Virally directed fluorescent imaging improves diagnostic sensitivity in the detection of minimal residual disease after potentially curative cytoreductive surgery

Completeness of cytoreduction is an independent prognostic factor after cure-intended surgery for peritoneal carcinomatosis. NV1066, a genetically engineered herpes simplex virus carrying the transgene for green fluorescent protein, selectively infects cancer cells. We sought to determine the feasibility of virally directed fluorescent imaging in the intraoperative detection of minimal residual disease after cytoreductive surgery. NV1066 infected human gastric cancer cells, OCUM-2MD3, and mesothelioma JMN cells at all doses. The infected cells expressed green fluorescent protein and were killed. OCUM-2MD3, and mesothelioma JMN cells at all doses. Peritoneal carcinomatosis was established in mice by injection of OCUM cells into the peritoneal cavity. Forty-eight hours after intraperitoneal injection of NV1066, two experienced surgeons resected all visible disease and identified mice free of disease. Eight of 13 mice thought to be free of disease were found to have residual disease as identified by green fluorescence (mean number of observations: 5; range: 1–9). Residual disease was most frequently observed in the retroperitoneum, pelvis, peritoneal surface, and liver. Specificity of NV1066 infection to tumor nodules was confirmed by immunohistochemistry and by polymerase chain reaction for viral gene. Virally directed fluorescent imaging, a novel molecular imaging technology, can be used for real-time visualization of minimal residual disease after cytoreductive surgery and can improve the completeness of cure-intended resection. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 14–18, 2005 (oral presentation) Supported in part by AstraZeneca Cancer Research and Prevention fellowship (P.S.A), training grant T 32 CA09501 (D.P.E and K.J.H.), grants RO1 CA 76416 and RO1 CA/DK80982 (Y.F.) from the National Institutes of Health, grant BC024118 from the U.S. Army (Y.F.), grant IMG0402501 from the Susan G. Komen Foundation (Y.F. and P.S.A), and grant 032047 from the Flight Attendant Medical Research Institute (Y.F. and P.S.A).     

The replication-competent oncolytic herpes simplex mutant virus NV1066 is effective in the treatment of esophageal cancer

BACKGROUND: The oncolytic herpes simplex-1 virus, NV1066, is a replication-competent virus that has been engineered to infect and lyse tumor cells selectively and to carry a transgene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine viral cytotoxicity in an esophageal cancer cell line and to determine whether EGFP expression could be used as a marker of viral infection. METHODS: BE3 esophageal adenocarcinoma cells were infected with NV1066 in vitro to determine cell kill and viral replication. EGFP expression was assessed by flow cytometry. The in vivo anti-tumor activity of NV1066 was tested in subcutaneous and intraperitoneal xenograft models. EGFP expression was localized in vivo by fluorescent microscopy and fluorescent laparoscopy. RESULTS: NV1066 effectively replicated within and killed BE3 cells in vitro and in vivo. EGFP expression identified infected tumor cells. After NV1066 treatment in vivo, EGFP expression localized to the tumor. In an intraperitoneal tumor model, EGFP could be visualized endoscopically using a laparoscope with a fluorescent filter. CONCLUSIONS: NV1066 has oncolytic activity against the BE3 cell line and may be a useful therapy against esophageal cancer. EGFP expression localizes the virus and may help to identify tumor deposits in vivo. Oncolytic activity with NV1066 against gastrointestinal cancers may potentially be tracked by endoscopy.     

