Basal Tumor Cell Isolation and Patient-Derived Xenograft Engraftment Identify High-Risk Clinical Bladder Cancers.

Strategies to identify tumors at highest risk for treatment failure are currently under investigation for patients with bladder cancer. We demonstrate that flow cytometric detection of poorly differentiated basal tumor cells (BTCs), as defined by the co-expression of CD90, CD44 and CD49f, directly from patients with early stage tumors (T1-T2 and N0) and patient-derived xenograft (PDX) engraftment in locally advanced tumors (T3-T4 or N+) predict poor prognosis in patients with bladder cancer. Comparative transcriptomic analysis of bladder tumor cells isolated from PDXs indicates unique patterns of gene expression during bladder tumor cell differentiation. We found cell division cycle 25C (CDC25C) overexpression in poorly differentiated BTCs and determined that CDC25C expression predicts adverse survival independent of standard clinical and pathologic features in bladder cancer patients. Taken together, our findings support the utility of BTCs and bladder cancer PDX models in the discovery of novel molecular targets and predictive biomarkers for personalizing oncology care for patients.

Translational strategies exploiting TNF-alpha that sensitize tumors to radiation therapy.

TNFerade is a radioinducible adenoviral vector expressing tumor necrosis factor-alpha (TNF-alpha) (Ad.Egr-TNF) currently in a phase III trial for inoperable pancreatic cancer. We studied B16-F1 melanoma tumors in TNF receptor wild-type (C57BL/6) and deficient (TNFR1,2-/- and TNFR1-/-) mice. Ad.Egr-TNF+IR inhibited tumor growth compared with IR in C57BL/6 but not in receptor-deficient mice. Tumors resistant to TNF-alpha were also sensitive to Ad.Egr-TNF+IR in C57BL/6 mice. Ad.Egr-TNF+IR produced an increase in tumor-associated endothelial cell apoptosis not observed in receptor-deficient animals. Also, B16-F1 tumors in mice with germline deletions of TNFR1,2, TNFR1 or TNF-alpha, or in mice receiving anti-TNF-alpha exhibited radiosensitivity. These results show that tumor-associated endothelium is the principal target for Ad.Egr-TNF radiosensitization and implicate TNF-alpha signaling in tumor radiosensitivity.

Iron release from corroded iron pipes in drinking water distribution systems: effect of dissolved oxygen.

Iron release from corroded iron pipes is the principal cause of "colored water" problems in drinking water distribution systems. The corrosion scales present in corroded iron pipes restrict the flow of water, and can also deteriorate the water quality. This research was focused on understanding the effect of dissolved oxygen (DO), a key water quality parameter, on iron release from the old corroded iron pipes. Corrosion scales from 70-year-old galvanized iron pipe were characterized as porous deposits of Fe(III) phases (goethite (alpha-FeOOH), magnetite (Fe(3)O(4)), and maghemite (alpha-Fe(2)O(3))) with a shell-like, dense layer near the top of the scales. High concentrations of readily soluble Fe(II) content was present inside the scales. Iron release from these corroded pipes was investigated for both flow and stagnant water conditions. Our studies confirmed that iron was released to bulk water primarily in the ferrous form. When DO was present in water, higher amounts of iron release was observed during stagnation in comparison to flowing water conditions. Additionally, it was found that increasing the DO concentration in water during stagnation reduced the amount of iron release. Our studies substantiate that increasing the concentration of oxidants in water and maintaining flowing conditions can reduce the amount of iron release from corroded iron pipes. Based on our studies, it is proposed that iron is released from corroded iron pipes by dissolution of corrosion scales, and that the microstructure and composition of corrosion scales are important parameters that can influence the amount of iron released from such systems.

Angiostatin effects on endothelial cells mediated by ceramide and RhoA.

Angiostatin is a cleavage product of plasminogen that has anti-angiogenic properties. We investigated whether the effects of angiostatin on endothelial cells are mediated by ceramide, a lipid implicated in endothelial cell signaling. Our results demonstrate that angiostatin produces a transient increase in ceramide that correlates with actin stress fiber reorganization, detachment and death. DNA array expression analysis performed on ceramide-treated human endothelial cells demonstrated induction of certain genes involved in cytoskeleton organization. Specifically, we report that treatment with angiostatin or ceramide results in the activation of RhoA, an important effector of cytoskeletal structure. We also show that treatment of endothelial cells with the antioxidant N-acetylcysteine abrogates morphological changes and cytotoxic effects of treatment with angiostatin or ceramide. These findings support a model in which angiostatin induces a transient rise in ceramide, RhoA activation and free radical production.

