Invasive glioblastoma cells acquire stemness and increased Akt activation.

Glioblastoma multiforme (GBM) is the most frequent and most aggressive brain tumor in adults. The dismal prognosis is due to postsurgery recurrences arising from escaped invasive tumor cells. The signaling pathways activated in invasive cells are under investigation, and models are currently designed in search for therapeutic targets. We developed here an in vivo model of human invasive GBM in mouse brain from a GBM cell line with moderate tumorigenicity that allowed simultaneous primary tumor growth and dispersal of tumor cells in the brain parenchyma. This strategy allowed for the first time the isolation and characterization of matched sets of tumor mass (Core) and invasive (Inv) cells. Both cell populations, but more markedly Inv cells, acquired stem cell markers, neurosphere renewal ability, and resistance to rapamycin-induced apoptosis relative to parental cells. The comparative phenotypic analysis between Inv and Core cells showed significantly increased tumorigenicity in vivo and increased invasion with decreased proliferation in vitro for Inv cells. Examination of a large array of signaling pathways revealed extracellular signal-regulated kinase (Erk) down-modulation and Akt activation in Inv cells and an opposite profile in Core cells. Akt activation correlated with the increased tumorigenicity, stemness, and invasiveness, whereas Erk activation correlated with the proliferation of the cells. These results underscore complementary roles of the Erk and Akt pathways for GBM proliferation and dispersal and raise important implications for a concurrent inhibitory therapy.

Preclinical assessment of a 980-nm diode laser ablation system in a large animal tumor model.

PURPOSE: To characterize the performance of a 980-nm diode laser ablation system in an in vivo tumor model. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. The ablation system consisted of a 15-W, 980-nm diode laser, flexible diffusing-tipped fiber optic, and 17-gauge internally cooled catheter. Ten immunosuppressed dogs were inoculated subcutaneously with canine-transmissible venereal tumor fragments in eight dorsal locations. Laser ablations were performed at 79 sites where inoculations were successful (99%) at powers of 10 W, 12.5 W, and 15 W, with exposure times between 60 and 180 seconds. In 20 cases, multiple overlapping ablations were performed. After the dogs were euthanized, the tumors were harvested, sectioned along the applicator tract, measured, and photographed. Measurements of ablation zone were performed on gross specimen. Histopathology and viability staining was performed with hematoxylin and eosin and nicotinamide adenine dinucleotide hydrogen staining. RESULTS: Gross pathologic examination confirmed a well circumscribed ablation zone with sharp boundaries between thermally ablated tumor in the center surrounded by viable tumor tissue. When a single applicator was used, the greatest ablation diameters ranged from 12 mm at the lowest dose (10 W, 60 seconds) to 26 mm at the highest dose (15 W, 180 seconds). Multiple applicators created ablation zones as large as 42 mm in greatest diameter (with the lasers operating at 15 W for 120 seconds). CONCLUSIONS: The new 980-nm diode laser and internally cooled applicator effectively create large ellipsoid thermal ablations in less than 3 minutes.

Selenium and vitamin E: cell type- and intervention-specific tissue effects in prostate cancer.

