BDIX Rats Research Articles

Inhibition of Tissue Factor (TF) Pathway Blunts Hepatic Tumor Development in a Rat Colorectal Cancer Model.

Occurrence of hepatic metastasis (HM) is the leading cause of death in colorectal cancer, making their prevention a major challenge. Tissue factor (TF), the principal initiator of coagulation after binding to its ligand, coagulation factor FVIIa, is expressed by most colorectal cancer cells, and has been demonstrated to be associated to tumor invasion and metastasis. The aim of this study was to analyze the effects of inhibition of the TF pathway in a rat model of HM of colorectal cancer.Methods DHDK12 Pro B cells (20.106 cells), a tumor cell line established from chemically induced colon carcinoma in BDIX rats, were injected in the portal vein of syngenic rats. Inhibition of TF was achieved in 19 rats (treat ment group) through intraperitoneal injection of active site-inactivated factor VIIa (FFR-FVIIa, Novo Nordisk, Denmark) (10μg/g, once a day from day 3 to day 8 after HM induction). Additionally, 18 rats underwent cancer cell infusion according to the same procedure, without FFR-FVIIa treatment (control group). Rats were sacrificed at day 14.Results In the control group, infusion of cancer cells resulted in the development of macroscopical hepatic tumors in 17 out of 18 rats. In rats treated by FFR-FVIIa, no macroscopical or histological hepatic tumors were visible on the liver surface in 16 out of 19 rats (p=0.002, versus control group).Conclusion These results suggest that blockage of TF with proteolytically inactive FFR-VIIa inhibits hepatic tumor development at an early stage of tumoral development and TF is a target for adjuvant therapy in the prevention of HM.

Effect of intratumoral injection of carboplatin combined with pluronic P85 or L61 on experimental colorectal carcinoma in rats.

Pluronic, a poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene oxide) block copolymer, has been shown to enhance the cytotoxic activity of anticancer drugs in various cell lines. In the current study the effect of Pluronic P85 (P85) and Pluronic L61 (L61) on the intratumoral chemotherapy of an experimental adenocarcinoma in rats was examined. A total of 120 subcutaneous tumors (4 per rat) were inoculated in 30 BDIX rats and were treated weekly for 4 weeks with intratumoral injection of carboplatin (CPt) alone or with either P85 or L61. Tumors were monitored weekly and were excised at the endpoint for histologic evaluation. The effect of Pluronic on levels of intracellular ATP was explored as a possible mechanism of sensitization. Results showed that tumors treated with low-dose CPt (2.8 mg/kg) and P85 or L61 exhibited significant reductions in tumor volume after 28 days relative to Day 0 (112.7% +/- 34.4%, n = 15; 131.3% +/- 55.6%, n = 8) compared with tumors treated with free drug (339.4% +/- 75.0%, n = 16). Control tumors treated with either P85 or L61 alone or with saline showed volume increases of 1079.4% +/- 143.6% (n = 16), 729.4% +/- 202.2% (n = 7), and 1119.2% +/- 6.1% (n = 16), respectively. Treatment with high-dose CPt (20.7 mg/kg) led to a 79.3% +/- 4.2% reduction in tumor volume, and no differences were noted with addition of P85 or L61. In vitro ATP measurements showed that 28.0 mg/kg of P85 significantly reduced levels of intracellular ATP to 44.7% +/- 1.5% of controls, whereas L61 at this concentration depleted ATP levels completely. Results confirm that Pluronic P85 and L61 act as potent sensitizers to carboplatin chemotherapy of the experimental colorectal carcinoma, leading to a significant reduction of tumor growth compared to carboplatin alone. ATP depletion is a possible mechanism for these observed differences.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication-competent SFV vector VA7-EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7-EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV-positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T-cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti-SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.