5-Fluorouracil and gemcitabine potentiate the efficacy of oncolytic herpes viral gene therapy in the treatment of pancreatic cancer

Oncolytic herpes viruses are attenuated, replication-competent viruses that selectively infect, replicate within, and lyse cancer cells and are highly efficacious in the treatment of a wide variety of experimental cancers. The current study seeks to define the pharmacologic interactions between chemotherapeutic drugs and the oncolytic herpes viral strain NV1066 in the treatment of pancreatic cancer cell lines. The human pancreatic cancer cell lines Hs 700T, PANC-1, and MIA P_aC_a-2 were treated in vitro with NV1066 at multiplicities of infection (MOI; ratio of the number of viral particles per tumor cell) ranging from 0.01 to 1.0 with or without 5-fluorouracil (5-FU) or gemcitabine. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Viral replication was measured using a standard plaque assay. Six days after combination therapy, 76% of Hs 700T cells were killed compared with 43% with NV1066 infection alone (MOI = 0.1) or 0% with 5-FU alone (2 βmol/L) (P < .01). Isobologram and combination-index analyses confirmed a strongly synergistic pharmacologic interaction between the agents at all viral and drug combinations tested (LD5 to LD95) in the three cell lines. Dose reductions up to 6- and 78-fold may be achieved with combination therapy for NV1066 and 5-FU, respectively, without compromising cell kill. 5-FU increased viral replication up to 19-fold compared with cells treated with virus alone. Similar results were observed by combining gemcitabine and NV1066. We have demonstrated that 5-FU and gemcitabine potentiate oncolytic herpes viral replication and cytotoxicity across a range of clinically achievable doses in the treatment of human pancreatic cancer cell lines. The potential clinical implications of this synergistic interaction include improvements in efficacy, treatment-associated toxicity, tolerability of therapeutic regimens, and quality of life. These data provide the cellular basis for the clinical investigation of combined oncolytic herpes virus therapy and chemotherapy in the treatment of pancreatic cancer. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 141-18, 2005 (oral presentation). Supported in part by training grant T 32 CA09501 (D.P.E. and K.J.H.), AACR-AstraZeneca Cancer Research and Prevention fellowship (P.S.A), grants RO1 CA 76416 and RO1 CA/DK80982 (Y.F.) from the National Institutes of Health, grant BC024118 from the U.S. Army (Y.F.), grant IMG0402501 from the Susan G. Komen Foundation (Y.F.), and grant 032047 from Flight Attendant Medical Research Institute (Y.F.).     

A Novel Genetically Modified Oncolytic Vaccinia Virus in Experimental Models is Effective Against a Wide Range of Human Cancers

Background

Replication-competent oncolytic viruses have shown great promise as a potential cancer treatment. This study aimed to determine whether a novel vaccinia virus, GLV-1h151, with genetic modifications enhancing cancer specificity and enabling virus detection, is effective against a range of human cancers and is safe when administered in preclinical models.

Methods

GLV-1h151 was modified with deletion of thymidine kinase enhancing specificity and insertion of the green fluorescent protein (GFP) gene. The virus was tested in several human cancer cell lines for cytotoxicity including breast, lung, pancreatic, and colorectal. Virus replication was assessed via visualization of GFP expression and bioluminescence, and viral plaque assays. Finally, GLV-1h151 was administered systemically or intratumorally in mice with pancreatic cancer xenografts (PANC-1) to assess virus biodistribution, toxicity, and effect on tumor growth.

Results

GLV-1h151 effectively infected, replicated in, and killed several cancer cell types. Detection and visualization of virus replication was successful via fluorescence imaging of GFP expression, which was dose dependent. When administered intravenously or intratumorally in vivo, GLV-1h151 regressed tumor growth (P < 0.001) and displayed a good biosafety profile. GLV-1h151 infection and replication in tumors was successfully visualized via GFP and bioluminescence, with virus presence in tumors confirmed histologically.

Conclusions

GLV-1h151 is effective as an oncolytic agent against a wide range of cancers in cell culture and is effective against pancreatic human xenografts displaying a good biosafety profile and ability to be detected via optical imaging. GLV-1h151 thus adds another potential medium for the killing of cancer and detection of virus in infected tissue.

