Prostate Tumor Model Research Articles

Noninvasive Bioluminescence Imaging of Normal and Spontaneously Transformed Prostate Tissue in Mice

Several transgenic mouse models of prostate cancer have been developed recently that are able to recapitulate many key biological features of the human condition. It would, therefore, be desirable to employ these models to test the efficacy of new therapeutics before clinical trial; however, the variable onset and non-visible nature of prostate tumor development limit their use for such applications. We now report the generation of a transgenic reporter mouse that should obviate these limitations by enabling noninvasive in vivo bioluminescence imaging of normal and spontaneously transformed prostate tissue in the mouse. We used an 11-kb fragment of the human prostate-specific antigen (PSA) promoter to achieve specific and robust expression of firefly luciferase in the prostate glands of transgenic mice. Ex vivo bioluminescence imaging and in situ hybridization analysis confirmed that luciferase expression was restricted to the epithelium in all four lobes of the prostate. We also show that PSA-Luc mice exhibit decreased but readily detectable levels of in vivo bioluminescence over extended time periods following androgen ablation. These results suggest that this reporter should enable in vivo imaging of both androgen-dependent and androgen-independent prostate tumor models. As proof-of-principle, we show that we could noninvasively image SV40 T antigen-induced prostate tumorigenesis in mice with PSA-Luc. Furthermore, we show that our noninvasive imaging strategy can be successfully used to image tumor response to androgen ablation in transgenic mice and, as a result, that we can rapidly identify individual animals capable of sustaining tumor growth in the absence of androgen.

Tumor Vascular Permeabilization by Vascular-Targeting Photosensitization: Effects, Mechanism, and Therapeutic Implications

Loss of vascular barrier function has been observed shortly following vascular-targeting photodynamic therapy. However, the mechanism involved in this event is still not clear, and the therapeutic implications associated with this pathophysiologic change have not been fully explored. The effect of vascular-targeting photodynamic therapy on vascular barrier function was examined in both s.c. and orthotopic MatLyLu rat prostate tumor models and endothelial cells in vitro, using photosensitizer verteporfin. Vascular permeability to macromolecules (Evans blue-albumin and high molecular weight dextran) was assessed with dye extraction (ex vivo) and intravital microscopy (in vivo) methods. Intravital microscopy was also used to monitor tumor vascular functional changes after vascular-targeting photodynamic therapy. The effects of photosensitization on monolayer endothelial cell morphology and cytoskeleton structures were studied with immunofluorescence staining. Vascular-targeting photodynamic therapy induced vascular barrier dysfunction in the MatLyLu tumors. Thus, tumor uptake of macromolecules was significantly increased following photodynamic therapy treatments. In addition to vascular permeability increase, blood cell adherence to vessel wall was observed shortly after treatment, further suggesting the loss of endothelial integrity. Blood cell adhesion led to the formation of thrombi that can occlude blood vessels, causing vascular shutdown. However, viable tumor cells were often detected at tumor periphery after vascular-targeting photodynamic therapy. Endothelial cell barrier dysfunction following photodynamic therapy treatment was also observed in vitro by culturing monolayer endothelial cells on Transwell inserts. Immunofluorescence study revealed microtubule depolymerization shortly after photosensitization treatment and stress actin fiber formation thereafter. Consequently, endothelial cells were found to retract, and this endothelial morphologic change led to the formation of intercellular gaps. Vascular-targeting photodynamic therapy permeabilizes blood vessels through the formation of endothelial intercellular gaps, which are likely induced via endothelial cell microtubule depolymerization following vascular photosensitization. Loss of endothelial barrier function can ultimately lead to tumor vascular shutdown and has significant implications in drug transport and tumor cell metastasis.

