Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model.

BACKGROUND: We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer. METHODS: We established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection. RESULTS: All mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049). CONCLUSIONS: Surgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.

MOZ-TIF2-induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2-mediated recruitment of CBP.

The MOZ-TIF2 fusion is associated with acute myeloid leukemia (AML) with inv(8)(p11q13). MOZ is a MYST family histone acetyltransferase (HAT), whereas TIF2 is a nuclear receptor coactivator that associates with CREB binding protein (CBP). Here we demonstrate that MOZ-TIF2 has transforming properties in vitro and causes AML in a murine bone marrow transplant assay. The C2HC nucleosome recognition motif of MOZ is essential for transformation, whereas MOZ HAT activity is dispensable. However, MOZ-TIF2 interaction with CBP through the TIF2 CBP interaction domain (CID) is essential for transformation. These results indicate that nucleosomal targeting by MOZ and recruitment of CBP by TIF2 are critical requirements for MOZ-TIF2 transformation and indicate that MOZ gain of function contributes to leukemogenesis.

Tumor-specific fluorescence antibody imaging enables accurate staging laparoscopy in an orthotopic model of pancreatic cancer.

BACKGROUND/AIMS: Laparoscopy is important in staging pancreatic cancer, but false negatives remain problematic. Making tumors fluorescent has the potential to improve the accuracy of staging laparoscopy. METHODOLOGY: Orthotopic and carcinomatosis models of pancreatic cancer were established with BxPC-3 human pancreatic cancer cells in nude mice. Alexa488-antiCEA conjugates were injected via tail vein 24 hours prior to laparoscopy. Mice were examined under bright field laparoscopic (BL) and fluorescence laparoscopic (FL) modes. Outcomes measured included time to identification of primary tumor for the orthotopic model and number of metastases identified within 2 minutes for the carcinomatosis model. RESULTS: FL enabled more rapid and accurate identification and localization of primary tumors and metastases than BL. Using BL took statistically significantly longer time than FL (p<0.0001, fold change and 95% CI for BL vs. FL: 8.12 (4.54,14.52)). More metastatic lesions were detected and localized under FL compared to BL and with greater accuracy, with sensitivities of 96% vs. 40%, respectively, when compared to control. FL was sensitive enough to detect metastatic lesions <1mm. CONCLUSIONS: The use of fluorescence laparoscopy with tumors labeled with fluorophore-conjugated anti-CEA antibody permits rapid detection and accurate localization of primary and metastatic pancreatic cancer in an orthotopic model. The results of the present report demonstrate the future clinical potential of fluorescence laparoscopy.

Knockdown of the ß(1) integrin subunit reduces primary tumor growth and inhibits pancreatic cancer metastasis.

To address the role of ß(1) integrins in pancreatic cancer progression, we stably knocked down ß(1) integrin subunit expression in human FG-RFP pancreatic cancer cells using lentiviral-based RNA interference. We then examined the effects of ß(1) integrin subunit knockdown on pancreatic cancer cell adhesion, migration and proliferation on tumor microenvironment-specific extracellular matrix proteins in vitro and on tumor progression in vivo using a clinically relevant fluorescent orthotopic mouse model of pancreatic cancer. Knockdown of the ß(1) integrin subunit inhibited cell adhesion, migration and proliferation on types I and IV collagen, fibronectin and laminin in vitro. In vivo, knockdown of the ß(1) integrin subunit reduced primary tumor growth by 50% and completely inhibited spontaneously occurring metastasis. These observations indicate a critical role for the ß(1) integrin subunit in pancreatic cancer progression and metastasis in particular. Our results suggest the ß(1) integrin subunit as a therapeutic target for the treatment of pancreatic cancer, especially in the adjuvant setting to prevent metastasis of this highly aggressive cancer.

Fluorescence laparoscopy imaging of pancreatic tumor progression in an orthotopic mouse model.

