SCCVII Tumors Research Articles

Optical Detection of Tumors In Vivo by Visible Light Tissue Oximetry

Endoscopy is a standard procedure for identifying tumors in patients suspected of having gastrointestinal (G.I.) cancer. The early detection of G.I. neoplasms during endoscopy is currently made by a subjective visual inspection that relies to a high degree on the experience of the examiner. This process can be difficult and unreliable, as tumor lesions may be visually indistinguishable from benign inflammatory conditions and the surrounding mucosa. In this study, we evaluated the ability of local ischemia detection using visible light spectroscopy (VLS) to differentiate neoplastic from normal tissue based on capillary tissue oxygenation during endoscopy. Real-time data were collected (i) from human subjects (N = 34) monitored at various sites during endoscopy (enteric mucosa, malignant, and abnormal tissue such as polyps) and (ii) murine animal subjects with human tumor xenografts. Tissue oximetry in human subjects during endoscopy revealed a tissue oxygenation (StO2%, mean +/- SD) of 46 +/- 22% in tumors, which was significantly lower than for normal mucosal oxygenation (72 +/- 4%; P < or = 0.0001). No difference in tissue oxygenation was observed between normal and non-tumor abnormal tissues (P = N.S.). Similarly, VLS tissue oximetry for murine tumors revealed a mean local tumor oxygenation of 45% in LNCaP, 50% in M21, and 24% in SCCVII tumors, all significantly lower than normal muscle tissue (74%, P < 0.001). These results were further substantiated by positive controls, where a rapid real-time drop in tumor oxygenation was measured during local ischemia induced by clamping or epinephrine. We conclude that VLS tissue oximetry can distinguish neoplastic tissue from normal tissue with a high specificity (though a low sensitivity), potentially aiding the endoscopic detection of gastrointestinal tumors.

The usefulness of continuous administration of hypoxic cytotoxin combined with mild temperature hyperthermia, with reference to effects on quiescent tumour cell populations

To evaluate the usefulness of continuous administration of hypoxic cytotoxins in terms of targeting acute hypoxia in solid tumours and the significance of combination with mild temperature hyperthermia (MTH) (40°C, 60 min), the cytotoxic effects of singly or continuously administered tirapazamine (TPZ) and TX-402 were examined in combination with or without MTH in vivo. Further, the effects were also analysed on total (= proliferating (P) + quiescent (Q)) and Q cell populations in solid tumours with the method for selectively detecting the Q cell response. C3H/He mice bearing SCC VII tumours received a continuous administration of 5-bromo-2′-deoxyuridine (BrdU) for 5 days to label all P cells. The tumour-bearing mice then received a single intra-peritoneal injection or 24 h continuous subcutaneous infusion of hypoxic cytotoxin, TPZ or TX-402, with or without MTH. On the other hand, to detect the changes in the hypoxic fraction (HF) in the tumours by MTH, another group of mice with or without MTH received a series of test doses of γ-rays while alive or after tumour clamping. After each treatment, the tumour cells were isolated and incubated with a cytokinesis blocker (= cytochalasin-B) and the micronucleus (MN) frequency in cells without BrdU labelling (=Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total tumour cells was determined from the tumours that were not pre-treated with BrdU. The sensitivity to TX-402 was slightly higher than that to TPZ in both total and Q tumour cells. Continuous administration elevated the sensitivity of both total and Q cells, especially total cells. MTH raised the sensitivity of Q cells more remarkably than that of total cells in both single and continuous administrations. It was thought to be probably because of the higher dose distribution of hypoxic cytotoxin in intermediately hypoxic areas derived mainly from chronic hypoxia through MTH. From the viewpoint of tumour control as a whole including both total and Q tumour cells, the continuous administration of hypoxic cytotoxin combined with MTH may be useful for sensitizing tumour cells in vivo.