Differential effects of CLDR and PDR brachytherapy on cell cycle progression in a syngeneic rat prostate tumour model

Purpose: The study consisted of two treatment arms comparing the effects of CLDR (continuous low dose rate) and PDR (pulsed dose rate) brachytherapy on cell cycle progression in a radioresistant rat prostate tumour model.Materials and methods: Interstitial PDR and CLDR brachytherapy (both 192-Ir, 0.75 Gy/h) were administered to Dunning prostate R3327-AT1 carcinomas transplanted subcutaneously into the thigh of Copenhagen rats. Increasing doses of up to 20 as well as up to 40 Gy were applied. Cell cycle distributions of the aneuploid tumour cell subpopulations were determined at 4 h (3 Gy), 24 h (18 Gy), 48 h (20 and 36 Gy), as well as during the subsequent redistribution period (20 and 40 Gy) at 72, 96, and 120 h. Tumours either implemented with an empty tubing system (n = 5) or under undisturbed growth (n = 5) served as controls. Three animals were irradiated per time point and exposure condition. At least two flow cytometrical analyses were carried out per animal.Results: The aneuploid cells possessed a constant DNA-Index of 1.9 ± 0.06. In contrast to sham-treated controls, the aneuploid cell fraction with G2/M DNA content was significantly increased (p < 0.05) after initiation of both, CLDR and PDR brachytherapy. However, CLDR resulted in an earlier accumulation of tumour cells in G2/M (24 h: 28% CLDR vs. 19% PDR, p < 0.05) with a concomitant reduction of cells in G1, whereas PDR yielded delayed, but then more pronounced cell cycle changes, particularly expressed during the redistribution period after both 20 and 40 Gy.Conclusion: CLDR and PDR brachytherapy showed differential effects on cell cycle progression. The induction of a significantly earlier but also less persistent G2/M cell cycle arrest after CLDR compared to PDR brachytherapy implies that a substantially higher fraction of tumour cells are irradiated in G2/M after CLDR.

Synthesis of branched antisense oligonucleotides having multiple specificities: Treatment of hormone insensitive prostate cancer

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.

The potential lymphangiogenic effects of hepatocyte growth factor/scatter factor in vitro and in vivo.

Lymphangiogenesis is key to the lymphatic spread of cancer cells. The current study examined the potential effect of hepatocyte growth factor (HGF), a factor known to have strong biological effects on endothelial cells, on the lymphangiogenic function of endothelial cells and the formation of lymphatic vessels using both in vitro and in vivo models. Human endothelial cells that have lymphatic characteristics, human prostate and breast cancer cells PC-3 and MDA MB 231, were used. Expression of lymphatic markers, podoplanin, Prox-1, vascular endothelial growth factor receptor 3 (VEGF-R3) and LYVE-1 was determined using reverse transcription polymerase reaction and quantitative PCR. In nude mice prostate and breast xenograft tumour models, either HGF or an HGF-producing fibroblast cell line MRC-5 was given with or without the HGF antagonist, NK4. The lymphangiogenic marker and lymphatic vessels in tumour tissues were also assessed using quantitative PCR and immunohistochemistry, respectively. In the mice tumour models, infusion of rhHGF significantly increased the levels of podoplanin and LYVE-1 in the tumour (p=0.05 for podoplanin and p<0.05 for LYVE-1 vs. without HGF in the prostate tumour model, p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF for the breast tumour model; p<0.05 for podoplanin and p<0.01 for LYVE-1 vs. without HGF in the breast tumour model). The increased level of LYVE-1 transcript was supported by an increase in the number of LYVE-1-positive lymphatic vessels in tumours, using immunohistochemical analysis. Co-injection of MRC5 cells also increased the levels of LYVE-1 and number of LYVE-1-positive vessels in tumour tissues. The effects of HGF and MRC5 were significantly reduced by the HGF antagonist, NK4. In the in vitro models, rhHGF significantly increased the level of both podoplanin and LYVE-1, as shown by quantitative PCR analysis. Hepatocyte growth factor has potential lymphangiogenic activities, and this may have important implications in the nodal spread of cancer cells.

