metastases to thyroid gland are 69.9 and 53 months, respectively. In 20% of cases the diagnosis of the NTM and its metastases to the thyroid was synchronous. Recent reports indicate that there is a higher frequency of sarcoma metastasizing to the thyroid gland than reported in prior years. Fine-needle aspiration biopsy (FNAB) of thyroid masses is useful in diagnosis of thyroid metastases. However, this requires information about the NTM so that the proper antibodies can be used for immunohistochemical analysis; therefore it is of lesser utility if the NTM is occult. In patients with preexisting thyroid pathology the FNAB diagnosis can be more difficult due to more than one lesion being present. CONCLUSIONS It is important to keep in mind that the thyroid gland can be a site of metastases for a variety of tumors when evaluating a thyroid nodule, especially in a patient with a prior history of malignancy. In patients with thyroid lesions and a history of malignant disease, regardless of time elapsed since the initial diagnosis of the primary neoplasm, disease recurrence or progression of malignancy must be considered until proven otherwise.

Factors influencing survival after resection for periampullary neoplasms

BACKGROUND The purpose of this study was to determine predictors of survival after resection for periampullary neoplasms. METHODS Over a 15-year period, 208 patients underwent laparotomy for periampullary neoplasms. Data were analyzed to assess predictors of survival. RESULTS Pathologic examination showed pancreatic cancer (n = 136; 65%), ampullary cancer (n = 28; 13%), distal common bile duct cancer (n = 10; 5%), duodenal cancer (n = 4; 2%), neuroendocrine tumor (n = 11; 5%), cystadenocarcinoma (n = 4; 2%), cystadenoma (n = 5; 2%), and other (n = 10; 5%). A total of 129 patients underwent pancreatic resection (71 Whipples, 35 total pancreatectomies, 21 distal pancreatectomies, and 2 partial pancreatectomies) whereas 79 patients were found to be unresectable and underwent palliative bypass and/or biopsy. Median survival was 20.4 months for resectable patients versus 4.5 months for unresectable patients (P<0.001). Of the 129 resected patients, factors significantly (P<0.05) favoring long-term survival on univariate analysis included well-differentiated histology, common bile duct or ampullary adenocarcinoma, early stage, tumor diameter <2 cm, negative margins, and absence of lymph node metastases, perineural, or vascular invasion. Age, sex, race, and type of procedure had no influence on survival. On multivariate analysis, only tumor differentiation appeared independently related to survival. Using Kendalls tau analysis, tumor type and grade correlated significantly with all other predictors. CONCLUSIONS Of all variables studied, tumor type and poor tumor differentiation in periampullary neoplasms appear to be markers that predict a constellation of other adverse findings.

Half-Antibody Functionalized Lipid−Polymer Hybrid Nanoparticles for Targeted Drug Delivery to Carcinoembryonic Antigen Presenting Pancreatic Cancer Cells

Current chemotherapy regimens against pancreatic cancer are met with little success as poor tumor vascularization significantly limits the delivery of oncological drugs. High-dose targeted drug delivery, through which a drug delivery vehicle releases a large payload upon tumor localization, is thus a promising alternative strategy against this lethal disease. Herein, we synthesize anti-carcinoembryonic antigen (CEA) half-antibody conjugated lipid-polymer hybrid nanoparticles and characterize their ligand conjugation yields, physicochemical properties, and targeting ability against pancreatic cancer cells. Under the same drug loading, the half-antibody targeted nanoparticles show enhanced cancer killing effect compared to the corresponding nontargeted nanoparticles.

Monotherapy with a Tumor-Targeting Mutant of S. typhimurium Inhibits Liver Metastasis in a Mouse Model of Pancreatic Cancer

Cancer of the exocrine pancreas is the fourth leading cause of cancer deaths in the United States. Currently, surgical resection is the only hope for cure. The majority of patients present with locally-advanced or metastatic disease. The most common site for distant metastasis is the liver. We report here a modified auxotrophic strain of S. typhimurium that can target and inhibit the growth of liver metastasis in a mouse model of pancreatic cancer. This strain of S. typhimurium is auxotrophic (leucine-arginine dependent) but apparently receives sufficient nutritional support from tumor tissue. To increase tumor targeting ability and tumor killing efficacy, this strain was further modified by re-isolation from a tumor growing in a nude mouse and termed A1-R. In the present study, we demonstrate the efficacy of locally- as well as systemically-administered A1-R on liver metastasis of pancreatic cancer. Mice treated with A1-R given locally via intrasplenic injection or systemically via tail vein injection had a much lower hepatic and splenic tumor burden compared with control mice. Systemic treatment with intravenous A1-R also increased survival time. All results were statistically significant. This study suggests the clinical potential of bacterial treatment of a critical metastatic target of pancreatic cancer.

Systemic targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma in nude mice with a tumor-selective strain of Salmonella typhimurium

We report here a new targeting strategy for primary bone tumor and lung metastasis with a modified auxotrophic strain of Salmonella typhimurium. We have previously developed the genetically-modified strain of S. typhimurium, selected for tumor targeting and therapy in vivo. Normal tissue is cleared of these bacteria even in immunodeficient athymic mice with no apparent side effects. In this study, the tumor-targeting strain of S. typhimurium, termed A1-R, was administered i.v. to nude mice which have primary bone tumor and lung metastasis. Primary bone tumor was obtained by orthotopic intratibial injection of 5 x 105 143B-RFP (red fluorescent protein) human osteosarcoma cells. One group of mice was treated with A1-R expressing GFP (green fluorescent protein) and another group was used a as control. A1-R (5 x 107 colony-forming units) was injected in the tail vein three times on weekly basis. On day 28, lung samples were excised and observed with the Olympus OV100 Small Animal Imaging System. The size of the primary tumor and RFP intensity of lung metastasis were measured. Primary bone tumor size (fluorescence area [mm2]) was 232 ± 70 in the untreated group and 95 ± 23 in the treated group (P