Radiobiologic effect of intermittent radiation exposure in murine tumors

In stereotactic irradiation using a linear accelerator, the effect of radiation may be reduced during intermittent exposures owing to recovery from sublethal damage in tumor cells. After our previous in vitro study suggesting this phenomenon, we investigated the issue in murine tumors. We used EMT6 and SCCVII tumors approximately 1 cm in diameter growing in the hind legs of syngeneic mice. Three schedules of intermittent radiation were investigated. First, 2 fractions of 10 Gy were given at an interval of 15-360 min to investigate the pattern of recovery from sublethal damage. Second, 5 fractions of 4 Gy were given with interfraction intervals of 2.5-15 min each. Third, 10 fractions of 2 Gy were given with interfraction intervals of 1-7 min each. Doses of 15-20 Gy were also given without interruption to estimate the dose-modifying factors. Tumors were excised 20 h later, and tumor cell survival was determined by an in vivo-in vitro assay. In the 2-fraction experiment, the increase in cell survival with elongation of the interval was much less than that observed in our previous in vitro study. In the 5- and 10-fraction experiments, no significant increase in cell survival was observed after the intermittent exposures. Moreover, cell survival decreased at most points of the 5-fraction experiments by interruption of radiation in both EMT6 and SCCVII tumors. In the 10-fraction experiment, cell survival also decreased when the interruption was 3 or 7 min in EMT6 tumors. The results of the present in vivo studies were different from those of our in vitro studies in which cell survival increased significantly when a few minutes or longer intervals were posed between fractions. This suggests that recovery from sublethal damage in vivo may be counterbalanced by other phenomena such as reoxygenation that sensitizes tumor cells to subsequent irradiation.

Forphenicinol enhances the antitumor effects of cyclophosphamide in a model of squamous cell carcinoma

We examined the interaction between forphenicinol (FPL) and cyclophosphamide (CPA) or ionizing radiation (IR) on the growth of murine squamous cell carcinoma tumors SCCVII. Primary tumors were established in C3H mice by injecting SCCVII tumor cells subcutaneously into the right hind limb. FPL (100 mg/kg for 8 days) and/or CPA (25 mg/kg twice) were administered by intraperitoneal injection. Tumors were irradiated to a total dose of 40 Gy (eight 5-Gy fractions). SCCVII tumor growth was inhibited by FPL (P=0.054), IR (P=0.003) and CPA (P<0.001) compared with control. The combination of FPL and CPA inhibited tumor growth additively compared with either treatment alone in both small- and large-volume tumors. FPL did not significantly enhance the antitumor effects of IR, however, when CPA+FPL were combined with IR, significant tumor growth inhibition was observed compared with FPL alone (P<0.001), CPA alone (P=0.002) and IR alone (P=0.002). Due to its low toxicity profile, FPL may be combined with CPA, IR and other cytotoxic therapies to potentially enhance the therapeutic ratio.

272. Induction of Anti-Tumoral Immune Response and IFN-Regulated Factors by Delivery of Control DNA

Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, CXCL9, MHCI and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: 1) TLR9 is not expressed in either SCCVII or 4T1 cell line, 2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and 3) the secretion factor (s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.

279. A Novel Functionalized Polymeric Formulation for use in Delivering Therapeutic Genes for the Treatment of Solid Tumors

We have described the synthesis, physico-chemical properties and gene therapy application of a novel water soluble gene delivery system PEG-PEI-Chol (PPC). PPC is composed of a low molecular weight branched polyethyleneimine, cholesterol and polyethylene glycol and designed to promote DNA condensation into nanoparticles, enhance membrane uptake and improve biocompatibility and pharmacokinetics, respectively. The chemical composition and molar ratios between the individual components of the polymer were verified by NMR. Formulation of PPC with DNA produced particles of |[sim]|100nm mean diameter and zeta potential of |[sim]|20mV. In vitro optimization studies using a luciferase reporter gene were performed in order to evaluate the effects of varying amounts of PEG and increasing nitrogen to phosphorus ratio (N:P). Use of the PPC delivery system with a plasmid encoding for interleukin-12 (IL-12) for treatment of solid tumors was examined in mice. Significant inhibition in tumor growth rate and/or increased incidence of complete tumor rejection was seen in murine models of breast cancer and head and neck cancer following intratumoral injections. A combination therapy approach was evaluated where the IL-12/PPC treatment was combined with an investigative polymeric formulation of paclitaxel against 4T1 mammary tumors and it was shown that combined therapy led to a synergistic inhibition of tumor growth. This form of paclitaxel had little effect on SCCVII head and neck tumors however, cyclophosphamide strongly inhibited SCCVII tumor growth. Further, when IL-12/PPC intratumoral injections were combined with cyclophosphamide administration, there was a slight additional inhibition in tumor growth over the cyclophosphamide treatment alone and there was a significant increase in the rate of complete tumor rejection. Future studies will be aimed at optimizing dose and dosing regimens of combination therapies for the treatment of solid tumors.