Inhibition of Lymphogenous Metastasis Using Adeno-Associated Virus-Mediated Gene Transfer of a Soluble VEGFR-3 Decoy Receptor

The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. It has recently been shown that the lymphangiogenic growth factor, vascular endothelial growth factor-C (VEGF-C), and its receptor, VEGF receptor-3 (VEGFR3), may play a pivotal role in the promotion of metastasis to regional lymph nodes. In this study, human prostate and melanoma tumor models that preferentially metastasize to the lymph nodes following s.c. tumor cell implantation were established from lymph node metastases via in vivo selection. Melanoma tumor cell sublines established from lymph node metastasis express higher amounts of VEGF-C than the parental tumor cells. The inhibition of tumor-derived VEGF-C with a soluble VEGFR3 decoy receptor, sVEGFR3-Fc, expressed via a recombinant adeno-associated viral vector, potently blocks tumor-associated lymphangiogenesis and tumor metastasis to the lymph nodes, when the treatment was initiated before the tumor implantation. In addition, sVEGFR3-Fc serum levels required for efficient blockade of lymph node metastases are strictly dependent on the VEGF-C levels generated by the primary tumor. Recombinant adeno-associated virus-mediated gene transfer of sVEGFR3-Fc may represent a feasible therapeutic strategy for blockade of lymphogenous metastasis.

Ki-67 Antisense-Therapie in murinen Nierenzellkarzinom-Modellen

The Ki-67 antigen is only expressed in proliferating cells. Previously, it was shown that Ki-67 derived antisense oligonucleotides (asONs) specifically inhibit the proliferation of tumor cells and tumour growth in vitro and in subcutaneous bladder and prostate tumor models. We intended to evaluate the effects of this therapeutic concept in two renal cell carcinoma (RCC) models. Human RCC cells (SK-RC 35) were initially transfected with FITC-labeled ONs and diffferent cationic lipids to analyze the transfection efficacy by flow cytometry (FACS). The potency of 14 different ONs sequences was compared by quantitative RT-PCR in vitro. For in vivo testing, ONs were administered to immunocompetent Balb/c mice bearing orthotopic RENCA tumors as well as to SCID mice bearing subcutaneous RCC SK-RC 35 xenografts. Tumor sizes and final tumor weights were documented. Additionally, several immunohistochemical staining procedures were performed. FACS analysis showed highly effective transfection conditions in vitro. Systemic administration of asONs significantly decreased the tumour growth in the RENCA model (p < 0.05) and in the SCID mouse model (p = 0.009). Immunohistochemical staining of tumor specimens revealed a marked down-regulation of target protein and a slight increase in apoptotic cells after antisense treatment while the microvessel count was not significantly altered. These results demonstrate that the Ki-67 antigen represents a suitable antiproliferative target and that asONs directed against this target are potent drugs that induce a significant inhibition of renal tumor growth in different mouse models.

Combining vascular and cellular targeting regimens enhances the efficacy of photodynamic therapy