Antitumor interaction of short-course endostatin and ionizing radiation.

PURPOSE: The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. METHODS: Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. RESULTS: In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. DISCUSSION: The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.

Angiostatin induces mitotic cell death of proliferating endothelial cells.

Angiostatin is an inhibitor of tumor angiogenesis that induces regression of experimental tumors and enhances the antitumor effects of radiation therapy. We report that the cytotoxic effects of angiostatin are restricted to the proliferating endothelial cell population. In addition, angiostatin and ionizing radiation (IR) interact by inducing death of dividing endothelial cells. We also show that angiostatin and IR interact to inhibit endothelial cell migration. These findings demonstrate that angiostatin targets the proliferating tumor vasculature and provide a mechanistic basis for the cytotoxic interaction of angiostatin and IR.

Surfactant sealing of membranes permeabilized by ionizing radiation.

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to gamma radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 +/- 3 min was determined between exposure to 160 Gy of gamma radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation.

The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the lation of angiogenesis Inhibition of VEGF-induced angiogenesis either by neutralizing antibodies or dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect. In vitro, the addition of VEGF decreases IR-induced killing of human umbilical vein endothelial cells, and the anti-VEGF treatment potentiates IR-induced lethality of human umbilical vein endothelial cells. Neither recombinant VEGF protein nor neutralizing antibody to VEGF affects the radiosensitivity of tumor cells These findings support a model in which induction of VEGF by IR contributes to the protection of tumor blood vessels from radiation-mediated cytotoxicity and thereby to tumor radioresistance.

Radiation-inducible gene therapy.

The radiation-inducible chimeric genetic construct Egr-TNF alpha introduced into human xenografts produces cytotoxicity of infected tumor cells resulting in tumor growth inhibition. The interaction between Egr-TNF and radiation is selectively cytotoxic for the tumor microvasculature resulting in vascular thrombosis and tumor necrosis. Gene therapy combined with radiation therapy offers great potential for the treatment of localized human cancers.

Oxidative cell membrane alteration. Evidence for surfactant-mediated sealing.

Exposure to very intense ionizing irradiation produces acute tissue sequelae including inflammation, pain, and swelling that often results in tissue fibrosis and/or necrosis. Acute tissue necrosis occurs in hours when sufficiently rapid damage to membrane lipids and proteins leads to altered membrane structure, disrupting the vital electrochemical diffusion barrier necessary for cell survival. This damage mechanism is thought to underlie the interphase death of lethally irradiated postmitotic cells such as neurons, but it has also been implicated in the rapid cell death of lymphocytes and acute vascular changes due to capillary epithelium dysfunction. It is not known whether sealing of radiation-permeabilized cell membranes will prolong survival of lethally irradiated cells or perhaps lead to repair of damaged nucleic acids. The purpose of this study is to begin to address the first question.

Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin.

Angiostatin, a proteolytic fragment of plasminogen, inhibits the growth of primary and metastatic tumors by suppressing angiogenesis. When used in combination with ionizing radiation (IR), angiostatin demonstrates potent antitumor synergism, largely caused by inhibition of the tumor microvasculature. We report here the temporal interaction of angiostatin and IR in Lewis lung carcinoma (LLC) tumors growing in the hind limbs of syngeneic mice. Tumors with an initial mean volume of 510 +/- 151 mm3 were treated with IR alone (20 Gy x 2 doses on days 0 and 1), angiostatin alone (25 mg/kg/day divided twice daily) on days 0 through 13, or a combination of the two as follows: (a) IR plus angiostatin (days 0 through 13); (b) IR plus angiostatin (days 0 and 1); and (c) IR followed by angiostatin beginning on the day after IR completion and given daily thereafter (days 2 through 13). By day 14, tumors in untreated control mice had grown to 6110 +/- 582 mm3, whereas in mice treated with: (a) IR alone, tumors had grown to 2854 +/- 338 mm3 (P < 0.05 compared with untreated controls); and (b) angiostatin alone, tumors had grown to 3666 +/- 453 mm3 (P < 0.05 compared with untreated controls). In combined-treatment groups, in mice treated with: (a) IR plus longer-course angiostatin, tumors reached 2022 +/- 282 mm3 (P = 0.036 compared with IR alone); (b) IR followed by angiostatin, tumors reached 2677 +/- 469 mm3 (P > 0.05 compared with IR alone); and (c) IR plus short-course angiostatin, tumors reached 1032 +/- 78 mm3 (P < 0.001 compared with IR alone). These findings demonstrate that the efficacy of experimental radiation therapy is potentiated by brief concomitant exposure of the tumor vasculature to angiostatin.