Photodynamic Therapy–Induced Cell Surface Expression and Release of Heat Shock Proteins: Relevance for Tumor Response

Almost instantaneously after the treatment of mouse SCCVII tumor cells with Photofrin-based photodynamic therapy (PDT), a fraction (15-25%) of total cellular heat shock protein 70 (HSP70) became exposed at the cell surface. The level of this surface-expressed HSP70 then remained unchanged for the next 6 hours and persisted at lower levels even at 18 hours after PDT. A similar induction of surface HSP70 expression was found with PDT-treated human umbilical vein endothelial cells. The same analysis for several other HSPs revealed the induced surface expression of HSP60 and GRP94, but not GRP78, on PDT-treated SCCVII cells. A fraction of total HSP70 existing in SCCVII cells at the time of PDT treatment was promptly (within 1 hour) released from cells after high treatment doses, whereas even lower PDT doses induced a substantial HSP70 release at later time intervals. Macrophages coincubated with PDT-treated SCCVII cells displayed elevated levels of both HSP70 and GRP94 on their surface and were stimulated to produce tumor necrosis factor alpha, whose production was inhibited by the presence of antibodies against either HSP70, Toll-like receptors 2 and 4, or specific NF-kappaB inhibitor in the coincubation medium. The induction of cell surface expression and release of HSPs by PDT may represent an important event in the response of tumors to this treatment modality with a critical role in the induced inflammatory and immune responses that contribute to the therapeutic outcome.

Increase in Tumor Oxygenation and Potentiation of Radiation Effects Using Pentoxifylline, Vinpocetine and Ticlopidine Hydrochloride

The purpose of the present study was to investigate the effects of Pentoxifylline (PTX), Vinpocetine (VPT) and Ticlopidine Hydrochloride (TCD), used commonly for vascular disorders in humans, on the pO2 in SCCVII tumors of C3H/HeJ mice and on the radioresponse of SCCVII tumors. The pO2 in the SCCVII tumors, which were measured 30 min after intraperitoneal (i.p.) injection of PTX (5 mg/kg), VPT (5 mg/kg), or TCD (10 mg/kg) using polarography, was compared to that in saline-treated control tumors. All the three drugs, PTX, VPT and TCD, yielded significant increase of the pO2 in the SCCVII tumors from 25.6 to 26.9 mmHg, from 18.6 to 22.9 mmHg, and from 22.6 to 25.9 mmHg, respectively. Frequency histogram of the pO2 distribution in the saline-treated SCCVII tumors did not show hypoxic fraction of less than 10 mmHg. The radioresponses of the drugs were investigated by tumor growth delay assay. In the drug-treated groups, the SCCVII tumors were irradiated with a single dose of 15 Gy 30 min after injection of the drugs at the same doses as those used in the experiments for intratumoral pO2 measurement. Compared with the irradiation alone group, significant tumor growth delays were observed in all the drug-treated groups. The time required to reach a four-fold increase in the initial tumor volume were 4 days in the saline-treated control group, 22 days in the irradiation (IR) alone group, 28 days in the PTX + IR group, 29 days in the VPT + IR group, and 32 days in TCD + IR group. In conclusion, VPT and TCD are potentially promising drugs for increasing the intratumoral pO2 although the mechanism for radiopotentiation observed in the present study is unknown due to small hypoxic fraction in the SCCVII tumors. Further studies on other mechanisms for radiopotentiation of PTX, VPT or TCD, besides of increasing the pO2 in the tumor, are needed.