Photodynamic therapy (PDT) can be designed to target either tumor vasculature or tumor cells by varying the drug-light interval. Photodynamic therapy treatments with different drug-light intervals can be combined to increase tumor response by targeting both tumor vasculature and tumor cells. The sequence of photosensitizer and light delivery can influence the effect of combined treatments. The R3327-MatLyLu rat prostate tumor model was used in this study. Photosensitizer verteporfin distribution was quantified by fluorescence microscopy. Tumor blood flow changes were monitored by laser-Doppler system and tumor hypoxia was quantified by the immunohistochemical staining for the hypoxic marker EF5. The therapeutic effects of PDT treatments were evaluated by the histologic examination and tumor regrowth assay. Fluorescence microscopic studies indicated that tumor localization of verteporfin changed from predominantly within the tumor vasculature at 15 min after injection, to being throughout the tumor parenchyma at 3 h after injection. Light treatment (50 J/cm(2)) at 15 min after verteporfin injection (0.25 mg/kg, i.v.) induced significant tumor vascular damage, as manifested by tumor blood flow reduction and increase in the tumor hypoxic fraction. In contrast, the vascular effect observed after the same light dose (50 J/cm(2)) delivered 3 h after administration of verteporfin (1 mg/kg, i.v.) was an initial acute decrease in blood flow, followed by recovery to the level of control. The EF5 staining revealed no significant increase in hypoxic fraction at 1 h after PDT using 3 h drug-light interval. The combination of 3-h interval PDT and 15-min interval PDT was more effective in inhibiting tumor growth than each individual PDT treatment. However, it was found that the combined treatment with the sequence of 3-h interval PDT before 15-min interval PDT led to a superior antitumor effect than the other combinative PDT treatments. Histologic studies confirmed that this combined treatment led to damage to both tumor vasculature and tumor cells. Importantly, the combined PDT treatment did not increase normal tissue damage and tissue recovered well at 60 days after treatment. Our results suggest that targeting both tumor vascular and cellular compartments by combining a long-interval PDT with a short-interval PDT can be an effective and safe way to enhance PDT damage to tumor tissue.

Effect of Tumor Host Microenvironment on Photodynamic Therapy in a Rat Prostate Tumor Model

Tumor host microenvironment plays an important role in tumor growth, metastasis, and response to cancer therapy. In this study, the influence of tumor host environment on tumor pathophysiology, photosensitizer distribution, and photodynamic therapy (PDT) treatment effect was examined in the metastatic at lymph node and lung (MatLyLu) rat prostate tumor. MatLyLu tumors implanted in different host environment [i.e., orthotopically (in the prostate) or s.c.] were compared for difference in vessel density, average vessel size, vascular permeability, tumor vascular endothelial growth factor production, and tumor oxygenation. Uptake of photosensitizer verteporfin in tumors in both sites was determined by fluorescence microscopy. To compare tumor response to PDT, both orthotopic and s.c. MatLyLu tumors were given the same doses of verteporfin and laser light treatment, and PDT-induced tumor necrotic area was measured histologically. Orthotopic MatLyLu tumors were found to grow faster, have higher vessel density and more permeable vasculature, have higher vascular endothelial growth factor protein levels, and have lower tumor hypoxic fraction than the s.c. tumors. Uptake of photosensitizer verteporfin in the orthotopic tumor was higher than in the s.c. tumors at 15 minutes after injection (1 mg/kg, i.v.), and became similar at 3 hours after injection. For the vascular targeting PDT treatment (0.25 mg/kg verteporfin, 50 J/cm(2) at 50 mW/cm(2), 15 minutes drug-light interval), there was no significant difference in PDT-induced tumor necrotic area between the orthotopic and s.c. tumors, with 85% to 90% necrosis in both types of tumors. However, tumor necrosis induced by the cellular targeting PDT (1 mg/kg verteporfin, 50 J/cm(2) at 50 mW/cm(2), 3 hours drug-light interval) was significantly different in the orthotopic (64%) versus the s.c. (29%) tumors. Tumor host environment can significantly affect photosensitizer verteporfin distribution and PDT treatment effect. Verteporfin-PDT regimen targeting tumor cells is more sensitive to such influence than the vascular targeting PDT. Our study showed the importance of tumor host environment in determining tumor physiologic properties and tumor response to PDT. To obtain clinically relevant information, orthotopic tumor model should be used in the experimental studies.