Instillation of Taurolidine/Heparin after Laparotomy Reduces Intraperitoneal Tumour Growth in a Colon Cancer Rat Model

Objective: To investigate whether irrigation of the abdominal cavity after laparotomy for caecum resection with taurolidine/heparin or adhesion prophylactic substances reduces intraperitoneal tumour growth or the local recurrence rate in a colon carcinoma rat model. Methods: 60 BDIX rats underwent caecum resection after intraperitoneal inoculation of 1 × 10⁴ colon carcinoma cells (DHD/K12/TRb). Intergel®, Interceed®, taurolidine/heparin or NaCl 0.9% were intraperitoneally applied after randomisation. Finally, the total number and total weight of intraperitoneal metastases were determined as well as the adhesion score according to Moreno. Metastatic tissue was examined histologically and immunohistochemically (E-cadherin, CD44, β₁-integrin). Results: Taurolidine/heparin significantly reduced not only the total number (3 vs. 11 in the control group) but also the total weight (65 vs. 330 mg) of intraperitoneal metastases in comparison to the control group (p = 0.003 and p = 0.005). E-Cadherin expression in the metastatic tissue of animals treated with taurolidine/heparin was significantly decreased (p = 0.016). Conclusion: Taurolidine/heparin effectively reduces intraperitoneal tumour growth when used as an intraoperative lavage. These results represent a good rationale for intraoperative adjuvant irrigation with taurolidine/heparin during resection of colon cancer.

Biodistribution of doxorubicin-alkylated poly( l-lysine citramide imide) conjugates in an experimental model of peritoneal carcinomatosis after intraperitoneal administration

Peritoneal spread is a common manifestation in ovarian and gastrointestinal cancer. Intraperitoneal (i.p.) chemotherapy after cytoreductive surgery is associated with high systemic or local toxicity. A macromolecular drug delivery system was evaluated with the aim of improving the therapeutic index of i.p. chemotherapy. Peritoneal carcinomatosis was generated in BDIX rats ( n = 55) by i.p. injection of 2 million DHDK12 cells. Fourteen days later, doxorubicin (DOX) and two DOX-alkylated poly( l-lysine citramide imide) conjugates bearing 9.5% and 20.5% (w/w) chemically bound drug, respectively, were given i.p. to rats at a single 2 mg DOX/kg dose. Free and polymer-bound DOX were assessed in plasma, peritoneal fluid, abdominal tissues and heart, 15 min, 2, 6, 24, 48 and 168 h after injection. According to pharmacokinetic profiles, the peritoneal fluid areas under the concentration versus time curves (AUCs) were 2 and 2.6 times greater for the conjugates (P-DOX20 and P-DOX10, respectively) than for the free drug, respectively. Conjugates crossed the peritoneal barrier slower than the free drug. For the tumor, AUCs were, respectively, 3 and 7 times higher for the conjugates than for free DOX. The elimination half-lives of the conjugates were higher than that calculated for the free drug. Only very small concentrations were detected in peripheral organs and in the heart. In contrast, significant retention and accumulation of the conjugates were found in abdominal organs, particularly in the tumor. There was no sign of macroscopic chemical peritonitis after injection of the polymer–DOX conjugates. In conclusion, the conjugates have higher elimination half-lives than free DOX and were preferentially retained in abdominal organs and in the peritoneal carcinomatosis. This feature is of clinical interest to target tumor deposits.

Determination of fluence rate and temperature distributions in the rat brain; implications for photodynamic therapy

Light and heat distributions are measured in a rat glioma model used in photodynamic therapy. A fiber delivering 632-nm light is fixed in the brain of anesthetized BDIX rats. Fluence rates are measured using calibrated isotropic probes that are positioned stereotactically. Mathematical models are then used to derive tissue optical properties, enabling calculation of fluence rate distributions for general tumor and light application geometries. The fluence rates in tumor-free brains agree well with the models based on diffusion theory and Monte Carlo simulation. In both cases, the best fit is found for absorption and reduced scattering coefficients of 0.57 and 28 cm(-1), respectively. In brains with implanted BT(4)C tumors, a discrepancy between diffusion and Monte Carlo-derived two-layer models is noted. Both models suggest that tumor tissue has higher absorption and less scattering than normal brain. Temperatures are measured by inserting thermocouples directly into tumor-free brains. A model based on diffusion theory and the bioheat equation is found to be in good agreement with the experimental data and predict a thermal penetration depth of 0.60 cm in normal rat brain. The predicted parameters can be used to estimate the fluences, fluence rates, and temperatures achieved during photodynamic therapy.