BACKGROUND: Secondary analyses of two randomized, controlled phase III trials demonstrated that selenium and vitamin E could reduce prostate cancer incidence. To characterize pharmacodynamic and gene expression effects associated with use of selenium and vitamin E, we undertook a randomized, placebo-controlled phase IIA study of prostate cancer patients before prostatectomy and created a preoperative model for prostatectomy tissue interrogation. METHODS: Thirty-nine men with prostate cancer were randomly assigned to treatment with 200 microg of selenium, 400 IU of vitamin E, both, or placebo. Laser capture microdissection of prostatectomy biopsy specimens was used to isolate normal, stromal, and tumor cells. Gene expression in each cell type was studied with microarray analysis and validated with a real-time polymerase chain reaction (PCR) and immunohistochemistry. An analysis of variance model was fit to identify genes differentially expressed between treatments and cell types. A beta-uniform mixture model was used to analyze differential expression of genes and to assess the false discovery rate. All statistical tests were two-sided. RESULTS: The highest numbers of differentially expressed genes by treatment were 1329 (63%) of 2109 genes in normal epithelial cells after selenium treatment, 1354 (66%) of 2051 genes in stromal cells after vitamin E treatment, and 329 (56%) of 587 genes in tumor cells after combination treatment (false discovery rate = 2%). Validation of 21 representative genes across all treatments and all cell types yielded Spearman correlation coefficients between the microarray analysis and the PCR validation ranging from 0.64 (95% confidence interval [CI] = 0.31 to 0.79) for the vitamin E group to 0.87 (95% CI = 0.53 to 0.99) for the selenium group. The increase in the mean percentage of p53-positive tumor cells in the selenium-treated group (26.3%), compared with that in the placebo-treated group (5%), showed borderline statistical significance (difference = 21.3%; 95% CI = 0.7 to 41.8; P = .051). CONCLUSIONS: We have demonstrated the feasibility and efficiency of the preoperative model and its power as a hypothesis-generating engine. We have also identified cell type- and zone-specific tissue effects of interventions with selenium and vitamin E that may have clinical implications.

Lysophosphatidic acid receptors determine tumorigenicity and aggressiveness of ovarian cancer cells.

BACKGROUND: Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown. METHODS: Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided. RESULTS: SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase. CONCLUSIONS: Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.

An in vivo orthotopic canine model to evaluate distribution of intraprostatic injectate: implications for gene therapy and drug delivery for prostate cancer.

OBJECTIVES: Complete and reliable infiltration of the entire prostate is important to improve the efficacy of intraprostatic gene therapy, as well as drug delivery. We sought to evaluate the optimal injection scheme and feasibility of magnetic resonance imaging (MRI) using a surrogate solution to assess the distribution of injectate in an orthotopic canine model. METHODS: Twelve dogs were anesthetized, and laparotomy was performed. Four dogs in each group were injected with a 1:10 dilution of 1% methylene blue and gadolinium-diethylenetriamine pentaacetic acid using 3, 10, and 20-core injection schemes. The dogs subsequently underwent MRI and then were killed. The prostates were harvested, sectioned, and photographed for methylene blue distribution. The cross-sectional area of the stained pathology and MRI slides was calculated, and correlation studies were performed. Statistical analysis was performed using the paired t test and Pearson's correlation test. RESULTS: The gadolinium distribution reflected the methylene blue distribution, with a Pearson's correlation coefficient of 0.97 (P <0.001). The fractional volume distribution in the 3, 10, and 20-core injection schemes was 10.2%, 28.5%, and 30.1%, respectively. The volume of distribution was significantly greater for the 10-core (P = 0.0024) and 20-core (P = 0.0025) injection schemes than for the 3-core scheme. However, no significant difference was found between the 10 and 20-core schemes (P = 0.52). CONCLUSIONS: MRI using gadolinium-diethylenetriamine pentaacetic acid is an excellent modality to evaluate the effective volume distribution of injectate in an in vivo orthotopic prostate model. The 10-core injection scheme seemed to be as good as the 20-core scheme in achieving adequate distribution of injectate within the prostate.

Liposome-incorporated Grb2 antisense oligodeoxynucleotide increases the survival of mice bearing bcr-abl-positive leukemia xenografts.