Construction of a bispecific antisense oligonucleotide containing multiple binding sites for the treatment of hormone insensitive prostate tumors

Antisense oligonucleotides (oligos) have demonstrated efficacy for the treatment of various cancers, infectious diseases and metabolic disorders. While most studies have utilized single oligos either administered alone, or more recently in combination therapy with other drugs, some investigators have administered more than one oligo in a combined administration or have designed oligos which target multiple proteins (those which share mRNA sequence homology). Antisense oligos inhibit mRNA translation through complementary base pair binding, often about the AUG initiation codon. This inhibition is further enhanced through destruction of the mRNA:oligo hybrid by RNAse H. Construction of an oligo with multiple binding sites located about the respective mRNA initiation codons could simultaneously block translation of more than one protein, even those unrelated in sequence. Such binding could produce a complex mRNA:oligo hybrid more prone to degradation and clearance. Furthermore, such a formulation would increase oligo specific activity and cellular uptake, reduce toxicity, and stabilize at 1:1 the ratio between multiple oligo active sites which otherwise must be comparably delivered (in amount) and individually targeted. The activity of these newly constructed oligos could be tested in both in vitro and in vivo prostate tumor models, utilizing the hormone sensitive LNCaP and the hormone insensitive PC-3 lines. In vitro testing would evaluate oligos administered either alone or in combination with other chemotherapeutics. In vivo testing would administer the oligos to tumors carried in athymic nude mice by either intratumoral inoculation or by using a diffusion pump. Antisense oligos which target proteins associated with growth factors or their receptors, could have a role in the treatment of human prostate cancers when administered with hormone deprivation therapy, or against tumors which have already become hormone insensitive. In the later case, such treatment could form the basis of a second tier of therapy based upon growth factor deprivation.

A New Prostate Carcinoma Binding Peptide (DUP-1) for Tumor Imaging and Therapy

Prostate carcinomas belong to the most widespread tumors, and their number is increasing. Imaging modalities used for diagnosis, such as ultrasound, computed tomography, and positron emission tomography, often produce poor results. Radiolabeled peptides with high sensitivity and specificity for prostate cancer would be a desirable tool for tumor diagnosis and treatment. We used phage display and the prostate-specific membrane antigen-negative cell line DU-145 to identify a peptide. The isolated DUP-1 was tested in vitro for its binding specificity, kinetics, and affinity. Internalization of the peptide was evaluated with confocal microscopy. The tumor accumulation in a nude mouse model was analyzed with 131I-labeled DUP-1 in PC-3 and DU-145 prostate tumors as well as in the rat prostate tumor model AT-1. The synthesized peptide showed rapid binding kinetics peaking at 10 minutes. It shows specific binding to prostate carcinoma cells but low binding affinity to nontumor cells. Peptide binding is competed with unlabeled DUP-1, and a time-dependent internalization into DU-145 cells was shown. Biodistribution studies of DUP-1 in nude mice with s.c. transplanted DU-145 and PC-3 tumors showed a tumor accumulation of 5% and 7% injected dose per gram, and bound peptide could not be removed by perfusion. The rat prostate tumor model showed an increase of radioactivity in the prostate tumor up to 300% in comparison with normal prostate tissue. DUP-1 holds promise as a lead peptide structure applicable in the development of new diagnostic tracers or anticancer agents that specifically target prostate carcinoma.

Analysis of sampling volume and tissue heterogeneity on the in vivo detection of fluorescence

The effect of sampling region size and tissue heterogeneity is examined using fluorescence histogram assessment in a rat prostate tumor model with benzoporphyrin derivative fluorophore. Spatial heterogeneity in the fluorescence signal occurs on both macroscopic and microscopic scales. The periphery of the tumor is more fluorescent than the center. Fluorescence is also highest nearest the blood vessels immediately after injection, but over time this fluorescence becomes uniform through the tumor tissue. Using microscopy analysis, the fluorescence intensity histogram distributions follow a normal distribution, yet as the sampling area is increased from the micron scale to the millimeter scale, the variance of the distribution decreases. The mean fluorescence intensity is accurately measured with a millimeter size scale, but this cannot provide accurate measurements of the microscopic variance of drug in tissue. Fiber probe measurements taken in vivo are used to confirm that the variance observed is smaller than would be expected with microscopic sampling, but that the average fluorescence can be measured with fibers. Sampling tissue with fibers smaller than the intercapillary spacing could provide a way to estimate the spatial variance more accurately. In summary, sampling fiber size affects the fluorescence intensities detected and use of multiple region microscopic sampling could provide better information about the distribution of values that occur.