BACKGROUND: The use of fluorescent proteins to label tumors is revolutionizing cancer research, enabling imaging of both primary and metastatic lesions, which is important for diagnosis, staging, and therapy. This report describes the use of fluorescence laparoscopy to image green fluorescent protein (GFP)-expressing tumors in an orthotopic mouse model of human pancreatic cancer. METHODS: The orthotopic mouse model of human pancreatic cancer was established by injecting GFP-expressing MiaPaCa-2 human pancreatic cancer cells into the pancreas of 6-week-old female athymic mice. On postoperative day 14, diagnostic laparoscopy using both white and fluorescent light was performed. A standard laparoscopic system was modified by placing a 480-nm short-pass excitation filter between the light cable and the laparoscope in addition to using a 2-mm-thick emission filter. A camera was used that allowed variable exposure time and gain setting. For mouse laparoscopy, a 3-mm 0° laparoscope was used. The mouse's abdomen was gently insufflated to 2 mm Hg via a 22-gauge angiocatheter. After laparoscopy, the animals were sacrificed, and the tumors were collected and processed for histologic review. The experiments were performed in triplicate. RESULTS: Fluorescence laparoscopy enabled rapid imaging of the brightly fluorescent tumor in the pancreatic body. Use of the proper filters enabled simultaneous visualization of the tumor and the surrounding structures with minimal autofluorescence. Fluorescence laparoscopy thus allowed exact localization of the tumor, eliminating the need to switch back and forth between white and fluorescence lighting, under which the background usually is so darkened that it is difficult to maintain spatial orientation. CONCLUSION: The use of fluorescence laparoscopy permits the facile, real-time imaging and localization of tumors labeled with fluorescent proteins. The results described in this report should have important clinical potential.

Development of the transgenic cyan fluorescent protein (CFP)-expressing nude mouse for "Technicolor" cancer imaging.

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non-transgenic nude mice with the transgenic CK/ECFP mouse in which the beta-actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA-1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color-coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color-coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment.

Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer.

BACKGROUND: Pancreatic cancer is a devastating disease characterized by dismal 5-year survival rates and limited treatment options. In an effort to provide useful models for preclinical evaluation of new experimental therapeutics, we and others have developed orthotopic mouse models of pancreatic cancer. The utility of these models for pre-clinical testing is dependent upon quantitative, noninvasive methods for monitoring in vivo tumor progression in real time. Toward this goal, we performed whole-body fluorescence imaging and ultrasound imaging to evaluate and to compare these noninvasive imaging modalities for assessing tumor burden and tumor progression in an orthotopic mouse model of pancreatic cancer. METHODS: The human pancreatic cancer cell line XPA-1, engineered for stable, high-level expression of red fluorescent protein (RFP), was implanted into the pancreas of nude mice using orthotopic implantation. The tumors were allowed to grow over a period of one to several weeks during which time the mice were imaged using both fluorescence imaging and ultrasound imaging to measure tumor burden and to monitor tumor growth. RESULTS: Whole-body fluorescence imaging and ultrasound imaging both allowed for the visualization and measurement of orthotopic pancreatic tumor implants in vivo. The imaging sessions were well-tolerated by the mice and yielded data which correlated well in the quantitative assessment of tumor burden. Whole-body fluorescence and two-dimensional ultrasound imaging showed a strong correlation for measurement of tumor size over a range of tumor sizes (R(2) = 0.6627, P = 0.003 for an exposure time of 67 msec and R(2) = 0.6553, P = 0.003 for an exposure time of 120 msec). CONCLUSION: Our findings suggest a complementary role for fluorescence imaging and ultrasound imaging in assessing tumor burden and tumor progression in orthotopic mouse models of human cancer.

The cyan fluorescent protein (CFP) transgenic mouse as a model for imaging pancreatic exocrine cells.

CONTEXT: The use of fluorescent proteins for in vivo imaging has opened many new areas of research. Among the important advances in the field have been the development of transgenic mice expressing various fluorescent proteins. OBJECTIVE: To report whole-body and organ-specific fluorescence imaging to characterize the transgenic cyan fluorescent protein mouse. DESIGN: Mice were imaged using two devices. Brightfield images were obtained with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). Fluorescence imaging was performed under the cyan fluorescent protein filter using the iBox Small Animal Imaging System (UVP, Upland, CA, USA). INTERVENTION: All animals were sacrificed immediately before imaging. They were imaged before and throughout multiple steps of a complete necropsy. Harvested organs were also imaged with both devices. Selected organs were then frozen and processed for histology, fluorescence microscopy, and H&E staining. Fluorescence microscopy was performed with an Olympus IMT-2 inverted fluorescence microscope. MAIN OUTCOME MEASURE: Determination of fluorescence intensity of different organs. RESULTS: Surprisingly, we found that there is differential enhancement of fluorescence among organs; most notably, the pancreas stands out from the rest of the gastrointestinal tract, displaying the strongest fluorescence of all organs in the mouse. Fluorescence microscopy demonstrated that the cyan fluorescent protein fluorescence resided in the acinar cells of the pancreas and not the islet cells. CONCLUSIONS: The cyan fluorescent protein mouse should lead to a deeper understanding of pancreatic function and pathology, including cancer.

