The role of infiltrating lymphocytes in the neo-adjuvant treatment of women with HER2-positive breast cancer.

BACKGROUND: Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. METHODS: We performed TIL analysis and T-cell analysis by IHC on the pretreatment and 'On-treatment' samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. RESULTS: In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10-3) but not TILs (p = 0.1) in their 'On-treatment' tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p < 0.01). CONCLUSIONS: The immune system may be 'primed' prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Employing mesenchymal stem cells to support tumor-targeted delivery of extracellular vesicle (EV)-encapsulated microRNA-379.

Adult Mesenchymal Stem Cells (MSCs) have a well-established tumor-homing capacity, highlighting potential as tumor-targeted delivery vehicles. MSCs secrete extracellular vesicle (EV)-encapsulated microRNAs, which play a role in intercellular communication. The aim of this study was to characterize a potential tumor suppressor microRNA, miR-379, and engineer MSCs to secrete EVs enriched with miR-379 for in vivo therapy of breast cancer. miR-379 expression was significantly reduced in lymph node metastases compared to primary tumor tissue from the same patients. A significant reduction in the rate of tumor formation and growth in vivo was observed in T47D breast cancer cells stably expressing miR-379. In more aggressive HER2-amplified HCC-1954 cells, HCC-379 and HCC-NTC tumor growth rate in vivo was similar, but increased tumor necrosis was observed in HCC-379 tumors. In response to elevated miR-379, COX-2 mRNA and protein was also significantly reduced in vitro and in vivo. MSCs were successfully engineered to secrete EVs enriched with miR-379, with the majority found to be of the appropriate size and morphology of exosomal EVs. Administration of MSC-379 or MSC-NTC cells, or EVs derived from either cell population, resulted in no adverse effects in vivo. While MSC-379 cells did not impact tumor growth, systemic administration of cell-free EVs enriched with miR-379 was demonstrated to have a therapeutic effect. The data presented support miR-379 as a potent tumor suppressor in breast cancer, mediated in part through regulation of COX-2. Exploiting the tumor-homing capacity of MSCs while engineering the cells to secrete EVs enriched with miR-379 holds exciting potential as an innovative therapy for metastatic breast cancer.

Mutant p53: a novel target for the treatment of patients with triple-negative breast cancer?

The identification and validation of a targeted therapy for patients with triple-negative breast cancer (TNBC) is currently one of the most urgent needs in breast cancer therapeutics. One of the key reasons for the failure to develop a new therapy for this subgroup of breast cancer patients has been the difficulty in identifying a highly prevalent, targetable molecular alteration in these tumors. Recently however, the p53 gene was found to be mutated in approximately 80% of basal/TNBC, raising the possibility that targeting the mutant p53 protein product might be a new approach for the treatment of this form of breast cancer. In this study, we investigated the anti-cancer activity of PRIMA-1 and PRIMA-1MET (APR-246), two compounds which were previously reported to reactivate mutant p53 and convert it to a form with wild-type (WT) properties. Using a panel of 18 breast cancer cell lines and 2 immortalized breast cell lines, inhibition of proliferation by PRIMA-1 and PRIMA-1MET was found to be cell-line dependent, but independent of cell line molecular subtype. Although response was independent of molecular subtype, p53 mutated cell lines were significantly more sensitive to PRIMA-1MET than p53 WT cells (p = 0.029). Furthermore, response (measured as IC50 value) correlated significantly with p53 protein level as measured by ELISA (p = 0.0089, r=-0.57, n = 19). In addition to inhibiting cell proliferation, PRIMA-1MET induced apoptosis and inhibited migration in a p53 mutant-dependent manner. Based on our data, we conclude that targeting mutant p53 with PRIMA-1MET is a potential new approach for treating p53-mutated breast cancer, including the subgroup with triple-negative (TN) disease.

Interrogation of gossypol therapy in glioblastoma implementing cell line and patient-derived tumour models.

BACKGROUND: Glioblastoma (GBM), being a highly vascularised and locally invasive tumour, is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. METHODS: Cell viability, endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. RESULTS: An endothelial/GBM cell viability inhibitory effect, as well as an anti-angiogenic and anti-invasive response, to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020, P=0.030), anti-angiogenic (P=0.040, P<0.0001) and pro-apoptotic (P=0.0083, P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response, respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed. CONCLUSIONS: Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis, invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy.

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression.

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival.

The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer.