Reevaluation of the Radiosensitizing Effects of Sanazole and Nimorazole In Vitro and In Vivo

Sanazole (AK-2123, 3-nitrotriazole derivative, N1-(3-methoxypropyl)-2-(3-nitro-1 H-1,2,4-triazol-1-yl)acetamide) and nimorazole (5-nitroimidazole derivative, 4-(2-(5-nitro-1H-1-imidazolyl)ethyl)morpholine) have been tested clinically as hypoxic cell radiosensitizers, mainly outside Japan. To determine if these sensitizers deserve clinical investigation in Japan, we reevaluated the radiosensitizing effects of these compounds in vitro and in vivo, in comparison with a fluorinated 2-nitroimidazole derivative KU-2285 (N1-(2-hydroxyethyl)-1,2-difluoro-3-(2-nitro-1 H-1-midazolyl)propanamide). KU-2285 is a known and established radiosensitizer, but is not suitable for clinical studies because of the high cost of synthesis. In vitro, the radiosensitizing effects of the three compounds on SCCVII (squamous cell carcinoma line in C3H mice) tumor cells were examined at 0.5 and 1 mM under aerobic or hypoxic conditions, using a colony assay. In vivo, SCCVII tumors grown subcutaneously in the hind legs of C3H/HeN mice were irradiated with or without prior intraperitoneal administration of 100, 200 or 400 mg/kg of the drugs. Thereafter, tumor growth delay was measured. In vitro, no sensitizing effect was observed under aerobic conditions at 1 mM. Under hypoxic conditions, the sensitizer enhancement ratio (SER) determined at 1% cell survival level for sanazole, nimorazole and KU-2285 was 1.55, 1.45 and 1.95, respectively, at 1 mM, and 1.40, 1.40 and 1.75, respectively, at 0.5 mM. In vivo, all three compounds had significant radiosensitizing effects; their effects appeared to decrease in the order of KU-2285, sanazole, and nimorazole. It was suggested that sanazole may be more suitable for clinical trials than nimorazole.

マイルド・ハイパーサーミアによる免疫能活性と抗腫瘍効果

It is believed that hyperthermia will show anti-tumor effects at the temperature over 42.5 degrees. But we found that the mild hyperthermia as under 42.5 degrees still shows anti-tumor effects due to the immunological activity. We used C3H mice bearing SCC-VII tumor. These mice were heated at the temperature of 39-41 degrees by 1 hour in controlled water bath. After giving mild hyperthermia, white blood cells (WBC), lymphocyte cells (LyC), NK cell activity were increased compared with pre-heat condition. The delays of the SCC-VII tumor growth in C3H mice was observed after mild-hyperthermia. It shows that the immunological activity were increased as activated WBC, LyC and NK cells. Also anti-tumor effects were produced by mild hyperthermia. It is supposed that quantitative stimulation of femoral region by mild hyperthermia has induced the activation of hemopoietic organs in bone marrow of femoral region and this has activated immunological potency, mild hyperthermia seems to be a useful and effective auxiliary therapy which is not only effective for the treatment of malignant tumors but also for the treatment of diseases related to immunological activity (e. g. viral hepatitis type C, collagen diseases, AIDS, postoperative physical restraint, auxiliary therapy during thermotherapy, elimination of immunodeficiency, etc.).

Effects of Hypoxic Cell Radiosensitizer Doranidazole (PR-350) on the Radioresponse of Murine and Human Tumor Cells in vitro and in vivo

We have investigated the radiosensitizing effect of doranidazole, a hypoxic cells radiosensitizer, using SCCVII tumor cells of C3H mice and CFPAC-1 and MIA PaCa-2 human pancreatic tumor cells. The radiosensitivity of hypoxic SCCVII cells in vitro increased with 1 mM doranidazole by a factor of 1.34 and 1.68, when determined by clonogenic survival and micronucleus (MN) formation, respectively. The radiation-induced growth delay of SCCVII tumors was significantly enhanced and the TCD(50/120) was reduced by a factor of 1.33 when 200 mg/kg doranidazole was injected, i.v., 20 min prior to tumor irradiation. The in vivo-in vitro excision assay showed that radiosensitivity of SCCVII cells in vivo increased by a factor of 1.47 with 200 mg/kg doranidazole. The radiation-induced growth delay of CFPAC-1 xenografts in nude mice was significantly enhanced and the TCD(50/90) was reduced by a factor of 1.30 by 200 mg/kg doranidazole. On the other hand, 200 mg/kg of doranidazole exerted no influence on the radiation-induced growth delay in MIA PaCa-2 xenografts. The tumor oxygenation status, as determined with an oxygen sensitive needle probe and the immunohistological study using pimonidazole, indicated that MIA PaCa-2 tumors are better oxygenated than CFPAC-1 tumors. The relatively well-oxygenated status in MIA PaCa-2 tumor may account for the lack of radiosensitization by doranidazole. It is concluded that the magnitude of radiosensitization of tumors by doranidazole is dependent on the oxygenation status of the tumors and that doranidazole may be useful in increasing the response of hypoxic human pancreatic tumor to IORT.