Injectable Polymer Depot Combined With Radiofrequency Ablation for Treatment of Experimental Carcinoma in Rat

The purpose of this study was to investigate whether an intralesional chemotherapy depot with or without a chemosensitizer could improve the efficacy of radiofrequency (RF) ablation in treatment of experimental carcinoma in rats. Eighteen BD-IX rats were inoculated with bilateral subcutaneous tumors via injection of DHD/K12TRb rat colorectal carcinoma cells in suspension. Four weeks after inoculation, one tumor in each rat was treated with RF ablation at 80 degrees C for 2 minutes and the other with RF ablation followed by intralesional chemotherapy with carboplatin. The drug was administered via 2 different in situ-forming poly(D,L-lactide-coglycolide) (PLGA) depot formulations either with or without a chemosensitizer. Treatment efficacy was assessed by comparing the change in tumor diameter compared with control, percent of coagulation necrosis and a rating of treatment completeness. Tumors treated with ablation and carboplatin + sensitizer (n = 9) showed a diameter decrease of 49.4 +/- 24.5% at the end point relative to ablation control, while those treated with ablation and carboplatin only (n = 8) showed a 7.1 +/- 12.6% decrease. Use of sensitizer also showed increased tissue necrosis (81.9 +/- 9.7% compared with 68.7 +/- 26.7% for ablation only) and double the number of complete treatments (6/9 or 66.7%) compared with ablation control (3/9 or 33.3%). From these results, we conclude that intralesional administration of a carboplatin and sensitizer-loaded polymer depot after RF ablation has the potential to improve the outcome of ablation by increasing effectiveness of local adjuvant chemotherapy in preventing progression of tumor unaffected by the ablation treatment.

Circulating Nucleic Acids in Plasma/Serum and Tumor Progression

The "genometastasis hypothesis" proposes that cell-free tumor nucleic acids might be able to transform host stem cells, and that this might be a pathway for the development of metastases. This theory is supported by previous experimental findings and is consistent with observations of other authors. It has been suggested that tumor DNA might be horizontally transferred by the uptake of apoptotic bodies and initiate the genetic changes that are necessary for tumor formation. In addition, apoptotic bodies have been proposed as possible vehicles that protect the nucleic acids circulating in the plasma from enzymatic degradation. In the present study, we analyzed the presence of apoptotic bodies in serum and its relationship with tumor progression in a heterotopic model of colon cancer in the rat. We injected DHD/K12-PROb cancer cells subcutaneously into BD-IX rats and divided the animals into three groups according to the time between the injection of tumor cells and euthanasia. A control group of healthy animals was included (n = 6). After euthanasia, macroscopic metastases were assessed and samples of blood were collected. To detect apoptotic bodies in the sera, each sample was mixed with FITC-conjugated annexin V antibody in combination with propidium iodide and then analyzed by flow cytometry. Detection of apoptotic bodies was only significantly increased in the sera of a few tumor-bearing animals in late stages of tumor development. Thus, such particles appear not to be the vehicle of the cell-free tumor nucleic acids that are detected at early stages of cancer.