Personalized medicine requires the identification of unambiguous prognostic and predictive biomarkers to inform therapeutic decisions. Within this context, the management of lymph node-negative breast cancer is the subject of much debate with particular emphasis on the requirement for adjuvant chemotherapy. The identification of prognostic and predictive biomarkers in this group of patients is crucial. Here, we demonstrate by tissue microarray and automated image analysis that the cocaine- and amphetamine-regulated transcript (CART) is expressed in primary and metastatic breast cancer and is an independent poor prognostic factor in estrogen receptor (ER)-positive, lymph node-negative tumors in two separate breast cancer cohorts (n=690; P=0.002, 0.013). We also show that CART increases the transcriptional activity of ERα in a ligand-independent manner via the mitogen-activated protein kinase pathway and that CART stimulates an autocrine/paracrine loop within tumor cells to amplify the CART signal. Additionally, we demonstrate that CART expression in ER-positive breast cancer cell lines protects against tamoxifen-mediated cell death and that high CART expression predicts disease outcome in tamoxifen-treated patients in vivo in three independent breast cancer cohorts. We believe that CART profiling will help facilitate stratification of lymph node-negative breast cancer patients into high- and low-risk categories and allow for the personalization of therapy.

Functional and prognostic relevance of the homeobox protein MSX2 in malignant melanoma.

BACKGROUND: The homeobox containing transcription factor MSX2 is a key regulator of embryonic development and has been implicated to have a role in breast and pancreatic cancer. METHODS: Using a selection of two- and three-dimensional in vitro assays and tissue microarrays (TMAs), the clinical and functional relevance of MSX2 in malignant melanoma was explored. A doxycyline-inducible over-expression system was applied to study the relevance of MSX2 in vitro. For TMA construction, tumour material from 218 melanoma patients was used. RESULTS: Ectopic expression of MSX2 resulted in the induction of apoptosis and reduced the invasive capacity of melanoma cells in three-dimensional culture. MSX2 over-expression was shown to affect several signalling pathways associated with cell invasion and survival. Downregulation of N-Cadherin, induction of p21 and inhibition of both BCL2 and Survivin were observed. Cytoplasmic MSX2 expression was found to correlate significantly with increased recurrence-free survival (P=0.008). Nuclear expression of MSX2 did not result in significant survival correlations, suggesting that the beneficial effect of MSX2 may be independent of its DNA binding activity. CONCLUSIONS: MSX2 may be an important regulator of melanoma cell invasion and survival. Cytoplasmic expression of the protein was identified as biomarker for good prognosis in malignant melanoma patients.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach.

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Assessment of candidate biomarkers of drug-induced hepatobiliary injury in preclinical toxicity studies.

This study was designed to assess the value of a set of potential markers for improved detection of liver injury in preclinical toxicity studies. Male Wistar rats were treated with drug candidates (BAY16, EMD335823, BI-3) that previously failed during development, in part due to hepatotoxicity, at two dose levels for 1, 3 and 14 days. Concentrations of lipocalin-2/NGAL and clusterin, which are frequently overexpressed and released from damaged tissues, and thiostatin, recently identified within PredTox as being elevated in urine in response to liver injury, were determined in rat urine and serum by ELISA. This was supplemented by confirmatory qRT-PCR and immunohistochemical analyses in the target organ. Serum paraoxonase-1 activity (PON1), which has been suggested as a marker of hepatotoxicity, was determined using a fluorometric assay. Clusterin and PON1 were not consistently altered in response to liver injury. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics (ROC) analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, i.e. ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment.

Vascular-targeted photodynamic therapy with BF2-chelated Tetraaryl-Azadipyrromethene agents: a multi-modality molecular imaging approach to therapeutic assessment.

BACKGROUND: Photodynamic therapy (PDT) is a treatment modality for a range of diseases including cancer. The BF(2)-chelated tetraaryl-azadipyrromethenes (ADPMs) are an emerging class of non-porphyrin PDT agent, which have previously shown excellent photochemical and photophysical properties for therapeutic application. Herein, in vivo efficacy and mechanism of action studies have been completed for the lead agent, ADMP06. METHODS: A multi-modality imaging approach was employed to assess efficacy of treatment, as well as probe the mechanism of action of ADPM06-mediated PDT. RESULTS: Tumour ablation in 71% of animals bearing mammary tumours was achieved after delivery of 2 mg kg(-1) of ADPM06 followed immediately by light irradiation with 150 J cm(-2). The inherent fluorescence of ADPM06 was utilised to monitor organ biodistribution patterns, with fluorescence reaching baseline levels in all organs within 24 h. Mechanism of action studies were carried out using dynamic positron emission tomography and magnetic resonance imaging techniques, which, when taken together, indicated a decrease in tumour vascular perfusion and concomitant reduction in tumour metabolism over time after treatment. CONCLUSION: The encouraging treatment responses in vivo and vascular-targeting mechanism of action continue to indicate therapeutic benefit for this new class of photosensitiser.

