Gastrin-releasing peptide receptor-based targeting using bombesin analogues is superior to metabolism-based targeting using choline for in vivo imaging of human prostate cancer xenografts.

PURPOSE: Prostate cancer (PC) is a major health problem. Overexpression of the gastrin-releasing peptide receptor (GRPR) in PC, but not in the hyperplastic prostate, provides a promising target for staging and monitoring of PC. Based on the assumption that cancer cells have increased metabolic activity, metabolism-based tracers are also being used for PC imaging. We compared GRPR-based targeting using the (68)Ga-labelled bombesin analogue AMBA with metabolism-based targeting using (18)F-methylcholine ((18)F-FCH) in nude mice bearing human prostate VCaP xenografts. METHODS: PET and biodistribution studies were performed with both (68)Ga-AMBA and (18)F-FCH in all VCaP tumour-bearing mice, with PC-3 tumour-bearing mice as reference. Scanning started immediately after injection. Dynamic PET scans were reconstructed and analysed quantitatively. Biodistribution of tracers and tissue uptake was expressed as percent of injected dose per gram tissue (%ID/g). RESULTS: All tumours were clearly visualized using (68)Ga-AMBA. (18)F-FCH showed significantly less contrast due to poor tumour-to-background ratios. Quantitative PET analyses showed fast tumour uptake and high retention for both tracers. VCaP tumour uptake values determined from PET at steady-state were 6.7 ± 1.4%ID/g (20-30 min after injection, N = 8) for (68)Ga-AMBA and 1.6 ± 0.5%ID/g (10-20 min after injection, N = 8) for (18)F-FCH, which were significantly different (p <0.001). The results in PC-3 tumour-bearing mice were comparable. Biodistribution data were in accordance with the PET results showing VCaP tumour uptake values of 9.5 ± 4.8%ID/g (N = 8) for (68)Ga-AMBA and 2.1 ± 0.4%ID/g (N = 8) for (18)F-FCH. Apart from the GRPR-expressing organs, uptake in all organs was lower for (68)Ga-AMBA than for (18)F-FCH. CONCLUSION: Tumour uptake of (68)Ga-AMBA was higher while overall background activity was lower than observed for (18)F-FCH in the same PC-bearing mice. These results suggest that peptide receptor-based targeting using the bombesin analogue AMBA is superior to metabolism-based targeting using choline for scintigraphy of PC.

Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues.

Prostate Cancer (PC) is a type of cancer that is often diagnosed at very early stages due to improved detection among man in the Western world. Current imaging techniques are not optimal to determine extent of minimal early stage PC even though this is of great clinical importance. Human PC and high-grade PIN have shown high Gastrin-Releasing Peptide Receptor (GRPR) expression, while normal prostate tissue and BPH revealed to be predominantly GRPR-negative. Radiolabelled Gastrin-Releasing Peptide (GRP) or bombesin (BN) analogues targeting the GRPR can be used as non-invasive tools to diagnose, monitor and potentially treat PC. These BN analogues have already proven to be able to image PC in both tumour-bearing mice and clinical patients showing no important side effects. It's desirable that new peptides require fast-track standardised comparative testing in relevant PC models to select the best performing BN analogues for further evaluation in patients. Although knowledge about GRPR expression and development of new BN analogues can be extended, it is time to study performance of BN analogues for peptide receptor based imaging in patients validating results of PC imaging using histopathology as a golden standard.

Human xenograft models as useful tools to assess the potential of novel therapeutics in prostate cancer.

With docetaxel as effective chemotherapy for hormone refractory prostate cancer (HRPC), the number of new treatment combinations for HRPC is expanding demanding a fast-track screening system. This review elaborates on the use of xenograft models to select the most promising combination therapies for entering into phase II clinical trials.

Endogenous androgen receptor proteomic profiling reveals genomic subcomplex involved in prostate tumorigenesis.