Darbepoietin Alfa Potentiates the Efficacy of Radiation Therapy in Mice with Corrected or Uncorrected Anemia

Darbepoietin alfa (DA) is a long-acting analogue of erythropoietin that has reduced receptor affinity and enhanced biological activity. Experiments were done to test the hypothesis that correction of anemia in tumor-bearing mice by DA would increase tumor oxygenation and potentiate radiation-induced tumor cell killing. A SCC VII tumor model was used to study tumor responses to fractionated radiation therapy in mice with anemia induced by total body irradiation. Administration of DA reduced the extent and duration of anemia and associated tumor hypoxia, protected the bone marrow cells and prevented the body weight loss from the effect of irradiation, and facilitated the recovery in a time-dependent manner, with the administration of DA prior to total body irradiation having the greatest protective effect. When combined with fractionated radiation therapy, DA increased the tumor growth delay time from 2.7 days for irradiation alone to 7.3 to 10.6 days for combination of DA and irradiation. The effect of DA on tumor responses to fractionated radiation therapy was observed when DA was given 18 to 4 days before starting radiation therapy, but DA was also equally effective as a radiosensitizer when given only 2 hours before fractionated irradiation therapy. Weekly dosing of DA was as efficacious for the enhancement of radiation responses of tumors as biweekly dosing. Similar results were obtained in the RIF-1 fibrosarcoma tumor model. These studies show that DA can effectively correct anemia in tumor-bearing mice and sensitize tumor cells to fractionated radiation therapy. Importantly, DA was also able to sensitize tumors to radiation in mice with uncorrected anemia and hypoxia, suggesting that the effect of DA on radiosensitivity was independent of these factors and a different mechanism of action may be responsible for this effect.

Induction of IFN-regulated factors and antitumoral surveillance by transfected placebo plasmid DNA

Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, MIG (approved gene symbol CXCL9), MHCI (MICA), and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor(s) such as IFN-beta. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: (1) TLR9 is not expressed in either SCCVII or 4T1 cell line, (2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and (3) the secretion factor(s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is refractory to the gene induction by DNA transfection. Our finding indicates that the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.

Application of a Macromolecular Contrast Agent for Detection of Alterations of Tumor Vessel Permeability Induced by Radiation

Permeability of tumor vasculature can be a major barrier to successful drug delivery, particularly for high molecular weight agents such as monoclonal antibodies and their diagnostic or therapeutic conjugates. In this study, changes in permeability of SCCVII tumor vessels after radiation treatment were evaluated by dynamic magnetic resonance imaging as a function of time after irradiation using a generation-8 polyamidoamine dendrimer (G8-Gd-D)-based magnetic resonance imaging contrast agent shown previously to be confined to tumor blood vessels. Tumor irradiation consisted of either single doses (2-15 Gy) or various daily fractionated doses (5 days). A single radiation dose of 15 Gy resulted in significant transient image enhancement of the tumor tissue with a maximum occurring between 7 and 24 hours after radiation treatment. No observable enhancement was recorded for fractionated radiation doses. Use of dynamic magnetic resonance imaging coupled with G8-Gd-D provides an exquisite methodology capable of defining the timing of enhanced permeability of macromolecules in tumors after irradiation. Such information might be applied to optimize the efficacy of subsequent or concurrent therapies including radiolabeled antibodies or other anticancer agents in combination with external beam therapies.