Establishment of a Syngeneic Model of Hepatic Colorectal Oligometastases

Regional and systemic therapies aimed at improving the outcome for patients with colorectal hepatic metastases have met with modest yet tangible success. Currently, liver resection remains the only curative treatment, but only a minority of patients are candidates for surgery. Animal models are an ideal way to study new treatments for patients with metastatic colorectal cancer. We propose a syngeneic animal model of hepatic colorectal metastases that simulates oligometastases, which is a clinical state considered amenable to regional therapeutic strategies. BDIX (BD-9) rats underwent intrasplenic injection of DHD/K12/TRb (Prob/K12) cells to create hepatic metastases via the portal system. After injection of 5 x 10(6) cells, rats underwent laparotomy to determine metastatic burden. Histological analysis confirmed the presence of metastases from resected tumors. Fifty-three animals were prospectively treated and observed for the development of oligometastases defined as between 1 and 10 hepatic lesions. Thirty-six (68%) of the animals developed detectable metastases while 32 (60%) developed oligometastases (average = 4.40 +/- 2.67). Four animals had overwhelming metastatic liver and peritoneal disease. All animals underwent peritoneal examination and thoracotomy to ensure localized disease. Histological analysis of five hepatectomy specimens confirmed the presence of metastatic cancer. Animals with oligometastases were healthy as evidenced by normal feeding and grooming behavior. An animal model of oligometastatic colorectal cancer to the liver can reproducibly mimic the stage IV state in humans conducive to regional therapy and can be used reliably to test novel treatments and mechanisms of metastatic colorectal cancer.

Attempts to improve locoregional chemotherapy for the prevention of colonic cancer liver metastasis in the rat

To improve prophylactic local treatment of hepatic metastasis from colonic cancer cells in the rat. The in vitro anticancer activity of 30 mn exposure to different drugs was first evaluated by dimethyl-thiazol-diphenyl-tetrazolium-bromide assay on confluent DHD/K12/PROb rat and HT29 human colonic cancer cells. Hepatic metastasis was induced by portal vein infusion of 12 x 106 PROb colonic cancer cells in syngenic BDIX rats. Hepatic and general tolerance to epirubicin was studied. Rats were treated with epirubicin delivered by either intravenous (IV), intraperitoneal (IP) or intraportal (Ipo) administration to compare their antitumoral effects. Hepatic distribution of epirubicin was assessed by fluorescence microscopy after IV, IP, Ipo, and combined administration. High pressure liquid chromatography was used to measure hepatic concentrations of epirubicin. Only pirarubicin was fully cytotoxic in vitro against the two types of tumor cells. No general or hepatic toxicity was observed. The preventive effect on hepatic metastasis was similar for IV, IP, and Ipo pirarubicin treatments. Hepatic pirarubicin concentrations obtained by Ipo administration were 4.1-fold higher than those obtained after IV administration (P=0.013). Three hours after IP and Ipo administration, hepatic remnants of pirarubicin were similar and significantly higher that those obtained after IV administration (P=0.074). Clamping the hepatic vein doubled hepatic pirarubicin concentrations after Ipo administration (P=0.048). Combined hepatic and intraportal administration was necessary to achieve diffuse, intense and homogeneous fluorescence throughout the entire liver. Homogeneous hepatic diffusion of pirarubicin was successfully achieved with combined hepatic vein and intraportal administration but systemic, intraperitoneal or intraportal administration had no preventive effect on hepatic metastasis. Other drugs could be tested using this approach to evaluate their efficacy and toxicity.

Surgery and Hematogenous Dissemination: Comparison Between the Detection of Circulating Tumor Cells and of Tumor DNA in Plasma Before and After Tumor Resection in Rats

To examine the effects of the surgical manipulation of tumors on the hematogenous dissemination of tumors, we compared rates of detection of tumor-derived DNA in the buffy coat and in plasma from tumor-bearing rats before and after tumor resection. We injected DHD/K12-PROb cells subcutaneously into BD-IX rats. Three weeks later, we removed the tumors surgically. Group PERI was sacrificed 3 hours after surgery, group POST-2 was sacrificed 2 weeks later, group POST-4 was sacrificed another 2 weeks later, and group POST-LONG was sacrificed when rats were close to death. In group PERI, four perioperative blood samples were taken. In the other groups, only one blood sample was taken per rat, immediately before euthanasia. We used polymerase chain reaction to detect tumor-derived DNA in buffy-coat, plasma, and lung samples. In group PERI, tumor DNA in plasma was more frequent than circulating tumor cells at all perioperative time points. The difference was statistically significant 3 hours after surgery (P = .035). In group POST-2, there was no detectable metastasis or tumor DNA in blood samples. There were lymphatic and lung metastases in most animals in group POST-4 and in all animals in group POST-LONG. In the last two groups, the frequencies of tumor DNA in the buffy coat and in plasma were similar. In our animal model, the hematogenous dissemination of tumors due to surgery seemed to be more closely related to tumor-derived cell-free DNA than to circulating tumor cells. In addition, the surgical resection of primary tumors did not inhibit the development of metastases.