Combination Antiangiogenic and Androgen Deprivation Therapy for Prostate Cancer

Androgen ablation therapy leads to mild regression or stabilization of prostate cancer, followed by progression to the fatal androgen-independent state. Whereas androgen ablation diminishes tumor angiogenesis by suppressing vascular endothelial growth factor (VEGF) production, androgen-independent disease is marked by androgen-independent VEGF expression. We examined combined androgen ablation and inhibition of VEGF signaling in an androgen-sensitive human prostate cancer xenograft model (LNCaP) that is known to develop androgen-independent growth after androgen ablation. N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine (ZD6474) is an orally active inhibitor of VEGF receptor tyrosine kinase activity, with additional activity against epidermal growth factor receptor tyrosine kinase. ZD6474 (50 mg/kg/d, per os) was administered to groups of castrated and noncastrated athymic mice bearing established (4-616 mm3) LNCaP xenografts. To evaluate the extent of tumor regrowth after ZD6474, treatment was stopped after 40 days of continuous dosing, and subsequent tumor growth was monitored. Prostate-specific antigen expression was assessed to determine the effect of ZD6474 on androgen-regulated genes. In comparison with orchiectomy, ZD6474 treatment produced greater tumor growth inhibition (P < 0.001), inducing complete cytostasis for the duration of dosing. An analysis of serum prostate-specific antigen concentration and tumor weight indicated that ZD6474 did not have a direct effect on androgen-related gene expression. Combination therapy (castration plus ZD6474) produced a comparable therapeutic effect to treatment with ZD6474 alone (in noncastrated mice), for the duration of ZD6474 administration. However, when ZD6474 treatment was discontinued, the rate of tumor regrowth was significantly less in the combination group. Tumors from mice receiving combined treatment were also found to be more necrotic than tumors from mice receiving either androgen ablation or ZD6474 alone. These data indicate that inhibition of VEGF signaling produces a highly significant inhibition of tumor growth in a human androgen-dependent prostate tumor model, which far exceeds that produced by androgen ablation alone. However, when ZD6474 treatment is removed, concurrent androgen ablation produces a greater inhibition of tumor regrowth than is observed in mice without androgen ablation. Increased necrosis observed in tumors from orchiectomized mice receiving ZD6474 also suggests benefit from combining anti-androgen and anti-VEGF signaling approaches.

Hypoxia in the Androgen-Dependent Shionogi Model for Prostate Cancer at Three Stages1

The objective of this study was to investigate a possible relationship between androgen status and hypoxia in the Shionogi murine prostate tumor model, which is widely used to study the effects of androgen withdrawal on hormone resistance and radiation response. Binding of the nitroimidazole hypoxia marker EF5 was assessed using the Cy3-tagged monoclonal antibody ELK3-51. Three hours after injection of EF5 (30 mg/kg), tumors from the following three stages were excised: androgen-dependent, regressed tumors 7 days after castration, and androgen-independent. Half of each tumor was disaggregated for analysis by flow cytometry and the remainder was flash frozen. Statistically significant differences (P < 0.01) were found between androgen-dependent, regressed and androgen-dependent tumors: approximately 30, approximately 2 and approximately 50% hypoxic cells, respectively. Frozen sections from androgen-dependent tumors exhibited highly variable EF5 binding; regressed tumors showed very little or no binding; each section from androgen-dependent tumors showed high levels and uniformly distributed binding of EF5. There was no correlation between the degree of hypoxia and tumor weight (P > 0.1). The results from this preliminary study indicate that hypoxia may play an important role with respect to the timing of irradiation in prostate cancer treatments and possibly may be a useful prognostic tool. In addition, hypoxia may also be relevant to progression in this disease after androgen ablation.

PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase.

Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K.

Antitumor efficacy and tumor-selective replication with a single intravenous injection of OAS403, an oncolytic adenovirus dependent on two prevalent alterations in human cancer.