Normal hematopoiesis after conditional targeting of RXRalpha in murine hematopoietic stem/progenitor cells.

Because of the retinoic acid receptor-alpha (RARalpha) gene's involvement in acute promyelocytic leukemia, the important role of RARs in hematopoiesis is now well established. However, relatively few studies of hematopoiesis have focused on the role of the retinoid X receptors (RXRs), the obligate heterodimeric partners of the RARs. We sought to establish whether conditional targeting of RXRalpha in early hematopoietic progenitors, ideally to the level of the hematopoietic stem cell (HSC), would compromise hematopoiesis. For hematopoietic targeting of RXRalpha, we characterized IFN-inducible MxCre mice for use in studying the role of RXRalpha in hematopoiesis. We established that MxCre executes recombination of loxP-flanked RXRalpha in hematopoietic progenitors immunophenotypically enriched for HSC, leading to widespread and sustained targeting of RXRalpha in hematopoietic cells. However, we found no evidence of hematologic compromise in mice lacking RXRalpha, suggesting that RXRalpha is dispensable for normal murine hematopoiesis. Nonetheless, RXRalpha null bone marrow cells cultured in methylcellulose form colonies more efficiently than bone marrow cells obtained from control mice. This result suggests that although RXRalpha is not required for murine hematopoiesis, there may be hematopoietic signaling pathways that respond selectively to RXRalpha or settings in which combined expression of RXR (alpha, beta, and gamma) is limiting.

Imaging the Interaction of Pancreatic Cancer and Stellate Cells in the Tumor Microenvironment during Metastasis.

BACKGROUND/AIM: Pancreatic stellate cells are involved in fibrosis of pancreatic cancer termed desmoplasia, which may contribute to both pancreatic cancer growth and metastasis, as well as to drug resistance. A better understanding of pancreatic cancer-cell interactions with stellate cells is therefore critical to our ability to develop effective anti-metastatic therapeutics for pancreatic cancer. MATERIALS AND METHODS: The human pancreatic cancer cell line XPA-1 was engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells were engineered to express RFP. The pancreatic cancer cells and stellate cells were co-cultured and their interaction was imaged in vitro. The pancreatic cancer cells and stellate cells were then co-injected in the spleen of transgenic cyan fluorescent protein (CFP) nude mice and imaged in liver, lung and diaphragm metastasis. RESULTS: The interaction of the pancreatic cancer cells expressing GFP in the nucleus and RFP in the cytoplasm and stellate cells expressing RFP was first imaged in vitro. The intimate relationship between the two cell types could be seen. Three hours after splenic co-injection, dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. By 28 days after splenic co-injection of the pancreatic cancer and stellate cells, liver metastases were observed in host CFP nude mice. Metastases were also observed in the lung and diaphragm. Stellate cells were observed along with the pancreatic cancer cells at all metastatic sites suggesting that stellate cells may be necessary for metastasis. With high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver and other metastases, further suggesting that stellate cells participate in metastasis formation. CONCLUSION: Pancreatic cancer cells and stellate stem cells form a very close relationship and accompany each other to distant metastatic sties. Our hypothesis is that pancreatic stellate cells form a niche for metastasis of pancreatic cancer.

Metachronous double parathyroid adenomas involving two different cell types: chief cell and oxyphil cell.