High sensitivity cytokine detection in acute coronary syndrome reveals up-regulation of interferon gamma and interleukin-10 post myocardial infarction.

Inflammation is an important element in the development and destabilization of atherosclerotic plaque. Using a high sensitivity multiplex assay, previously untested in the context of atherosclerotic disease, we determined serum concentrations of GM-CSF, IFNgamma, IL-1beta, IL-2, IL-10, IL-12p70, TNF alpha, IL-6, and IL-8 in 48 Myocardial Infarction (MI) patients, 14 Unstable Angina (UA) patients and 12 healthy controls. IFNgamma levels were significantly higher in MI compared to UA (p=0.0091) and Control groups (p=0.0014). IL-10 also showed higher expression levels between MI, UA groups and Controls (p=0.0299).This up-regulation may reflect the extent of plaque instability and/or rupture in MI patients.Our observations provide evidence that IFNgamma and IL-10 merit further investigation in atherosclerotic disease states as potential markers of disease and therapeutic targets.

Applications of SELDI-MS technology in oncology.

Considerable interest, speculation and controversy have been generated utilising surface-enhanced laser desorption/ionization in conjunction with mass spectrometry (SELDI-MS) for the diagnosis, prognosis and therapeutic monitoring of cancer and offers an attractive approach to cancer biomarker discovery from tissues and biological fluids. This technology utilises a combination of mass spectrometry and chromatography to facilitate protein profiling of complex biological mixtures. Compared to some other more traditional proteomic platforms, such as 2D polyacrylamide gel electrophoresis, it has a high-throughput capability and can resolve low-mass proteins. However, a considerable number of challenging issues related to the design of studies, including reproducibility, sensitivity, specificity, variation in sample collection, processing and storage, have been reported as problematic with this technology; albeit some of these concerns could perhaps also be lauded against other proteomic approaches that have attempted to address complex protein mixtures, such as plasma. Applications, successes and limitations of SELDI-MS in both clinical and basic science arenas will be reviewed in this article.

Cancer invasion and metastasis: changing views.

The formation of distant metastasis is the main cause of morbidity and mortality in patients with cancer. The aim of this article is to review recent advances in molecular and clinical aspects of metastasis. Traditionally, genes encoding extracellular matrix (ECM) processing proteases, adhesion proteins, and motility factors were thought to be amongst the main mediators of metastasis. Recently, however, genes activated during the early stages of tumourigenesis were implicated in the process. Conversely, genes thought to be primarily involved in metastasis such as urokinase plasminogen (uPA) and certain matrix metalloproteases (MMPs) are now known to also play a role in the early steps of tumour progression, perhaps by stimulating cell proliferation and/or promoting angiogenesis. Paradoxically, certain endogenous protease inhibitors such as PAI-1 and TIMP-1 appear to promote cancer metastasis rather than inhibiting the process. These recent advances in our understanding should lead to the development of new molecular markers for predicting the likely formation of metastasis as well as the identification of new targets for anti-metastatic therapies.

Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles.

Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.

Molecular links between mammary gland development and breast cancer.

During its lifetime, the mammary gland undergoes many phases of development and differentiation. Much of this occurs during puberty, when the ductal epithelium expands by branching morphogenesis, invading the surrounding fat pad to form an organised mammary tree. Throughout its existence, the epithelium will go through several cycles of proliferation and cell death during pregnancy, lactation and involution. Many of the signalling mechanisms which control the initial invasion of the fat pad by the epithelium, and regulate its continuing plasticity, can be harnessed or corrupted by tumour cells in order to support their aberrant growth and progression towards invasion. This is true not just for the epithelial cells themselves but also for cells in the surrounding microenvironment, including fibroblasts, macrophages and adipocytes. This review examines the complex web of signalling and adhesion interactions controlling branching morphogenesis, and how their alteration can promote malignancy. Current in vivo and in vitro mammary gland models are also discussed. (Part of a Multi-author Review).

Novel "plum pudding" gels as potential drug-eluting stent coatings: controlled release of fluvastatin.