753 poster Effect of liposome-encapsulated hemoglobin NRC on the hypoxia of SCCVII tumor and on its response to radiotherapy

753 poster Effect of liposome-encapsulated hemoglobin NRC on the hypoxia of SCCVII tumor and on its response to radiotherapy

Tumor enhancement using Mn-metalloporphyrin in mice: magnetic resonance imaging and histopathologic correlation.

To determine the signal enhancement characteristics of tumors after administration of a metalloporphyrin derivative, HOP-9P (13, 17-bis (1-carboxypropionyl) carbamoylethyl-3, 8-bis (1-phenylpropyloxyethyl)-2, 7, 12, 18-tetramethyl-porphyrinato manganese (III)) and to determine whether HOP-9P is tumor-necrosis specific. Ten C3H/He mice bearing a SCC VII tumor in the right flank were examined using T1-weighted conventional spin echo magnetic resonance (MR) imaging before contrast injection, and five minutes, one hour, and 24 hours after intravenous administration of 0.1 mmol/kg of HOP-9P. Following the imaging schedule, the mice were sacrificed, and sectioned in the same axial planes as the MR images. Based on an MR imaging-histopathologic correlation, mean signal intensities were measured, and signal-to-noise ratios (SNR) were calculated for both pure viable component and admixture of necrotic and viable component of the tumor. Mean SNR of the pure viable component peaked at one hour (35.0 +/- 3.8) and maintained that level until 24 hours (34.6 +/- 3.6). Mean SNR of the admixture of necrotic and viable component peaked at 24 hours (44.3 +/- 12.1). Although different enhancement patterns were seen between the pure viable component and the admixture of necrotic and viable component, HOP-9P enhanced both of the two components.

348. IL-21 and IL-15 Gene Therapy Induces Powerful Antitumor Effect Against Head and Neck Squamous Cell Carcinoma

Top of pageAbstract Interleukin (IL)-21 is a member of the 4-helix-bundle cytokine family capable of supporting proliferation of mature T and B lymphocytes. The cytokine also facilitates expansion and maturation of natural killer (NK) cells in synergy with IL-15. We previously reported that intravascular transfection of IL-21 and IL-15 genes augmented antitumor immunity, resulting in a marked therapeutic outcome in a hepatic metastasis model of lymphoma in mice (Kishida et al. Molecular Therapy, 8:552, 2003). It remains to be elucidated, however, whether the combinatory cytokine gene therapy could also inhibit a pre-established solid tumor in vivo. In the present study, we investigated the efficacy of intravascular delivery of IL-21 and IL-15 genes in suppressing head and neck squamous cell carcinoma (HNSCC) implant in mice. SCCVII, a murine HNSCC cell line, was subcutaneously inoculated into the flank of syngenic C3H/He mice to develop palpable subcutaneous tumors. A plasmid vector encoding murine IL-21 was intravenously injected into the tumor-bearing mice via their tail vein, alone or in combination with an IL-15 expression vector construct. The genetic transfer of either IL-21 or IL-15 elicited a significant, but moderate, antitumor effect compared with the treatments with an empty plasmid. In contrast, a remarkable antitumor effect was obtained by repetitive transfection with both the IL-21 and IL-15 genes, leading to a significant prolongation of the survival periods of the animals (P<.05, IL-21+IL-15 vs IL-21 alone; P<0.05, IL-21+IL-15 vs IL-15 alone; P<0.005, IL-21+IL-15 vs control), although a single treatment with both of the cytokine genes failed to show such therapeutic results. Importantly, any detectable toxic effect such as body weight loss and capillary leaking was not revealed in the mice that had received these cytokine genes. IL-21 significantly elevated cytotoxic T lymphocyte (CTL) killing activity against the SCCVII in the spleen of the tumor-bearing mice (P<0.005, IL-21+IL-15 vs control; P<0.05, IL-21 alone vs control), while IL-21 and IL-15 augmented NK cytotoxicity (P<0.0005, IL-21+IL-15 vs control; P<0.001, IL-21+IL-15 vs IL-21 alone; P<0.005, IL-21+IL-15 vs IL-15 alone). The co-transfection with IL-21 and IL-15 genes slightly, but significantly, elevated the production of interferon (IFN)-γ in the liver compared with the delivery of either IL-21, IL-15, or control plasmids, whereas IFN-γ concentration in the sera was not significantly affected. Furthermore, IL-21 gene transfection induced production of immunoglobulin specific for SCCVII tumor antigen, as demonstrated by indirect cellular ELISA and flowcytometric analyses of the sera from the genetically modified tumor bearers (P<0.05, IL-21+IL-15 vs control; P<0.005, IL-21 alone vs control). Taken together, the present study strongly suggest that the IL-21 and IL-15 genes synergistically elicited powerful cellular and humoral antitumor immune responses, which effectively suppressed HNSCC-derived pre-established tumor without any detectable adverse effect in vivo.