High Intra-abdominal Pressure Enhances the Penetration and Antitumor Effect of Intraperitoneal Cisplatin on Experimental Peritoneal Carcinomatosis

To investigate the role of increased intra-abdominal pressure (IAP) on the intratumoral accumulation and the antitumor effect of intraperitoneal cisplatin in rats with advanced peritoneal carcinomatosis. To evaluate the tolerance of IAP in pigs, as it is a large animal with a body size equivalent to humans. To investigate if an active convection, driven by a positive IAP, increases cisplatin penetration and antitumor effectiveness in a model of advanced peritoneal carcinomatosis in rats. BDIX rats with macroscopic peritoneal tumors received cisplatin administered as intravenous injection (IV), conventional intraperitoneal injection (IP), or sustained intraperitoneal injection of cisplatin given in a large volume of solvent for maintaining IAP for 1 hour. Platinum tissue concentration was measured by atomic absorption spectroscopy (AAS), and platinum distribution into the tumor nodules was assessed by the particular-induced x-ray emission (PIXE) method. The antitumor effect was assessed in a survival experiment. The hemodynamic, local, and systemic tolerance of IAP, with or without cisplatin, was evaluated in Large White pigs. The maximum tolerated IAP was 22 mm Hg for 1 hour in nonventilated rats. IAP, in comparison with IV or conventional IP injections, resulted in the increased concentration and depth of diffusion of platinum into diaphragm and peritoneal tumor nodules. Consequently, IAP treatment induced an extended survival of rats treated at an advanced stage of carcinomatosis. In 7 50- to 70-kg ventilated pigs, a 40-mm Hg IAP was well tolerated when maintained stable for 2 hours. Renal failure occurred in pigs receiving a total dose of 200 and 400 mg of cisplatin with IAP, but a dose of 100 mg was well tolerated. Intraperitoneal chemotherapy with increased IAP, in comparison with conventional IP or IV chemotherapy, improved the tumor accumulation and the antitumor effect of cisplatin in rats bearing advanced peritoneal carcinomatosis. In preclinical conditions, the tolerance of sustained IAP was manageable in ventilated pigs.

Minimally Invasive Photodynamic Therapy (PDT) for Ablation of Experimental Rat Glioma

The feasibility of using ALA-mediated photodynamic therapy (PDT) tumor ablation as a minimally invasive treatment alternative for malignant brain tumors was evaluated in a rodent model. Treatment efficacy and side effects were evaluated with MRI, histopathology and survival rates. BT (4)C orthotopic brain tumors were induced in BD-IX rats. At various time intervals following tumor induction the animals were given 5-aminolevulinic acid (ALA) and 4 hours later optical fibers were inserted directly into the tumor without mechanical debulking or cranial decompression. A 3-day course of steroid treatment was initiated immediately prior to PDT. All untreated animals inevitably died within one month after tumor implantation (28.5 +/- 2.5 days). Complete tumor eradication was achieved in only 1/17 rats, but a significant increase in survival was obtained in the group of animals receiving 125 mg/kg ALA and 26 Joules of light fluence. Histopathology revealed large areas of central tumor necrosis, although clusters of viable tumor cells were often found at the tumor periphery. Pronounced edema in the necrotic tumor center as well as in the surrounding brain, and along white matter tracts was evident in all the brains studied from PDT-treated animal. This study suggests that ALA-mediated PDT may become a promising alternative therapy for the minimally invasive treatment of brain tumors. A judicious choice of PDT regimens that minimizes inflammatory responses through the use multiple fractionated long-term treatment protocols would likely be required.