This study evaluated novel structural motifs known as "plum pudding" gels as potential drug-eluting stent coatings. Controlled delivery of a HMG-CoA reductase inhibitor (statin) from the intravascular stent surface represents a potential therapeutic modality for prevention of in-stent restenosis (ISR). In this study, gels were comprised of fluvastatin-loaded thermoresponsive microgel particles containing the relatively hydrophilic N-isopropylacrylamide (NiPAAm), mixed with the more hydrophobic N-tert-butylacrylamide (NtBAAm) in different wt/wt ratios: 85/15, 65/35, and 50/50, randomly dispersed in a 65/35 or 85/15 NiPAAm/NtBAAm copolymer matrix. Fluvastatin release from 5 microm copolymer films was greatest from the most hydrophilic systems and least from the more hydrophobic systems. Release from hydrophobic matrices appeared to be via Fickian diffusion, enabling use of the Stokes-Einstein equation to determine diffusion coefficients. Release from hydrophilic matrices was non-Fickian. Eluted drug retained its bioactivity, assessed as selective inhibition of human coronary artery smooth muscle cell proliferation. When stainless steel stent wires were coated (25 microm thickness) with fluvastatin-loaded 65/35 microgels in an 85/15 copolymer matrix, drug elution into static and perfused flow environments followed similar elution profiles. In contrast to elution from copolymer films cast on flat surfaces, diffusion from stent wires coated with hydrophilic and hydrophobic systems both followed Fickian patterns, with slightly larger diffusion coefficients for elution from the flow system. We conclude that manipulation of the relative hydrophobicities of both microgel and matrix components of "plum pudding" gels results in tightly regulated release of fluvastatin over an extended time period relevant to initiation and propagation of ISR.

A potent nonporphyrin class of photodynamic therapeutic agent: cellular localisation, cytotoxic potential and influence of hypoxia.

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics.

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.

Oligonucleotide microarray analysis of gene expression in neuroblastoma displaying loss of chromosome 11q.

A number of distinct subtypes of neuroblastoma exist with different genetic abnormalities that are predicative of outcome. Whole chromosome gains are usually associated with low stage disease and favourable outcome, whereas loss of 1p, 3p and 11q, unbalanced gain of 17q and MYCN amplification (MNA) are indicative of high stage disease and unfavourable prognosis. Although MNA and loss of 11q appear to represent two distinct genetic subtypes of advanced stage neuroblastoma, a detailed understanding of how these subtypes differ in terms of global gene expression is still lacking. We have used metaphase comparative genomic hybridization (CGH) analysis in combination with oligonucleotide technology to identify patterns of gene expression that correlate with specific genomic imbalances found in primary neuroblastic tumours and cell lines. The tumours analysed in this manner included a ganglioneuroma, along with various ganglioneuroblastoma and neuroblastoma of different stages and histopathological classifications. Oligonucleotide microarray-based gene expression profile analysis was performed with Affymetrix HU133A arrays representing approximately 14 500 unique genes. The oligonucleotide microarray results were subsequently validated by quantitative real-time PCR, immunohistochemical staining, and by comparison of specific gene expression patterns with published results. Hierarchical clustering of gene expression data distinguished tumours on the basis of stage, differentiation and genetic abnormalities. A number of genes were identified whose patterns of expression were highly correlated with 11q loss; supporting the concept that loss of 11q represents a distinct genetic subtype of neuroblastoma. The implications of these results in the process of neuroblastoma development and progression are discussed.

Extended delivery of the antimitotic agent colchicine from thermoresponsive N-isopropylacrylamide-based copolymer films to human vascular smooth muscle cells.

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of an antimitotic agent to human vascular smooth muscle cells (HASMC) over an extended period of time. Copolymer films were prepared using a 50:50 (w/w) ratio of N-isopropylacrylamide (NiPAAm) monomer to the more hydrophobic N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (0.1 micromol per film) at room temperature. Colchicine release from films was sustained over a 14-day period. At 24 h postloading, the concentration of colchicine in the medium overlying films was 2.12 +/- 0.16 microM; this fell to 0.20 +/- 0.01 microM at 7 days and decreased further to 0.12 +/- 0.01 microM after 14 days. Colchicine released from copolymer films inhibited proliferation when subsequently placed on HASMC: at 0.1 microM, released colchicine reduced proliferation to 18.5 +/- 0.8% of control cells (p < 0.001, n = 9). The antiproliferative effect of released colchicine was comparable to that of native colchicine, as observed in separate experiments. Furthermore, colchicine released from 50:50 polymer films inhibited the proliferation of cells grown in the same environment as the copolymer. Inhibition of cell proliferation was not due to the release of cytotoxic particles from the copolymer because medium incubated with copolymer for 5 days and then applied to HASMC did not alter cell viability. In conclusion, this study demonstrates that 50:50 NiPAAm:NtBAAm copolymers can deliver bioactive concentrations of the antimitotic agent colchicine to human vascular cells over an extended period of time.