Androgen receptor (AR) is a key player in prostate cancer development and progression. Here we applied immunoprecipitation mass spectrometry of endogenous AR in LNCaP cells to identify components of the AR transcriptional complex. In total, 66 known and novel AR interactors were identified in the presence of synthetic androgen, most of which were critical for AR-driven prostate cancer cell proliferation. A subset of AR interactors required for LNCaP proliferation were profiled using chromatin immunoprecipitation assays followed by sequencing, identifying distinct genomic subcomplexes of AR interaction partners. Interestingly, three major subgroups of genomic subcomplexes were identified, where selective gain of function for AR genomic action in tumorigenesis was found, dictated by FOXA1 and HOXB13. In summary, by combining proteomic and genomic approaches we reveal subclasses of AR transcriptional complexes, differentiating normal AR behavior from the oncogenic state. In this process, the expression of AR interactors has key roles by reprogramming the AR cistrome and interactome in a genomic location-specific manner.

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Androgen deprivation of the PC-310 [correction of prohormone convertase-310] human prostate cancer model system induces neuroendocrine differentiation.

Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating neuropeptides via a regulated secretory pathway (RSP). We studied NE differentiation after androgen withdrawal in the androgen-dependent prostate cancer xenograft PC-310. Expression patterns of chromogranin A, secretogranin III, and prohormone convertase-1 were analyzed at both protein and mRNA level to mark the kinetics of NE differentiation both in vivo and in vitro. PC-310 tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, and 21 days postcastration. PC-310C cultures initiated from collagenase-treated tumor tissue could be maintained up to four passages, and androgen-deprivation experiments were performed similarly. PC-310 tumor volumes decreased by 50% in 10 days postcastration. Proliferative activity and prostate-specific antigen (PSA) serum levels decreased to zero postcastration, whereas PSA levels in PC-310C culture media first decreased and subsequently increased after 5 days. In vivo, androgen receptor (AR) expression decreased initially but returned to control level from 5 days postcastration on. CgA, secretogranin III, and secretogranin V expression increased in vivo from 5 days postcastration on. Subsequently, prohormone convertase-1 and peptidyl alpha-amidating monooxygenase as well as the vascular endothelial growth factor were expressed from 7 days postcastration on, and, finally, growth factors such as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days postcastration. The PC-310 tumors did not show colocalization of the AR on the NE cells in the tumor residues after 21 days. As in the PC-310 xenograft, NE differentiation was induced and AR expression relapsed after prolonged androgen suppression in PC-310C. For PC-310C cells, this relapse was associated with the secretion of PSA. PC-310C is the first culture of human prostatic cancer cells having the NE phenotype. The PC-310 model system is a potential androgen-dependent model for studying the role of NE cells in the progression of clinical prostate cancer. Androgen deprivation of NE-differentiated prostate cancer may induce the formation of both NE- and AR-positive dormant tumor residues, capable of actively producing NE growth factors via a RSP, possibly leading to hormone refractory disease.

Differentially expressed genes in androgen-dependent and -independent prostate carcinomas.

Differential gene expression between androgen-dependent (LNCaP-FGC) and androgen-independent (LNCaP-LNO) prostate cancer cells has been investigated using RNA arbitrarily primed and differential display PCR of mRNA. Four differentially expressed cDNA transcripts were identified, of which differential expression was confirmed by Northern blot analysis. Sequence analysis revealed two unknown (JC19 and GC79) and two known genes [B-cell translocation gene 1 and UDP-glucuronosyltransferase 2B15 (UGT2B15)]. JC19, GC79, and B-cell translocation gene 1 were more highly expressed in LNCaP-FGC cells compared with LNCaP-LNO cells, whereas UGT2B15 was only expressed in LNCaP-LNO cells. Androgens and 1,25-dihydroxyvitamin D3 were able to down-regulate UGT2B15 mRNA in LNCaP-LNO cells. For GC79 mRNA, down-regulation was only observed with androgens in LNCaP-FGC cells. Expression of JC19 mRNA was studied using a panel of human prostate cancer xenografts. In androgen-dependent xenografts, expression of JC19 mRNA was much higher compared with androgen-independent xenografts, in which significant expression was hardly detectable. The mRNA expression pattern in the xenografts is in good agreement with that observed in the cell culture system. In conclusion, the differential display technique used in the present study allows analysis of gene expression in vitro and in vivo and can be used for the identification of important genes involved in androgen-independent prostate cancer development.

Imaging heterogeneity of peptide delivery and binding in solid tumors using SPECT imaging and MRI.