OBJECTIVE: To describe a patient with the rare occurrence of metachronous double parathyroid adenomas involving two different cell types. METHODS: We present a case report and a summary of the related literature. RESULTS: Double parathyroid adenomas are rare, occurring in 1.7 to 9% of patients with primary hyperparathyroidism. Most double parathyroid adenomas are synchronous lesions; few metachronous double parathyroid adenomas have previously been reported. Most parathyroid adenomas are of the chief cell variety, with oxyphil cell adenomas occurring in less than 1% of cases. In a 73-year-old man with no prior history or known family history of endocrine disease, primary hyperparathyroidism developed, and cervical ultrasonography demonstrated a mass in the right side of the neck. Subsequent parathyroidectomy revealed a right superior chief cell adenoma. Postoperatively, the patient's parathyroid hormone and serum calcium levels returned to normal and remained so for at least 9 years. Twelve years after the first operation, recurrent hyperparathyroidism prompted repeated surgical exploration of the neck, which disclosed a large left superior parathyroid mass. Surgical excision and histologic examination revealed the lesion to be an oxyphil cell adenoma. CONCLUSION: To our knowledge, this is the first reported case of metachronous double parathyroid adenomas involving two different cell types: chief cell and oxyphil cell.

A dual-color genetically engineered mouse model for multispectral imaging of the pancreatic microenvironment.

OBJECTIVES: To develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment. METHODS: Cre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression after exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses. RESULTS: Pdx1-Cre-mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. Green fluorescent protein expression was mosaic in Pdx1-Cre-mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata. CONCLUSIONS: This mouse model enables multispectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.

Detection of colon cancer metastases with fluorescence laparoscopy in orthotopic nude mouse models.

OBJECTIVE: To improve detection of colon cancer metastases using fluorescence laparoscopy (FL). DESIGN: An orthotopic mouse model of human colon cancer was established by intracecal injection of HCT-116 human colon cancer cells expressing green fluorescent protein into 12 mice. One group modeled early disease and the second modeled late metastatic disease. For the early-disease model, 2 weeks after implantation, 6 mice underwent 2 modalities of laparoscopy: bright field laparoscopy (BL) and FL. The number of metastases identified within each of the 4 abdominal quadrants was recorded with both laparoscopy modalities. This process was repeated in the late-metastatic disease group 4 weeks after implantation. All animals were then humanely sacrificed and imaged using open fluorescence laparoscopy (OL) as a positive control to identify metastases. SETTING: Basic science laboratory. PARTICIPANTS: Twelve female, 6-week-old nude mice. INTERVENTIONS: Detection of tumor foci by FL compared with BL. MAIN OUTCOME MEASURES: Number of tumors identified in each quadrant. RESULTS Fluorescence laparoscopy enabled superior visualization of colon cancer metastases compared with BL in the early (P = .03) and late (P = .002) models of colon cancer. Compared with OL, BL was significantly inferior in the early (P = .04) and late (P < .001) groups. Fluorescence laparoscopy was not significantly different from OL in the early (P = .85) or late (P = .46) group. Thus, FL allowed identification of micrometastases that could not be distinguished from surrounding tissue using BL. CONCLUSIONS: The use of FL enables identification of metastases that could not be visualized using standard laparoscopy. This report illustrates the important clinical potential for FL in the surgical treatment of cancer.

Fluorescence-guided surgery allows for more complete resection of pancreatic cancer, resulting in longer disease-free survival compared with standard surgery in orthotopic mouse models.

BACKGROUND: Negative surgical margins are vital to achieve cure and prolong survival in patients with pancreatic cancer. We inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes and reduce recurrence rates in orthotopic mouse models of human pancreatic cancer. STUDY DESIGN: A randomized active-control preclinical trial comparing bright light surgery (BLS) to FGS was used. Orthotopic mouse models of human pancreatic cancer were established using the BxPC-3 pancreatic cancer cell line expressing red fluorescent protein (RFP). Two weeks after orthotopic implantation, tumors were resected with BLS or FGS. Pre- and postoperative images were obtained with the OV-100 Small Animal Imaging System to assess completeness of surgical resection in real time. Postoperatively, noninvasive whole body imaging was done to assess recurrence and follow tumor progression. Six weeks postoperatively, mice were sacrificed to evaluate primary pancreatic and metastatic tumor burden at autopsy. RESULTS: A more complete resection of pancreatic cancer was achieved using FGS compared with BLS: 98.9% vs 77.1%, p = 0.005. The majority of mice undergoing BLS (63.2%) had evidence of gross disease with no complete resections; 20% of mice undergoing FGS had complete resection and an additional 75% had only minimal residual disease (p = 0.0001). The mean postoperative tumor burden was significantly less with FGS compared with BLS: 0.08 ± 0.06 mm(2) vs 2.64 ± 0.63 mm(2), p = 0.001. The primary tumor burden at termination was significantly less with FGS compared with BLS: 19.3 ± 5.3 mm(2) vs 6.2 ± 3.6 mm(2), p = 0.048. FGS resulted in significantly longer disease-free survival than BLS (p = 0.02, hazard ratio = 0.39, 95% CI 0.17, 0.88). CONCLUSIONS: Surgical outcomes were improved in pancreatic cancer using fluorescence-guidance. This novel approach has significant potential to improve surgical treatment of cancer.