A potentially promising treatment of metastatic cancer is the systemic delivery of oncolytic adenoviruses. This requires engineering viruses which selectively replicate in tumors. We have constructed such an oncolytic adenovirus, OAS403, in which two early region genes are under the control of tumor-selective promoters that play a role in two key pathways involved in tumorigenesis. The early region E1A is controlled by the promoter for the E2F-1 gene, a transcription factor that primarily upregulates genes for cell growth. The E4 region is under control of the promoter for human telomerase reverse transcriptase, a gene upregulated in most cancer cells. OAS403 was evaluated in vitro on a panel of human cells and found to elicit tumor-selective cell killing. Also, OAS403 was less toxic in human hepatocyte cultures, as well as in vivo when compared to an oncolytic virus that lacked selective E4 control. A single intravenous injection of 3 x 10(12) vp/kg in a Hep3B xenograft mouse tumor model led to significant antitumor efficacy. Additionally, systemic administration in a pre-established LNCaP prostate tumor model resulted in over 80% complete tumor regressions at a tolerable dose. Vector genome copy number was measured in tumors and livers at various times following tail vein injection and showed a selective time-dependent increase in tumors but not livers over 29 days. Furthermore, efficacy was significantly improved when OAS403 treatment was combined with doxorubicin. This virus holds promise for the treatment of a broad range of human cancers including metastatic disease.

Directionally specific paracrine communication mediated by epithelial FGF9 to stromal FGFR3 in two-compartment premalignant prostate tumors.

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

1021. VP22 Mediates Tumor Vasculature Specific Targeting

Prostate cancer (CaP) is the most frequently diagnosed cancer in men and the second leading cause of cancer related deaths in American men. In 2003, CaP was expected to account for 220,900 cancer diagnoses and 28,900 cancer deaths. Despite recent advances in the early detection and treatment of locally advanced prostate cancer, the prognosis for patients with the advanced form of prostate cancer is grave. The explosion of research in the genetic and molecular events paralleling the development of the lethal form of the disease has lead to the identification of multiple therapeutic targets. Targeting the molecular mechanisms underlying CaP development and metastasis offers a novel approach towards the development of effective therapeutic modalities. As part of our long term goal the major focus of the current study was to integrate real-time intravital imaging of prostate tumor dynamics along with retroviral and adenoviral gene transfer approach for maximizing the therapeutic efficacy and bystander effect of apoptosis inducers by exploiting VP22-mediated tumor vasculature specific targeting. We have used intravital imaging for the real-time monitoring of tumor progression, angiogenesis and adenoviral mediated gene transfer in athymic nude mouse models of prostate cancer. The human prostate cancer cell lines, LNCaP, C4-2 and PC-3 were transduced with an enhanced green fluorescent protein expressing, gibbon ape leukemia virus (GALV) envelope pseudotyped retroviral vector MFG-EGFP-GALV. Flow cytometry revealed that the retroviral transduction efficiency is cell line dependent and was in the order PC-3 (72.46%)>LNCaP (44.37%)>C4-2 (34.47%). A fluorescent prostate tumor model was developed by subcutaneous injection of PC-3 cells in six weeks old, uncastrated, male athymic nude mice. The tumor bearing mice were injected with Dextran-Texas Red dye and subjected to sequential intravital imaging on days 17 and 24 using a dual photon Bio-Rad MRC-1024 microscope. Higly fluorescent EGFP expressing tumor cells were observed along with neo-vascularization of the tumor. Infiltration of Dextran-Texas Red dye in between the EGFP expressing tumor cells was observed thereby indicating leaky tumor vasculature. We have used this technique for the real time intravital imaging of Herpes simplex virus tegument protein VP22 mediated intercellular trafficking dynamics and novel tumor vasculature specific targeting in androgen dependent and androgen independent prostate tumor models following intra-tumoral as well as systemic delivery. For the very first time, we would like to report VP22-mediated tumor vasculature specific targeting while sparing the normal vasculature associated with hepatic and muscular tissues. Our striking and novel findings suggest that hybrid VP22-apoptosis inducers might act as a double edged sword by maximizing apoptosis induction and the associated bystander effect on one hand and selectively targeting the tumor vasculature destruction thereby potentiating the anti-angiogenic effects.