BACKGROUND: As model system, a solid-tumor patient-derived xenograft (PDX) model characterized by high peptide receptor expression and histological tissue homogeneity was used to study radiopeptide targeting. In this solid-tumor model, high tumor uptake of targeting peptides was expected. However, in vivo SPECT images showed substantial heterogeneous radioactivity accumulation despite homogenous receptor distribution in the tumor xenografts as assessed by in vitro autoradiography. We hypothesized that delivery of peptide to the tumor cells is dictated by adequate local tumor perfusion. To study this relationship, sequential SPECT/CT and MRI were performed to assess the role of vascular functionality in radiopeptide accumulation. METHODS: High-resolution SPECT and dynamic contrast-enhanced (DCE)-MRI were acquired in six mice bearing PC295 PDX tumors expressing the gastrin-releasing peptide (GRP) receptor. Two hours prior to SPECT imaging, animals received 25 MBq (111)In(DOTA-(ßAla)2-JMV594) (25 pmol). Images were acquired using multipinhole SPECT/CT. Directly after SPECT imaging, MR images were acquired on a 7.0-T dedicated animal scanner. DCE-MR images were quantified using semi-quantitative and quantitative models. The DCE-MR and SPECT images were spatially aligned to compute the correlations between radioactivity and DCE-MRI-derived parameters over the tumor. RESULTS: Whereas histology, in vitro autoradiography, and multiple-weighted MRI scans all showed homogenous tissue characteristics, both SPECT and DCE-MRI showed heterogeneous distribution patterns throughout the tumor. The average Spearman's correlation coefficient between SPECT and DCE-MRI ranged from 0.57 to 0.63 for the "exchange-related" DCE-MRI perfusion parameters. CONCLUSIONS: A positive correlation was shown between exchange-related DCE-MRI perfusion parameters and the amount of radioactivity accumulated as measured by SPECT, demonstrating that vascular function was an important aspect of radiopeptide distribution in solid tumors. The combined use of SPECT and MRI added crucial information on the perfusion efficiency versus radiopeptide uptake in solid tumors and showed that functional tumor characteristics varied locally even when the tissue appeared homogenous on current standard assessment techniques.

Effects of adrenal androgens on the transplantable human prostate tumor PC-82.

The potential of the adrenal androgens androstenedione (A) and dehydroepiandrosterone (DHEA) to stimulate prostate tumor growth was investigated in the hormone-dependent human prostate tumor model PC-82, propagated in nude mice. Substitution of castrated mice bearing growth-arrested tumors with DHEA for 28 days, resulting in peripheral levels of 9.2 +/- 1.7 nmol/liter (mean +/- SEM), led to a decline of tumor burden comparable to that observed in castrated controls. Intratumor testosterone (T) and 5 alpha-dihydrotestosterone were similar to those detected in the castrated group. In contrast, high-dose A supplementation (peripheral level of 13.5 +/- 1.3 nmol/liter) in androgen ablated tumor-bearing mice resulted in tumor growth, although less pronounced than in T-resubstituted mice (T level of 18.8 +/- 1.5 nmol/l). Intraprostatic levels of androgens were not different between both groups. Substitution of castrated PC-82 tumor-bearing mice with low-dose A (2.5 +/- 0.4 nmol/l) neither stimulated growth of tumors nor did it lead to regression of PC-82 tumors. Proliferative activity as estimated by BrdU incorporation (S-phase cells) was not induced in these tumors. In conclusion, DHEA does not have a stimulatory effect on growth of PC-82 tumor tissue, but A is capable of inducing PC-82 tumor growth, most likely through peripheral conversion of A into T and 5 alpha-dihydrotestosterone.

Regulation of androgen receptor expression in the human heterotransplantable prostate carcinoma PC-82.