     

Noninvasive detection of bladder cancer in an orthotopic murine model with green fluorescence protein cytology

PURPOSE: Orthotopic models of bladder cancer mimic the normal microenvironment and provide an opportunity to study new therapies for superficial bladder cancer. The use of green fluorescent protein (GFP) transduced cells provides a sensitive way of monitoring this disease. We investigated whether examining voided urine for GFP expressing cells would indicate the presence of GFP producing tumors in an orthotopic bladder tumor model in nude mice. MATERIALS AND METHODS: The human bladder cancer cell lines KU-7, UM-UC-3 and UM-UC-14 were used. GFP transductants were generated after transfection with pEGFP-N3, followed by G418 selection. After the cells were inoculated in an orthotopic model of superficial bladder cancer voided urine was collected on slides weekly for 3 weeks and observed for GFP expressing cells by fluorescence microscopy. Bladder tumor imaging for GFP was performed in surgically exposed bladders to determine the tumor incidence. RESULTS: KU-7 GFP cells produced tumors in all 16 mice on whole bladder GFP imaging. UM-UC-3 and UM-UC-14 GFP cells produced tumors in 8 of 12 (67%) and 18 of 25 (72%) mice, respectively. The rate of GFP positive cells in spontaneously voided urine varied by cell line and increased with time but it was generally less than the rate of detection by whole bladder GFP imaging. All mice with GFP expressing cells in the urine had GFP expressing bladder tumors. CONCLUSIONS: Examining urine for GFP expressing cells is less sensitive than imaging surgically exposed bladders but it is 100% specific.     

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 10⁷ G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation. Presented in part at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Enhancing Surgical Vision by Using Real-Time Imaging of α<Subscript>v</Subscript>β<Subscript>3</Subscript>-Integrin Targeted Near-Infrared Fluorescent Agent

Background  

This study was designed to improve the surgical procedure and outcome of cancer surgery by means of real-time molecular imaging feedback of tumor spread and margin delineation using targeted near-infrared fluorescent probes with specificity to tumor biomarkers. Surgical excision of cancer often is confronted with difficulties in the identification of cancer spread and the accurate delineation of tumor margins. Currently, the assessment of tumor borders is afforded by postoperative pathology or, less reliably, intraoperative frozen sectioning. Fluorescence imaging is a natural modality for intraoperative use by directly relating to the surgeon’s vision and offers highly attractive characteristics, such as high-resolution, sensitivity, and portability. Via the use of targeted probes it also becomes highly tumor-specific and can lead to significant improvements in surgical procedures and outcome.     

一种可用活体荧光成像技术检测的前列腺原位肿瘤模型的建立

目的建立可用活体荧光成像技术动态观察前列腺原位肿瘤发生及生长的裸鼠模型。方法 10只BALB/c裸鼠前列腺背侧包膜内注入携带红色荧光蛋白（RFP）的前列腺癌PC-3细胞（PR7细胞）悬液20μL,第2、4、6、8周分别用非侵入式活体分子影像系统观察前列腺原位肿瘤发生及其转移情况。尸检取前列腺原位肿瘤、肺、肝、肾、股骨、盆腔淋巴结等组织,制成冰冻切片,通过荧光显微镜下观察及HE染色等方法分别检测肿瘤发生及有无转移等情况。结果第2周开始可用非侵入式活体分子影像系统检测到前列腺原位肿瘤形成,第4周开始可于裸鼠下腹部触及原位肿瘤,第6～8周裸鼠逐渐出现恶液质。荧光信号随原位肿瘤长大而增强,但并未见明显转移灶。尸检也未发现转移灶。结论前列腺包膜内注入携带RFP的前列腺癌PR7细胞可成功建立可用活体荧光成像技术监测的前列腺原位肿瘤模型,该模型可作为研究前列腺肿瘤发生发展及治疗方式的较理想的动物模型。     

Herpes simplex virus based gene therapy enhances the efficacy of mitomycin C for the treatment of human bladder transitional cell carcinoma