The Interaction Mode of Premalignant Schwann and Immune Effector Cells during Chemically Induced Carcinogenesis in the Rat Peripheral Nervous System Is Strongly Influenced by Genetic Background

Contrary to rats of the highly sensitive inbred strain BDIX, BDIV rats are resistant to the induction of malignant schwannomas by N-ethyl-N-nitrosourea, arising predominantly in the trigeminal nerves. A point mutation of the neu/erbB-2 gene diagnostic of N-ethyl-N-nitrosourea-induced rat schwannomas is an early marker of Schwann precursor cells at high risk of subsequent malignant transformation. Neu-mutant cells initially arise at a similar frequency in sensitive and resistant animals. However, these cells disappear from the trigeminal nerves of resistant rats while giving rise to highly malignant schwannomas in susceptible animals. The resistance of BDIV rats obviously includes mechanisms to recognize and eliminate premalignant cells. The involvement of a cellular immune response was investigated in trigeminal nerves of both strains at different times after neonatal carcinogen exposure. An inflammatory reaction involving sequentially CD4(+) macrophages and T helper cells, CD8(+) cytotoxic T cells, and ED1(+) and ED2(+) macrophages was detected as a consequence of N-ethyl-N-nitrosourea treatment as early as postnatal day 40, briefly after the emergence of premalignant neu-mutant Schwann cells. It persisted throughout the observation period (40-250 days). However, there were no gross differences in immune cell counts between tumor-susceptible and tumor-resistant rats, except for a moderate increase of ED2(+) macrophages in N-ethyl-N-nitrosourea-treated BDIX rats only. Differential interactions of immune effector cells with premalignant Schwann cells may thus be involved in genetically determined tumor susceptibility or resistance, which could include functional differences of immune effector cells and/or a differential capability of premalignant Schwann cells to escape or counteract the cellular immune response.

Influence of light fluence rate on the effects of photodynamic therapy in an orthotopic rat glioma model

Failure of treatment for high-grade gliomas is usually due to local recurrence at the site of resection, indicating that a more aggressive local therapy could be beneficial. Photodynamic therapy (PDT) is a local treatment involving the administration of a tumor-localizing photosensitizing drug, in this case aminolevulinic acid (ALA). The effect depends on the total light energy delivered to the target tissue, but may also be influenced by the rate of light delivery. In vitro experiments showed that the sensitivity to ALA PDT of BT4C multicellular tumor spheroids depended on the rate of light delivery (fluence rate). The BT4C tumors were established intracranially in BD-IX rats. Microfluorometry of frozen tissue sections showed that photosensitization is produced with better than 200:1 tumor/normal tissue selectivity after ALA injection. Four hours after intraperitoneal ALA injection (125 mg/kg), 26 J of 632 nm light was delivered interstitially over 15 (high fluence rate) or 90 (low fluence rate) minutes. Histological examination of animals treated 14 days after tumor induction demonstrated extensive tumor necrosis after low-fluence-rate PDT, but hardly any necrosis after high-fluence-rate treatment. Neutrophil infiltration in tumor tissue was increased by PDT, but was similar for both treatment regimens. Low-fluence-rate PDT administered 9 days after tumor induction resulted in statistically significant prolongation of survival for treated rats compared with nontreated control animals. Treatment with ALA PDT induced pronounced necrosis in tumors only if the light was delivered at a low rate. The treatment prolonged the survival for tumor-bearing animals.

Antitumor efficacy improved by local delivery of species-specific endostatin

Conflicting results have been reported concerning the antitumor efficacy of the angiogenesis inhibitor endostatin. This may be due to differences in the biological distribution of endostatin between studies or to the varying biological efficacies of the different protein forms that were examined. To address this issue, the authors used a local delivery approach in which each tumor cell secreted endostatin, providing uniform endostatin levels throughout the tumors. This allowed a direct assessment of the biological efficacy of soluble endostatin in vivo. The authors genetically engineered BT4C gliosarcoma cells so that they would stably express and secrete either the human or murine form of endostatin. Endostatin-producing cells or mock-infected cells were implanted intracerebrally in syngeneic BD-IX rats. The antitumor efficacy of endostatin was evaluated on the basis of survival data and tumor volume comparisons. In addition, microvascular parameters were assessed. The authors confirmed the continuous release of endostatin by the BT4C cells. A magnetic resonance imaging-assisted comparison of tumor volumes revealed that local production of murine endostatin significantly inhibited tumor growth. Notably, 40% of the animals in this treatment group experienced long-term survival without histologically verifiable tumors 7 months after cell implantation. After local treatment with murine endostatin, tumor blood plasma volumes were reduced by 71%, microvessel density counts by 84%, and vascular area fractions by 75%. In contrast, human endostatin did not inhibit tumor growth significantly in this model. Centrally located regions of necrosis were present in tumors secreting both the human and the murine species-specific form of endostatin. The results suggest that endostatin inhibits tumor angiogenesis in vivo in a species-specific manner.