In vivo effects of androgen withdrawal and substitution on human androgen receptor (hAR) expression were evaluated in the androgen-dependent human prostatic carcinoma tumor line PC-82. By application of several antibodies reactive with different epitopes of the hAR molecule, hAR protein expression was studied in tumor transplants by immunohistochemistry and immunoblotting. hAR messenger RNA (mRNA) levels were quantitated in PC-82 tumor tissue with a S1-nuclease protection assay. Most PC-82 tumor cells (> 97%) from testosterone-supplemented mice displayed nuclear hAR protein expression immunohistochemically. The almost complete reduction of nuclear hAR immunoreactivity within 5 days after androgen withdrawal (< 10%) was restored after androgen substitution within 1 day. The immunochemical data were confirmed by Western blot analysis. In contrast, no significant changes were observed in hAR mRNA content of PC-82 cells after 5 days of androgen withdrawal. Correlating hAR expression with proliferative activity of PC-82 tumor tissue during endocrine manipulation, a rapid, castration-induced decline of the percentage of bromodeoxyuridine-labeled cells accompanied the loss of hAR. Androgen substitution in castrated male mice restored the proliferative activity. However, this increase of proliferative activity lagged at least 24 h behind the normalization of the hAR protein level. In contrast to the steroid receptor down-regulation by homologous ligands observed in other experimental models, our data support the concept of hAR up-regulation by androgen. Since the hAR mRNA content of PC-82 tumor tissue was hardly affected by castration, expression of the hAR in PC-82 is thought to be modulated by translational and/or posttranslational mechanisms.

Ki-67 expression and BrdUrd incorporation as markers of proliferative activity in human prostate tumour models.

The validity of the use of the monoclonal antibodies Ki-67 and anti-BrdUrd to evaluate proliferative activity of human prostate tumour models was studied. Growth of the transplantable PC-82 and PC-EW prostate tumours, as assessed by tumour volume measurements, was significantly correlated with the proliferative activity as reflected by BrdUrd incorporation into DNA (r = 0.64 and r = 0.78, respectively). The proliferative activity of PC-82 tumours detected by Ki-67 antigen expression paralleled the pattern observed with BrdUrd (r = 0.51) and a significant correlation (r = 0.60) between the results obtained with both markers was found. In growing PC-82 and PC-EW tumours only small variations in the Ki-67 and BrdUrd indices were observed. In contrast, Ki-67 expression in regressing PC-82 tumours varied considerably (2.7 +/- 2.2%). The BrdUrd index in regressing PC-82 tumours showed less variation (1.3 +/- 0.2%), but part of the BrdUrd-positive cells were found in the stromal (murine) part of the regressing tissue. It is concluded that the Ki-67 and BrdUrd proliferation markers are reliable parameters to monitor changes in growth of prostate tumour lines, but that in slow growing or regressing tumours Ki-67 and BrdUrd data should be interpreted with caution.

A novel gene on human chromosome 2p24 is differentially expressed between androgen-dependent and androgen-independent prostate cancer cells.

Identification of genes involved in the transition from androgen-dependent to androgen-independent prostate cancer is important to extend our current knowledge of the disease. Using differential display RT-PCR analysis between androgen-dependent and androgen-independent prostate cancer cells, we have identified a novel gene, designated GC109. GC109 harbours a putative Cys-His cluster, a nuclear localisation signal, a leucine zipper and a ret finger protein (rfp)-like domain. GC109 mRNA expression in normal human tissues was found not to be restricted to the prostate. However, using a variety of 15 human cancer cell lines, GC109 mRNA was preferentially expressed in androgen-dependent LNCaP-FGC, compared with androgen-independent LNCaP-LNO, DU145 and PC3 human prostate cancer cells. Finally, the GC109 gene was mapped on human chromosome 2p24. Based on its protein domain structure and chromosomal localisation, we hypothesise that GC109 may be involved in chromosomal rearrangements in prostate cancer.

Effects of low testosterone levels and of adrenal androgens on growth of prostate tumor models in nude mice.

Two transplantable, androgen dependent prostate tumor models of human origin, PC-82 and PC-EW, were used to study the effect of low androgen levels and adrenal androgens on prostate tumor cell proliferation. Tumor load of the PC-82 and PC-EW tumors could be maintained constant when plasma testosterone levels were 0.8 and 0.9 nmol/l, respectively, corresponding with an intratissue 5 alpha-dihydrotestosterone level of 3-4 pmol/g tissue. This critical androgen level for prostate tumor growth stimulation amounted to 2-3 times the castration level and proved to be similar for both tumor models. Relatively high levels of androstenedione resulted in physiological levels of plasma testosterone causing androgen concentrations in PC-82 tumor tissue exceeding the critical level for tumor growth. These results indicate that submaximal suppression of androgens can stop tumor growth in these prostate tumor models.

Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.

Validation of transrectal ultrasonographic volumetry for orthotopic prostate tumours in mice.

Orthotopic human prostate tumour models in athymic nude mice are regarded as being most suitable for fundamental and pre-clinical research on prostate cancer. The anatomic localization of the tumour in the pelvis, however, provides little possibility for monitoring tumour growth or regression. To assess time-related changes in orthotopic tumour volume, we applied transrectal ultrasonography (TRUS) to the murine prostate. This technique has the advantages of allowing accurate monitoring of tumours during therapeutic manipulations and a reduction of animal use due to a reduction of sacrificing endpoints. To validate the TRUS method, the mouse prostate reconstitution model, RM-9, and the prostate-specific antigen (PSA) producing human prostate cancer xenograft PC-346 were used. Volumetric calliper measurements were performed with a 30 MHz ultrasound probe designed for intra-arterial use in humans. Tumour weight, determined at various time-points, was found to be closely related to actual tumour weight (R = 0.99) and, in the PC-346 model, to the level of PSA in the plasma. Furthermore, the interobserver variation for TRUS was low for tumours above 50 mg. Thus, TRUS for murine prostate tumours proves to be an accurate, reproducible and sensitive method.

Cross-resistance between taxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer.

INTRODUCTION: Treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. METHODS: To identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. RESULTS: We observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. CONCLUSIONS: In conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.

Improving radiopeptide pharmacokinetics by adjusting experimental conditions for bombesin receptor-targeted imaging of prostate cancer.

AIM: Prostate cancer (PC) is a major health problem. The Gastrin-Releasing Peptide Receptor (GRPR) offers a promising target for staging and monitoring of PC since it is overexpressed in PC and not in normal prostatic tissue. To improve receptor-mediated imaging we investigated the impact of various experimental conditions on pharmacokinetics using the Indium-111 labelled bombesin (BN) analogue AMBA. Besides frequently used androgen-resistant PC-3 also the clinically more relevant androgen sensitive VCaP celline was used as human PC xenograft in nude mice. METHODS: Non-purified [111In]AMBA was compared with HPLC-purified [111In]AMBA. Effect of specific activity was studied administering 0.1MBq [111In]AMBA supplemented with different amounts of AMBA (1-3000pmol). GRPR was saturated with Tyr4-BN 1 and 4h prior to injection of [111In]AMBA. RESULTS: GRPR-positive tissue showed a significant 2 to 3-fold increase in absolute uptake after HPLC-purification while keeping a stable tumor-to-pancreas ratio. Lowering specific activity resulted in decline in uptake to 43% in tumor, 49% in kidney and 92% in pancreas between 10 and 3000 pmol. Tumor-to-pancreas ratio improved six-fold from 0.1±0 after 10 pmol up to 0.6±0.2 after 3000 pmol (P<0.01). When saturating GRPR 4h prior to [111In]AMBA injection tumor-to-pancreas ratio improved from 0.10±0.3 to 0.22±0.2 (P<0.01) and tumor-to-kidney ratio increased from 0.92±0.16 to 3.45±0.5 (P<0.01). CONCLUSION: Besides specific peptide characteristics also the experimental conditions, such as HPLC-purification, variations in specific activity and saturation of the GRPR prior to [111In]AMBA administration essentially affect radiopeptide pharmacokinetics. Experimental conditions therefore need to be carefully selected in order to compose ideal standardised protocols for optimal targeting.

A transductionally retargeted adenoviral vector for virotherapy of Her2/neu-expressing prostate cancer.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody(®) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Disconnecting the yin and yang relation of epidermal growth factor receptor (EGFR)-mediated delivery: a fully synthetic, EGFR-targeted gene transfer system avoiding receptor activation.

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently labeled polyplexes after only 30 min of incubation. EGF pretreatment of cells induced enhanced cellular internalization of all polyplex types tested, pointing at generally enhanced macropinocytosis. EGF polyplexes diminished cell surface expression of EGFR for up to 4 hr, whereas GE11 polyplexes did not. In a clinically relevant orthotopic prostate cancer model, intratumorally injected GE11 polyplexes were superior in inducing transgene expression when compared with untargeted polyplexes.