PURPOSE: Oncolytic replication competent herpes simplex virus type-1 (HSV) mutants have the ability to replicate in and kill malignant cells. We have previously reported the ability of replication competent HSV to control bladder cancer growth in an orthotopic murine model. We hypothesized that the combination of a chemotherapeutic agent used for intravesical treatment, namely mitomycin C (MMC) (Bristol-Myers Squibb Oncology, Princeton, New Jersey), and oncolytic HSV would exert a synergistic effect for the treatment of human transitional cell carcinoma. MATERIALS AND METHODS: We used mutant HSV NV1066 (Medigene, San Diego, California), which is deleted for viral genes ICP0 and ICP4, and selectively infects cancer cells, to treat the transitional cell carcinoma lines KU19-19 and SKUB. Cell survival was determined by lactate dehydrogenase assay for each agent as well as for drug-viral combinations from days 1 to 5. The isobologram method and the combination index method of Chou-Talalay were used to assess the synergistic effect. RESULTS: NV1066 enhanced MMC mediated cytotoxicity at all combinations tested for KU19-19 and SKUB. The combination of the 2 agents demonstrated a synergistic effect and allowed dose reduction by 12 and 10.4 times (NV1066), and by 3 and 156 times (MMC) for the treatment of KU19-19 and SKUB, respectively, while achieving an estimated 90% cell kill. CONCLUSIONS: These data provide the cellular basis for the clinical investigation of combined MMC and oncolytic HSV therapy for the treatment of bladder cancer.     

Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions.

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3-4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.     

Fluorescence‐guided surgery of human prostate cancer experimental bone metastasis in nude mice using anti‐CEA DyLight 650 for tumor illumination

The present report demonstrates efficacy of fluorescence‐guided surgery (FGS) to resect and prevent recurrence of experimental skeletal metastasis in a nude‐mouse model of human prostate cancer. Green fluorescent protein (GFP)‐expressing PC‐3 human prostate cancer cells were injected into the intramedullary cavity of the tibia in 25 nude mice. One week after implantation, monoclonal antibodies, specific for carcinoembryonic antigen (CEA), labeled with DyLight 650, were injected into the tail vein of 13 mice. Thirteen mice underwent FGS and 12 mice underwent bright‐light surgery (BLS). Weekly GFP fluorescence imaging of the mice was performed to observe tumor recurrence. The extent of residual tumor after BLS was 13‐fold greater than after FGS (p < 0.001). Time‐course imaging visualized rapid growth of the residual tumor in the BLS group, whereas the FGS group showed only slight tumor growth and significantly improved disease‐free survival of the treated mice. Our study demonstrated that FGS significantly reduced residual tumor as well as the recurrence of experimental prostate‐cancer bone metastasis. The present results suggest that FGS will be effective for resection of skeletal metastases in selected patients with prostate cancer. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:559–565, 2016.     

G207, modified herpes simplex virus type 1, kills human pancreatic cancer cells in vitro

Pancreatic cancer is often fetal, and farther effective therapeutic options are needed. This study was designed to assess whether the replication-restricted herpes simplex virus, G207, was effective in killing human pancreatic cancer cells in vitro. G207, a multimutated strain of herpes simplex virus type 1 carrying lacZ reporter gene, is capable of efficient cytolytic growth in many dividing cells, including certain tumor cells, but not in nondividing cells. Three human pancreatic cell lines, AsPC-1, MIA PaCa-2, and BxPC-3, were infected with G207 at different multiplicities of infection. After 24 hours, expression of the lacZ reporter gene was tested using a histochemical X-gal assay. In addition, cell lines were infected with G207 for 24 to 48 hours; then the virus obtained from cell pellets and media supernatant was used to infect Vero cells to obtain G207 titers by plaque assay. To assess whether increasing viral immediate early gene expression would improve cytolysis and virus production, similar experiments were performed with the addition of 0.5 mmol/L of hexamethylene bisacetamide (HMBA) 1 hour after viral infection. Finally, MTS cell viability assays were performed to measure viable cells at 24 to 96 hours post infection. The X-gal assay data revealed a viral dose-dependent β-galactosidase expression, indicating G207 infectivity and expression of the lacZ reporter gene. Plaque assays demonstrated a viral dose-dependent increase in plaque formation, indicating viral production from all three cell lines. In addition, HMBA data indicated a modest increase in viral production. The MTS assay data indicated a dose-dependent cytotoxicity for G207 in the cell lines tested. G207 infects, replicates in, and is cytotoxic to the above-listed human pancreatic cell lines in vitro and warrants therapeutic evaluation in models of pancreatic cancer. Supported by the Wilmot Cancer Research Fellowship Program funded by the James P. Wilmot Foundation. Presented at the Thirty-Ninth Annual Meeting of The Society for Surgery of the Alimentary Tract, New Orleans, La., May 17–20,1998.     