We previously demonstrated that liposome-incorporated antisense oligodeoxynucleotide specific for the grb2 mRNA (L-Grb2) inhibited Grb2 protein expression and the proliferation of bcr-abl-positive leukemia cell lines. To determine whether L-Grb2 has the potential of being a therapeutic modality against bcr-abl-positive leukemia, we studied the tissue distribution of L-Grb2 in normal mice before studying its effects in mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 was widely distributed in the body. The highest tissue concentrations of L-Grb2 were found in the spleen and liver, which are the organs where the tumor mass of bcr-abl-positive leukemia is mainly found. At 4 h post-injection, the amount of L-Grb2 detected per g of tissue was 64 microg in spleen and 50 microg in liver. Intravenous injection of bcr-abl-positive 32D mouse leukemia cells into radiated NOD/scid mice caused a lethal leukemia syndrome; we determined whether L-Grb2 could prolong the survival of mice bearing such xenografts. One day after leukemia cell inoculation, mice received twice weekly intravenous injections of L-Grb2. At an injection dose of 15 mg of L-Grb2 per kg of mouse body weight, 80% of mice treated with L-Grb2 survived to 48 days (end of study) whereas 0% of mice treated with the same dose of liposomal control oligonucleotide survived; the mean survival duration of these groups was 44 and 20 days, respectively. Our data indicate that L-Grb2 prolonged the survival of mice bearing bcr-abl-positive leukemia xenografts. L-Grb2 may be used as a novel cancer therapeutic modality.

Control of graft-versus-host disease with maintenance of the graft-versus-leukemia effect in a murine allogeneic transplant model using retrovirally transduced murine suicidal lymphocytes.

OBJECTIVE: Limited clinical trials have validated the hypothesis of controlling graft-versus-host disease (GVHD) arising from stem cell transplant utilizing suicidal T-lymphocytes that have been transduced to express the HSV-TK gene. However, clinical utility has been limited by diminished T-cell function arising from the production process. To evaluate strategies for harnessing the graft-versus-leukemia (GVL) effect while improving the safety and function of suicidal lymphocytes, we have developed techniques to produce fully functional, retrovirally transduced, HSV-TK-positive murine T cells (TK+TC). METHODS: Utilizing a murine major histocompatibility complex-matched transplant model, we evaluated the ability of TK+TC to generate a GVL effect and the ability to control GVHD in experiments where we varied the dose of TK+TC, ganciclovir (GCV) dose, the start of GCV administration (day 4, 7, 10, 13, 15, or 19) posttransplantation, and the GCV administration route (osmotic pump versus intraperitoneal). RESULTS: At TK+TC doses in excess of the standard lethal dose (SLD) of unmanipulated T-cells, GCV administration completely (2 x SLD) and partially (4 x SLD) controlled GVHD. Additionally, GVHD remained reversible despite delaying administration of GCV for a week after GVHD developed. Importantly, GVHD was controlled with a 1-log but not 2-log reduction in GCV dose, and this "partial suicide" preserved more circulating TK+TC compared with standard-dose GCV. Survival of leukemia-positive mice receiving TK+TC and GCV was significantly increased compared with control cohorts not receiving GCV or transplanted with unmanipulated T cells, thereby demonstrating a GVL effect. CONCLUSION: Retrovirally transduced suicidal lymphocytes generate a potent GVL effect while simultaneously enabling control of GVHD, which results in improved leukemia and GVHD-free survival.

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia.

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.

Nanoparticle based systemic gene therapy for lung cancer: molecular mechanisms and strategies to suppress nanoparticle-mediated inflammatory response.

