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Purpose: High-grade glioma (HGG) treatment is limited by the inability of otherwise potentially efficacious drugs to penetrate the blood–brain barrier. We evaluate the unique intracavity delivery mode and translational potential of a blend of poly(DL-lactic acid-co-glycolic acid; PLGA) and poly(ethylene glycol; PEG) paste combining temozolomide and etoposide to treat surgically resected HGG. Experimental Design: To prolong stability of temozolomide prodrug, combined in vitro drug release was quantitatively assessed from low pH–based PLGA/PEG using advanced analytic methods. In vitro cytotoxicity was measured against a panel of HGG cell lines and patient-derived cultures using metabolic assays. In vivo safety and efficacy was evaluated using orthotopic 9L gliosarcoma allografts, previously utilized preclinically to develop Gliadel. Results: Combined etoposide and temozolomide in vitro release (22 and 7 days, respectively) was achieved from a lactic acid–based PLGA/PEG paste, used to enhance stability of temozolomide prodrug. HGG cells from central-enhanced regions were more sensitive to each compound relative to primary lines derived from the HGG-invasive margin. Both drugs retained cytotoxic capability upon release from PLGA/PEG. In vivo studies revealed a significant overall survival benefit in postsurgery 9L orthotopic gliosarcomas, treated with intracavity delivered PLGA/PEG/temozolomide/etoposide and enhanced with adjuvant radiotherapy. Long-term survivorship was observed in over half the animals with histologic confirmation of disease-free brain. Conclusions: The significant survival benefit of intracavity chemotherapy demonstrates clinical applicability of PLGA/PEG paste-mediated delivery of temozolomide and etoposide adjuvant to radiotherapy. PLGA/PEG paste offers a future platform for combination delivery of molecular targeted compounds.

Objective: Postnatal angiogenesis is critical in vascular homeostasis and repair. m 6 A (N6-methyladenosine) RNA methylation is emerging as a new layer for fine-tuning gene expression. Although the contribution of the m 6 A-catalyzing enzyme, METTL3 (methyltransferase-like 3), in cancer biology has been described, its role in endothelial cell (EC) function, particularly during angiogenesis, remains unclear. Approach and Results: To characterize the relevance of METTL3 in angiogenesis regulation, we performed gain- and loss-of-function studies in vitro. We demonstrated that depletion of METTL3 in ECs reduced the level of m 6 A and impaired EC function, whereas adenovirus-mediated METTL3 overexpression increased angiogenesis. Mechanistically, we showed that METTL3 depletion in ECs decreased mature angiogenic microRNAs let-7e-5p and the miR-17-92 cluster, and increased the expression of their common target, Tsp1 (thrombospondin 1). Conversely, Ad.METTL3 increased the expression of let-7e-5p and miR-17-92 cluster and reduced protein levels of Tsp1 in ECs. Moreover, overexpression of let-7e-5p and miR-18a-5p restored the angiogenic potential of METTL3-depleted ECs. We corroborated our data in vivo employing 3 mouse models. When tested in an in vivo Matrigel plug assay, METTL3-depleted ECs had diminished ability to vascularize the plug, whereas overexpression of METTL3 promoted angiogenesis. Local Ad.METTL3 gene transfer increased postischemic neovascularization in mice with either unilateral limb ischemia or myocardial infarction. Conclusions: METTL3 regulates m 6 A RNA methylation in ECs. Endogenous METTL3 is essential for EC function and angiogenesis, potentially through influencing let-7e and miR-17-92 cluster processing. Thus, the therapeutic modulation of METTL3 should be considered as a new approach for controlling angiogenic responses in the clinical setting.

Background Therapeutic intervention in metastatic medulloblastoma is dependent on elucidating the underlying metastatic mechanism. We investigated whether an epithelial–mesenchymal transition (EMT)-like pathway could drive medulloblastoma metastasis. Methods A 3D Basement Membrane Extract (3D-BME) model was used to investigate medulloblastoma cell migration. Cell line growth was quantified with AlamarBlue metabolic assays and the morphology assessed by time-lapse imaging. Gene expression was analyzed by qRT-PCR and protein expression by immunohistochemistry of patient tissue microarrays and mouse orthotopic xenografts. Chromatin immunoprecipitation was used to determine whether the EMT transcription factor TWIST1 bound to the promoter of the multidrug pump ABCB1. TWIST1 was overexpressed in MED6 cells by lentiviral transduction (MED6-TWIST1). Inhibition of ABCB1 was mediated by vardenafil, and TWIST1 expression was reduced by either Harmine or shRNA. Results Metastatic cells migrated to form large metabolically active aggregates, whereas non-tumorigenic/non-metastatic cells formed small aggregates with decreasing metabolic activity. TWIST1 expression was upregulated in the 3D-BME model. TWIST1 and ABCB1 were significantly associated with metastasis in patients (P = .041 and P = .04, respectively). High nuclear TWIST1 expression was observed in the invasive edge of the MED1 orthotopic model, and TWIST1 knockdown in cell lines was associated with reduced cell migration (P < .05). TWIST1 bound to the ABCB1 promoter (P = .03) and induced cell aggregation in metastatic and TWIST1-overexpressing, non-metastatic (MED6-TWIST1) cells, which was significantly attenuated by vardenafil (P < .05). Conclusions In this study, we identified a TWIST1–ABCB1 signaling axis during medulloblastoma migration, which can be therapeutically targeted with the clinically approved ABCB1 inhibitor, vardenafil.