Fluorophore-conjugated anti-CEA Antibody for the Intraoperative Imaging of Pancreatic and Colorectal Cancer

Introduction  Colorectal and pancreatic cancers together comprise the third and fourth most common causes of cancer-related death in the United States. In both of these cancers, complete detection of primary and metastatic lesions at the time of surgery is critical to optimal surgical resection and appropriate patient treatment. Materials and Methods  We have investigated the use of fluorophore-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibody to aid in cancer visualization in nude mouse models of human colorectal and pancreatic cancer. Anti-CEA was conjugated with a green fluorophore. Subcutaneous, orthotopic primary and metastatic human pancreatic and colorectal tumors were easily visualized with fluorescence imaging after administration of conjugated anti-CEA. The fluorescence signal was detectable 30 min after systemic antibody delivery and remained present for 2 weeks, with minimal in vivo photobleaching after exposure to standard operating room lighting. Tumor resection techniques revealed improved ability to resect labeled tumor tissue under fluorescence guidance. Comparison of two different fluorophores revealed differences in dose–response and photobleaching in vivo. Conclusion  These results indicate that fluorophore-labeled anti-CEA offers a novel intraoperative imaging technique for enhanced visualization of tumors in colorectal and pancreatic cancer when CEA expression is present, and that the choice of fluorophore significantly affects the signal intensity in the labeled tumor. These data were presented at the Society for Surgery of the Alimentary Tract meeting as part of the Digestive Diseases Week, San Diego CA, May 21 2008. Sharmeela Kaushal and Michele K. McElroy shared authorship. Work supported in part by: Cancer Therapeutics Training Program (T32 CA121938) National Institutes of Health (CA109949-03) American Cancer Society (RSG-05-037-01-CCE).     

Comparison of pulmonary nodule detection rates between preoperative CT imaging and intraoperative lung palpation

Background

Recent advances in computed tomographic (CT) imaging have improved the detection rate of pulmonary metastasis. The aim of this study was to test the hypothesis that the pulmonary nodule detection rate for preoperative CT imaging and intraoperative palpation are now equivalent.

Methods

A retrospective review of 108 pulmonary metastasectomies in 84 patients was performed. The number of nodules detected on preoperative CT imaging by radiologist report was compared with the number of malignant nodules identified on pathology. Secondary outcome measures were operative approach and primary malignancy.

Results

Sarcoma metastases were the most common indication for resection (n = 54 [50%]). Thirty-three percent of metastasectomies were performed using a thoracoscopic approach. When thoracotomy was used, significantly more nodules were palpated and resected than were identified on preoperative CT imaging (3.24 vs 2.12, P < .001). Significantly more of these nodules were confirmed malignant on final pathology (2.40 vs 1.60, P = .01). This difference was not seen for thoracoscopic resections.

Conclusions

Although the sensitivity of CT imaging has improved, a significant number of malignant pulmonary nodules are detected intraoperatively that are not identified on preoperative imaging. Patients undergoing pulmonary metastasectomy require careful intraoperative palpation of lung parenchyma, and therefore open thoracotomy remains the standard of care.     