974. Adeno-Associated Viral (AAV)-Mediated Gene Transfer of Soluble Vascular Endothelial Growth Factor Receptor 3 (sVEGFR3) Inhibits Development of Lymph Node Metastases

The metastatic spread of tumor cells represents a critical point in tumor progression. The presence of metastases in regional lymph nodes is a strong indicator of poor patient survival in many types of cancer. Production of the lymphangiogenic factor, vascular endothelial growth factor C (VEGF-C), has been detected in tumors and the significance of VEGF-C mediated lymphangiogenesis for tumors has been demonstrated. VEGF-C stimulates tumor lymphangiogenesis and metastasis to regional lymph nodes by binding to VEGF receptor 3 (VEGFR3). A number of studies have demonstrated that blocking VEGFR3 signaling suppresses tumor lymphangiogenesis and lymph node metastasis. In this study, we investigated whether AAV-mediated gene delivery of a soluble VEGFR3 fusion protein, sVEGFR3-Fc, inhibits development of lymph node metastases in prostate (PC-3mlg2) and melanoma (A375mln1) tumor models. Tumor cell lines were established from lymph node metastases of mice bearing PC-3 and A375 subcutaneous tumors, respectively. These tumor cell lines were transduced with the luciferase gene enabling the detection of lymphatic and systemic metastases by luminescence imaging. A single injection of AAV-sVEGFR3-Fc inhibited tumor cell metastasis to the regional lymph nodes. The inhibition was approximately 70% in PC-3mlg2 and A375mln1 models, as evident by the reduced number of tumor-bearing lymph nodes when compared with the animals injected with an AAV null vector. We are currently performing dose response studies in both tumor models to determine the optimum VEGFR3 serum levels required for efficient block of lymphogenic metastasis.

Enhanced therapeutic effects of doxorubicin and paclitaxel in combination with liposome-entrapped ends-modified raf antisense oligonucleotide against human prostate, lung and breast tumor models

Raf-1 protein kinase plays an important role in cell growth, proliferation and cell survival. We have previously described the use of liposome-entrapped antisense raf oligonucleotide (LErafAON) to inhibit Raf-1 expression resulting in tumor growth inhibition and radiosensitization. The present study was undertaken to evaluate the chemosensitization effects of LErafAON in combination with doxorubicin or paclitaxel on a panel of human tumor xenografts. LErafAON (25.0 mg/kg i.v. x 10) displayed significant antitumor activity (P<0.05) when administered as a single agent in prostate (PC-3), lung (A549) and breast (MDA-MB 231) carcinoma models. Doxorubicin (1.0-4.0 mg/kg i.v. per week x 3) and paclitaxel (1.0-4.0 mg/kg i.v. on alternate days x 3) were administered as single agents at non-toxic doses that led to only minimal to moderate antitumor activity. However, a combination of LErafAON with doxorubicin or paclitaxel led to significantly enhanced antitumor activity in all the tumor types tested (PC-3, P<0.03; A549, P<0.035; MDA-MB 231, P<0.045) as compared with LErafAON alone or chemotherapeutic agents alone treated groups. This effect of chemosensitization appeared to be sequence-specific because a mismatch control oligonucleotide continued to show significant tumor growth. Additionally, no inhibition in Raf-1 expression in MDA-MB 231 tumor tissue was observed with mismatch oligonucleotide treatment. On the other hand, LErafAON treatment led to >75% inhibition of Raf-1 expression in tumor tissue. These preclinical observations support the use of LErafAON in combination with chemotherapeutic agents to improve the treatment of human cancers.