Cancer gene therapy for the treatment of lung cancer has shown promise in the laboratory and in Phase I/II clinical trials. However, it is currently limited to treating localized tumors due to host-immunity against the gene delivery vector and the transgene. Therefore, there is a tremendous effort to develop and test alternate gene delivery vectors that are efficient, non-immunogenic, and applicable for systemic therapy. One such gene delivery vehicle is the non-viral vector, DOTAP:cholesterol (DOTAP:Chol) nanoparticle. Preclinical studies from our laboratory has shown that DOTAP:Chol. nanoparticles are effective systemic gene delivery vectors that efficiently deliver tumor-suppressor genes to disseminated lung tumors. Based on our findings we have recently initiated a Phase-I trial for systemic treatment of lung cancer using a novel tumor suppressor gene, FUS1. Although DOTAP:Chol. nanoparticles complexed to DNA (DNA-nanoparticles) are efficient vectors for systemic therapy, induction of an inflammatory response in a dose-dependent fashion has also been observed thereby limiting its use. A better understanding of the underlying mechanism for DNA-nanoparticles-mediated inflammatory response will allow us to develop strategies to suppress inflammation and expand the therapeutic window in treating human cancer. In the present study we conducted experiments examining the mechanism of nanoparticle-mediated inflammatory response in vitro and in vivo. We demonstrate that systemic administration of DNA-nanoparticles induced multiple signaling molecules both in vitro and in vivo that are associated with inflammation. Use of small molecule inhibitors against the signaling molecules resulted in their suppression and thereby reduced inflammation without affecting transgene expression. Our results provide a rationale to use small molecule inhibitors to suppress nanoparticle-mediated inflammation when administered systemically. Further development and testing will allow us to incorporate this strategy into future clinical trials that is based on systemic non-viral vector gene therapy.

Imaging taxane-induced tumor apoptosis using PEGylated, 111In-labeled annexin V.

UNLABELLED: 99mTc-Labeled annexin V has been used for the imaging of tumor apoptosis induced by chemotherapy. However, owing to the short half-life of annexin V, multiple injections of the radiotracer are necessary to capture the peak apoptotic activity. In this study, we evaluated the imaging properties of an (111)In-labeled, long-circulating annexin V. METHODS: Both polyethylene glycol (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) were simultaneously introduced to annexin V or ovalbumin through the use of a heterofunctional PEG precursor. Imaging studies were performed in mice bearing subcutaneously inoculated human mammary MDA-MB-468 tumors. The mice were treated with poly(L-glutamic acid)-paclitaxel, monoclonal antibody C225, or a combination of poly(L-glutamic acid)-paclitaxel and C225, followed by intravenous injection of (111)In-DTPA-PEG-annexin V. Images were acquired 48 h after the injection of the radiotracer. Autoradiography and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) staining were performed on adjacent tumor slices for the localization of apoptotic cells. The imaging properties of unPEGylated annexin V and PEGylated ovalbumin were also determined to permit assessment of the specificity of (111)In-DTPA-PEG-annexin V. RESULTS: Tumor apoptotic index increased from 1.67% +/- 0.31% at baseline to 7.60% +/- 0.72% and 11.07% +/- 1.81%, respectively, 4 d after treatment with poly(L-glutamic acid)-paclitaxel or combined poly(L-glutamic acid)-paclitaxel and C225. Tumor uptake (percentage of injected dose per gram of tumor [%ID/g]) of PEGylated (111)In-DTPA-PEG-annexin 4 d after treatment was significantly higher in tumors treated with poly(L-glutamic acid)-paclitaxel (10.76 +/- 1.38 %ID/g; P = 0.001) and with combined poly(L-glutamic acid)-paclitaxel and C225 (9.84 +/- 2.51 %ID/g; P = 0.029) than in nontreated tumors (6.14 +/- 0.67 %ID/g), resulting in enhanced visualization of treated tumors. (111)In-DTPA-PEG-annexin V distributed into the central zone of tumors, whereas (111)In-DTPA-annexin V was largely confined to the tumor periphery. Furthermore, uptake of (111)In-DTPA-PEG-annexin V by tumors correlated with apoptotic index (r = 0.87, P = 0.02). Increase in tumor uptake of the nonspecific PEGylated protein (111)In-DTPA-PEG-ovalbumin was also observed after poly(L-glutamic acid)-paclitaxel treatment (55.6%), although this increase was less than that observed for (111)In-DTPA-PEG-annexin V (96.7%). CONCLUSION: Increased uptake of and improved visualization with (111)In-DTPA-PEG-annexin V in solid tumors after chemotherapy are mediated through both specific binding to apoptotic cells and nonspecific retention of macromolecular contrast agents in the tumors. (111)In-Labeled, PEGylated annexin V may be used to assess tumor response to chemotherapy.