In situ analysis of breast cancer progression in murine models using a macroscopic fluorescence imaging system

BACKGROUND AND OBJECTIVE: The goal of this study was to use an inexpensive macroscopic imaging system to monitor tumor progression in mouse models in real-time with minimal intervention. STUDY DESIGN/MATERIALS AND METHODS: Illumination is provided via a xenon arc lamp and a fiber optic probe which delivers white light or quasi-monochromatic excitation via specific bandpass filters. Fluorescence emission from SCID and nude mice following mammary fat pad injection of red fluorescence protein (RFP)-expressing human breast cancer cell lines was recorded and quantified using a single lens reflex (SLR) digital camera. RESULTS: This simple system enabled the verification of successful tumor take and temporal quantification of tumor progression in mouse models. CONCLUSION: The macroscopic fluorescence imaging system represents an inexpensive and portable tool to facilitate non-invasive in situ cancer detection with the potential to monitor fluorescent tumor formation and investigation of the efficacy of potential cancer therapeutics.     

Characterization of PacMetUT1, a recently isolated human prostate cancer cell line

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.     

A novel bioluminescent tumor model of human renal cancer cell lines: an in vitro and in vivo characterization

PURPOSE: Bioluminescent imaging permits sensitive in vivo detection and quantification of cells engineered to emit light. We developed a bioluminescent human renal cancer cell line for in vitro and in vivo studies. MATERIAL AND METHODS: The 2 human renal cell carcinoma cell lines SN12-C and SN12-L1 were stably transfected to constitutively express luciferase using a retroviral shuttle. The bioluminescent signal was correlated with tumor cell numbers in vitro. Parental and transfected cells were compared by growth kinetics and histology. Tumor burden after heterotopic injection in immune deficient mice was monitored up to 39 days. The kinetics of the bioluminescent signal was evaluated for 1 to 60 minutes following luciferin injection. RESULTS: Bioengineered renal cancer cell lines stably expressed luciferase. The growth kinetics of the cells in vitro and the histology of tumors resulting from implantation of these cells were unaffected by retroviral transfection with the luciferase gene. As few as 1,000 cells could be reliably detected. The intensity of the bioluminescent signal correlated with the number of tumor cells in vitro. Photon emission in vivo and ex vivo correlated significantly with tumor weight at sacrifice. After intraperitoneal injection of luciferin there was a time dependent change in the intensity of the bioluminescent signal with maximum photon emission at 20 minutes (optimal 17 to 25). CONCLUSIONS: Luciferase transfected human renal cancer lines allow reliable, rapid, noninvasive and longitudinal monitoring of tumor growth in vivo. The ability to assess tumor development in vivo with time is economical and effective compared to end point data experiments.     

Vascular Endothelial Growth Factor C Promotes Lymph Node Metastasis in a Rectal Cancer Orthotopic Model

Purpose Vascular endothelial growth factor C (VEGF-C), a novel member of the vascular endothelial growth factor family, is a relatively specific lymphangiogenic growth factor. It has been suggested that increased expression of VEGF-C in primary tumors is correlated with lymph node metastasis. We conducted this study to determine whether VEGF-C directly affects lymphangiogenesis and lymph node metastasis in colorectal cancer.Methods For an accurate analysis and clear visualization of metastases, the rectal cancer cell line, DLD1, was engineered to stably express green fluorescent protein (GFP) (DLD1/GFP). We implanted DLD1/GFP cells overexpressing VEGF-C orthotopically into the rectal walls of nude mice.Results Lymph node metastasis was confirmed in all (100%) of the mice bearing DLD1/GFP-VEGF-C tumors, but in only 25% of the mice bearing control tumors. There were more lymph node metastases per mouse in the mice bearing DLD1/GFP-VEGF-C tumors than in the mice bearing control tumors. There were no differences in cell growth and motility in vitro or in the resulting tumor volume from the implanted cells between the two groups. Immunohistochemical staining revealed that VEGF-C induced the growth of lymphatic vessels, which were enlarged in the tumor periphery and contained tumor cell emboli.Conclusion These results suggest that VEGF-C-induced lymphangiogenesis mediates tumor spread and the formation of lymph node metastasis.