Ultra-short echo time spectroscopic imaging in rats: implications for monitoring lipids in glioma gene therapy

We demonstrate the feasibility of using ultra-short echo time (TE = 2 ms) magnetic resonance spectroscopic imaging (MRSI) to detect intracranial mobile lipids in the rat brain. High-performance outer volume suppression and pre-localization were demonstrated in phantoms and by the total absence of signals arising from extra-cranial lipids in MRSI spectra from control rats. The sequence performance was tested on glioma-bearing BDIX rats. Fast-relaxing lipid signals were spatially varied within a glioma during herpes simplex virus thymidine kinase-mediated gene therapy, demonstrating the potential application of this method.

Repetitive Photodynamic Therapy of Malignant Brain Tumors

The probability of achieving local control with current single-shot, intraoperative photodynamic therapy (PDT) treatments of intracerebral gliomas seems improbable due to the length of time required to deliver adequate light fluences to depths of 1-2 cm in the resection margin. Additionally, due to the short doubling time of many malignant gliomas, the kill rate per cell doubling indicates that it seems unlikely that a single treatment would be sufficient to prevent tumor recurrence. Multiple repetitive treatments would therefore seem required. In this publication we primarily review our work examining the effects of repetitive PDT on malignant brain tumor cells both in vitro and in vivo. The in vitro therapy response of human and rat glioma spheroids to 5-aminolevulinic acid (ALA)-mediated PDT in repetitive form was investigated. The results indicated that PDT repeated at relatively long intervals (weeks) was more effective at inhibiting spheroid growth than either daily fractionated PDT or single-treatment regimes. The in vivo response to repetitive treatment was evaluated in a rodent glioma model where BT4C cell line tumors were established in the brains of inbred BD-IX rats. Microfluorometry of frozen tissue sections showed that PpIX is produced with a 10-20:1 tumor to normal tissue selectivity ratio 4 hr after ALA injection. Preliminary evidence of increased efficacy of repetitive PDT and low fluence rate treatment is presented.

Gender‐specific polygenic control of ethylnitrosourea‐induced oncogenesis in the rat peripheral nervous system

The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.

The endothelin B (ETB) receptor agonist IRL 1620 is highly vasoconstrictive in two syngeneic rat tumour lines: potential for selective tumour blood flow modification

The vascular effects of the endothelin B (ETB) receptor agonist IRL 1620 were investigated in the rat P22 carcinosarcoma and a range of normal tissues in BDIX rats. Tissue blood flow rate was calculated from measurements of tissue uptake of radiolabelled iodoantipyrine. A comparison of vascular effects in the P22 tumour and the HSN sarcoma growing in CBH/CBi rats was made using laser Doppler flowmetry, showing similar effects of IRL 1620, with red cell flux rapidly decreasing by 50–60% and then returning to control levels within approximately 30 min. This corresponded to similar levels but different spatial organisation of ETB binding sites in the two tumours, as measured by autoradiography. The decrease in tumour blood flow and an increase in vascular resistance suggest that the vascular component of ETB receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ETA receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ETB receptors. IRL 1620 caused vasoconstriction in normal skeletal muscle, kidney and small intestine of the BDIX rat as well as in tumour, but did not affect blood flow in other tissues. These effects could be useful for limiting toxicity of certain chemotherapeutic agents. Fully functional ETB receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ETB receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620.