The Influence of Medicinal Mushroom Preparations on Mouse Tumors

Extracts from various medicinal mushrooms (Lentinus edodes, Grifola frondosa, and Coriolus (Trametes) versicolor), and particularly their proper blends, are of significant interest in selecting the best of them for treatment, or even prevention, of different pathological aberrations, including cancer. In this study, mushroom preparation effects on the proliferation of tumors and normal cells, respectively, as well as the survival rate of the mice with transplanted tumors, were determined. The incorporation of radioactive 3H thymidine in tumor cells (mouse squamous cell carcinoma [SCCVII], fibrosarcoma [FsaR], and melanoma [B16F10]) was inhibited in the presence of particular mushroom preparations. The effect was dose dependent and was different for tumor cell cultures used. On the other hand, the incorporation of radioactive 3H thymidine in normal Chinese hamster lung fibroblasts (V79 cell) increased, and the effect was generally dose dependent. The application of mushroom preparation to the mice bearing either SCCVII or FsaR tumor could influence their survival. On day 30 after SCCVII tumor cell transplantation, there was a 20% survival rate of mice in the control group, but 60-80% survived if particular mushroom extracts were applied. There were no survivors in the control group on the 36th day, but 40-60% were alive in the experimental groups. Furthermore, there was a 60% survival rate for mice with FsaR tumor when treated with mushroom preparations and a 20% survival rate in the control group on day 36 after tumor cell transplantation.Thus, the findings that the mushroom preparation tested induced a selective inhibition of tumor cell proliferation observed in vitro and a pronounced prolongation of the survival rate of tumor-bearing mice are of particular interest. These findings, together with the data that these compounds are not toxic to the organism, deserve further attention.

DNA-targeted 1,2,4-benzotriazine 1,4-dioxides: potent analogues of the hypoxia-selective cytotoxin tirapazamine.

Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

713 Prospective study of 18FDG-PET in patients with cervical lymph node metastases from an unknown primary tumor

PET allows non-invasive mapping of tumor hypoxia, but the combination of low resolution, slow tracer adduct-formation and slow clearance of unbound tracer remains problematic. Using a murine tumor with a hypoxic fraction within the clinical range and a tracer post-injection sampling time that results in clinically obtainable tumor-to-reference tissue activity ratios, we have analyzed to what extent inherent limitations actually compromise the validity of PET-generated hypoxia maps.Mice bearing SCCVII tumors were injected with the PET hypoxia-marker fluoroazomycin arabinoside (FAZA), and the immunologically detectable hypoxia marker, pimonidazole. Tumors and reference tissue (muscle, blood) were harvested 0.5, 2 and 4 h after FAZA administration. Tumors were analyzed for global (well counter) and regional (autoradiography) tracer distribution and compared to pimonidazole as visualized using immunofluorescence microscopy.Hypoxic fraction as measured by pimonidazole staining ranged from 0.09 to 0.32. FAZA tumor to reference tissue ratios were close to unity 0.5 h post-injection but reached values of 2 and 6 when tracer distribution time was prolonged to 2 and 4 h, respectively. A fine-scale pixel-by-pixel comparison of autoradiograms and immunofluorescence images revealed a clear spatial link between FAZA and pimonidazole-adduct signal intensities at 2 h and later. Furthermore, when using a pixel size that mimics the resolution in PET, an excellent correlation between pixel FAZA mean intensity and density of hypoxic cells was observed already at 2 h post-injection.Despite inherent weaknesses, PET-hypoxia imaging is able to generate quantitative tumor maps that accurately reflect the underlying microscopic reality (i.e., hypoxic cell density) in an animal model with a clinical realistic image contrast.