An LED light source and novel fluorophore combinations improve fluorescence laparoscopic detection of metastatic pancreatic cancer in orthotopic mouse models.

BACKGROUND: The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations. STUDY DESIGN: Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis. RESULTS: Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. CONCLUSIONS: Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer.

Submillimeter-resolution fluorescence laparoscopy of pancreatic cancer in a carcinomatosis mouse model visualizes metastases not seen with standard laparoscopy.

BACKGROUND: Staging laparoscopy can visualize peritoneal and liver metastases in pancreatic cancer otherwise undetectable by preoperative imaging. However, false-negative rates may be as high as 18%-26%. The aim of the present study was to improve detection of metastatic pancreatic cancer with the use of fluorescence laparoscopy (FL) in a nude-mouse model with the tumors expressing green fluorescent protein (GFP). METHODS: The carcinomatosis mouse model of human pancreatic cancer was established by intraperitoneal injections of green fluorescent protein-expressing MiaPaca-2 human pancreatic cancer cells into 6-week-old female athymic mice. Two weeks later, mice underwent diagnostic laparoscopy. Laparoscopy was performed first under standard brightfield lighting, followed by fluorescent lighting. The number of metastatic foci identified within the four quadrants of the peritoneal cavity was recorded. After laparoscopy, the animals were sacrificed, opened, and imaged with the OV-100 Small Animal Imaging system as a positive control to identify metastasis. Tumors were collected and processed for histologic review. RESULTS: FL enabled visualization of pancreatic cancer metastatic foci not visualized with standard brightfield laparoscopy (BL). Under FL, in 1 representative mouse, 26 separate micrometastatic lesions were identified. In contrast, only very large tumors were seen using BL. Use of the OV-100 images, as positive controls, confirmed the presence of tumor foci. FL thus allowed identification and exact localization of submillimeter tumor foci. Such small-sized tumor foci were not distinguished from surrounding tissue under BL. All malignant lesions were histologically confirmed. CONCLUSIONS: The use of FL enables the identification of tumor foci that cannot be seen with standard laparoscopy. The technology described in this report has important potential for the clinical development of FL.

Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer.

There is a need for a clinically relevant mouse model of thyroid cancer that enables real-time, non-invasive monitoring of tumor growth, progression, and drug response over time. Human thyroid cancer cell lines NPA (papillary) and KAK-1 (anaplastic) were stably transfected to express either red or green fluorescent protein. Cancer cells were injected into the thyroid glands of 8-week-old athymic mice. The animals were imaged with whole-body fluorescence imaging weekly and sacrificed when premorbid. At necropsy, the primary tumor was resected en bloc with the respiratory system for processing and analysis. Histology was performed on fixed tissue specimens for review of morphologic findings. Both anaplastic and papillary thyroid cancer cell lines led to robust development of orthotopic fluorescent tumors in nude mice. Injection of 5×10(5) cancer cells was sufficient for tumor development. Tumors were visualized for both cell lines via non-invasive imaging as early as 3 weeks post-implantation and were monitored over time. Time to premorbid condition varied between mice and was associated with a primary tumor growth pattern (early local compression of the esophagus vs. late metastatic disease) rather than tumor size. At necropsy, tumor fluorescence demonstrated metastases in the lungs, lymph nodes and vessels that were not visible under white light. Thus an orthotopic mouse model of thyroid cancer has been developed that replicates the major clinical features of thyroid cancer and enables real-time, non-invasive monitoring of tumor progression. This model should permit preclinical evaluation of novel thyroid cancer therapeutics.