Modulation of apoptotic response of a radiation-resistant human carcinoma by Pseudomonas exotoxin-chimeric protein.

Strategies to sensitize human tumors that are resistant to apoptosis have been clinically unsuccessful. We demonstrate that a structurally modified chimeric Pseudomonas exotoxin, PEdelta53L/TGF-alpha/KDEL, with binding specificity for the epidermal growth factor receptor, markedly enhances sensitivity of human xenografts to radiation killing. Exposure to PEdelta53L/TGF-alpha/KDEL decreases the apoptotic threshold through protein synthesis inhibition and simultaneous production of ceramide in tumor cells that lack functional p53 protein. In contrast, no increase in local or systemic toxicity was observed with the chimeric toxin and radiation. We conclude that biochemical targeting of the chimeric toxin and physical targeting of ionizing radiation may increase the therapeutic ratio in the treatment of human cancers with alterations of p53 expression. This strategy offers a high therapeutic potential for Pseudomonas exotoxin A chimeric proteins and irradiation.

Combined effects of angiostatin and ionizing radiation in antitumour therapy.

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumour progression. Angiostatin, a proteolytic fragment of plasminogen that was first isolated from the serum and urine of tumour-bearing mice, inhibits angiogenesis and thereby growth of primary and metastatic tumours. Radiotherapy is important in the treatment of many human cancers, but is often unsuccessful because of tumour cell radiation resistance. Here we combine radiation with angiostatin to target tumour vasculature that is genetically stable and therefore less likely to develop resistance. The results show an antitumour interaction between ionizing radiation and angiostatin for four distinct tumour types, at doses of radiation that are used in radiotherapy. The combination produced no increase in toxicity towards normal tissue. In vitro studies show that radiation and angiostatin have combined cytotoxic effects on endothelial cells, but not tumour cells. In vivo studies show that these agents, in combination, target the tumour vasculature. Our results provide support for combining ionizing radiation with angiostatin to improve tumour eradication without increasing deleterious effects.

Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation.

Approximately 30% of cancer deaths result from the failure to control local and regional tumors. The goal of radiotherapy is to maximize local and regional tumor cell killing while minimizing normal tissue destruction. Attempts to enhance radiation-mediated tumor cell killing using halogenated pyrimidines, antimetabolites, and other DNA-damaging agents or sensitizers of hypoxic tumor cells have met with only modest clinical success. In an unique strategy to modify tumor radiosensitivity, we used an inhibitor of the protein kinase C group A and B isoforms, chelerythrine chloride (chelerythrine), to enhance the killing effects of ionizing radiation (IR). Protein kinase C activity plays a central role in cellular proliferation, differentiation, and apoptosis. Chelerythrine increases sphingomyelinase activity and enhances IR-mediated cell killing through induction of apoptotic tumor cell death in a radioresistant tumor model both in vitro and in vivo. Although previous reports have suggested that IR-mediated apoptosis correlates with tumor volume reduction, we demonstrate for the first time that lowering the apoptotic threshold increases tumor cell killing in vivo.

Loss of ceramide production confers resistance to radiation-induced apoptosis.

Ionizing radiation mediates cell death, in part, through chromosomal damage following one or more cell divisions. X-rays also induce programmed cell death (apoptosis) in some cell types both in vitro and in vivo. Both neutral and acidic sphingomyelinases, which generate the lipid second messenger ceramide, are reported to induce apoptosis following ionizing radiation and other death signals such as tumor necrosis factor alpha and Fas ligand. Herein we report that a loss of ceramide production from a neutral sphingomyelinase generates a radioresistant phenotype as measured by a marked decrease in apoptosis. A WEHI-231 subline made deficient in ceramide production was found to be resistant to apoptosis compared with the parental subline following treatment with X-rays. The resistant subline underwent two to three subsequent cell divisions following X-irradiation, confirming that X-rays induce cell death through both mitotic and apoptotic mechanisms. These data suggest that loss of ceramide production following X-rays represents an extranuclear mechanism for the development of radioresistance. Modulation of extranuclear signals may increase tumor cell killing following radiation and represent new cellular targets for cancer therapy.

DNA double-strand break rejoining rates, inherent radiation sensitivity and human tumour response to radiotherapy.

The relationship between DNA double-strand break rejoining rates, inherent radiation sensitivity and tumour response to radiation therapy was determined for a group of 25 squamous cell carcinoma (SCC) and eight sarcoma (SAR) tumours. DNA double-strand break frequencies were measured by neutral filter elution in first passage following explant tumour samples after in vitro exposure to 100 Gy of 60Co gamma-rays. There was no significant difference between SCC and SAR tumour cells in their sensitivity to break induction, but in a 1 h time period SAR tumour cells rejoined significantly fewer breaks than SCC tumour cells, consistent with the greater sensitivity of SAR and suggesting that differences in rates of break rejoining account for the different distributions of radiosensitivities seen when different tumour types are compared. The percentage of breaks rejoined in 1 h in these tumour samples correlated well with D(o) and with the beta component of the survival curve, measured in vitro by clonogenic assay in tumour cell lines established from the tumour samples, but not with SF2 or the alpha component of the survival curve. The rates of DNA double-strand break rejoining therefore appear to influence the exponential portion of survival curves and probably the interactions between breaks. The percentage of breaks rejoined in 1 h was higher in SCC tumours that subsequently failed radiotherapy and, although the differences were not significant, they suggest that rates of break rejoining are an important component of tumour response to radiation therapy.

Genetic radiotherapy overcomes tumor resistance to cytotoxic agents.

We report that radiation enhances gene therapy of a radioresistant tumor by upregulating the induction of a chimeric gene encoding a radiosensitizing protein, tumor necrosis factor alpha (TNF-alpha). We ligated the radiation-inducible CArG elements of the radiation-inducible Egr-1 promoter/enhancer region upstream to the transcriptional start site of the human TNF cDNA (pE425-TNF). This construct was transfected using cationic liposomes into the variant murine fibrosarcoma cell line, P4L. The P4L cell line was both radioresistant (D0 = 188) and resistant to TNF. After a single intratumoral injection of 10 micrograms of pE425-TNF in cationic liposomes and two 20-Gy doses of irradiation, mean tumor volumes were significantly reduced in P4L tumors as compared to those receiving either pE425-TNF in liposomes or radiation alone (P = 0.01). TNf protein in P4L tumors was induced by radiation as high as 29 times control levels and remained detectable for 14 days. Our data indicate that combined gene therapy using liposomes, together with ionizing radiation to locally activate the induction of a radiosensitizing protein, is successful at overcoming resistance to both TNF and radiation.

Clonal interactions in a human squamous cell carcinoma.

F.2a and B.2, cell clones of the human squamous cell carcinoma SCC-12, were examined to characterize their interactions through the expression of growth factors. F.2a was nontumorigenic yet B.2 was fully tumorigenic when injected into the flanks of athymic nude mice. Combination injections of F.2a and a subtumorigenic level of B.2 produced tumors. F.2a and B.2 overexpressed the 4.8-kb transcript for transforming growth factor-alpha (TGF-alpha) as well as the 10.5- and 5.8- kb transcripts for the epidermal growth factor receptor. Neither clone expressed the transcript for epidermal growth factor, while both expressed transcripts for insulin-like growth factor-I (IGF-I) of 8.15, 4.9, and 1.6 kb and transcripts for its receptor of 8.5 and 6.5 kb. Conditioned medium (CM) from either clone stimulated the growth of itself and the other clone in tissue culture. Both clones produced intracellular TGF-alpha detectable by immunohistochemical staining, but not detectable in CM by enzyme-linked immunosorption assay. IGF-I was detected at low levels in CM by radioimmunoassay. Neutralizing antibodies to TGF-alpha but not IGF-I partially inhibit the growth of both clones in tissue culture. These results suggest that (1) TGF-alpha is active in autocrine signaling (2) IGF-I is not directly stimulatory, and (3) additional factors, as yet unidentified, are present in CM and enhance tumor growth. It is concluded that human squamous cell carcinoma SCC-12 is composed of tumorigenic and nontumorigenic clones which interact to enhance growth.