The chemotherapy resistance of esophageal adenocarcinomas (EAC) is underpinned by cancer cell extrinsic mechanisms of the tumor microenvironment (TME). We demonstrate that by targeting the tumor-promoting functions of the predominant TME cell type, cancer associated fibroblasts (CAF) with phosphodiesterase type 5 inhibitors (PDE5i) we can enhance the efficacy of standard-of-care chemotherapy. In ex-vivo conditions PDE5i prevented the transdifferation of normal fibroblasts to CAF and abolished the tumor-promoting function of established EAC CAF. Using shotgun proteomics and single cell RNA-Seq we revealed PDE5i specific regulation of pathways related to fibroblast activation and tumor promotion. Finally, we confirmed the efficacy of PDE5i in combination with chemotherapy in close-to-patient and in vivo PDX-based model systems. These findings demonstrate that CAF drive chemotherapy resistance in EAC and can be targeted by repurposing PDE5i, a safe and well tolerated class of drug administered to millions of patients world-wide to treat erectile dysfunction.

© 2020 Wiley-VCH GmbH The size, shape, and underlying chemistries of drug delivery particles are key parameters which govern their ultimate performance in vivo. Responsive particles are desirable for triggered drug delivery, achievable through architecture change and biodegradation to control in vivo fate. Here, polymeric materials are synthesized with linear, hyperbranched, star, and micellar-like architectures based on 2-hydroxypropyl methacrylamide (HPMA), and the effects of 3D architecture and redox-responsive biodegradation on biological transport are investigated. Variations in “stealth” behavior between the materials are quantified in vitro and in vivo, whereby reduction-responsive hyperbranched polymers most successfully avoid accumulation within the liver, and none of the materials target the spleen or lungs. Functionalization of selected architectures with doxorubicin (DOX) demonstrates enhanced efficacy over the free drug in 2D and 3D in vitro models, and enhanced efficacy in vivo in a highly aggressive orthotopic breast cancer model when dosed over schedules accounting for the biodistribution of the carriers. These data show it is possible to direct materials of the same chemistries into different cellular and physiological regions via modulation of their 3D architectures, and thus the work overall provides valuable new insight into how nanoparticle architecture and programmed degradation can be tailored to elicit specific biological responses for drug delivery.

Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen novel ruthenium(II) organometallic arene complexes have been evaluated for activity (in vitro and in vivo) in models of human ovarian cancer, and cross-resistance profiles established in cisplatin and multi-drug-resistant variants. A broad range of IC50 values was obtained (0.5 to >100 μM) in A2780 parental cells with two compounds (RM175 and HC29) equipotent to carboplatin (6 μM), and the most active compound (HC11) equipotent to cisplatin (0.6 μM). Stable bi-dentate chelating ligands (ethylenediamine), a more hydrophobic arene ligand (tetrahydroanthracene) and a single ligand exchange centre (chloride) were associated with increased activity. None of the six active ruthenium(II) compounds were cross-resistant in the A2780cis cell line, demonstrated to be 10-fold resistant to cisplatin/carboplatin by a mechanism involving, at least in part, silencing of MLH1 protein expression via methylation. Varying degrees of cross-resistance were observed in the P-170 glycoprotein overexpressing multi-drug-resistant cell line 2780AD that could be reversed by co-treatment with verapamil. In vivo activity was established with RM175 in the A2780 xenograft together with non-cross-resistance in the A2780cis xenograft and a lack of activity in the 2780AD xenograft. High activity coupled to non cross-resistance in cisplatin resistant models merit further development of this novel group of anticancer compounds. British Journal of Cancer (2002) 86, 1652–1657. DOI: 10.1038/sj/bjc/6600290 www.bjcancer.com © 2002 Cancer Research UK

Glioblastoma is a malignant brain tumour with a median survival of 14.6 months from diagnosis. Despite maximal surgical resection and concurrent chemoradiotherapy, reoccurrence is inevitable. To try combating the disease at a stage of low residual tumour burden immediately post-surgery, we propose a localised drug delivery system comprising of a spray device, bioadhesive hydrogel (pectin) and drug nanocrystals coated with polylactic acid-polyethylene glycol (NCPPs), to be administered directly into brain parenchyma adjacent to the surgical cavity. We have repurposed pectin for use within the brain, showing in vitro and in vivo biocompatibility, bio-adhesion to mammalian brain and gelling at physiological brain calcium concentrations. Etoposide and olaparib NCPPs with high drug loading have shown in vitro stability and drug release over 120 h. Pluronic F127 stabilised NCPPs to ensure successful spraying, as determined by dynamic light scattering and transmission electron microscopy. Successful delivery of Cy5-labelled NCPPs was demonstrated in a large ex vivo mammalian brain, with NCPP present in the tissue surrounding the resection cavity. Our data collectively demonstrates the pre-clinical development of a novel localised delivery device based on a sprayable hydrogel containing therapeutic NCPPs, amenable for translation to intracranial surgical resection models for the treatment of malignant brain tumours.

Background and aimsEsophageal adenocarcinoma (EAC) is chemoresistant in the majority of cases. The tumor-promoting biology of cancer associated fibroblasts (CAF) make them a target for novel therapies. Phosphodiesterase type 5 inhibitors (PDE5i) have been shown to regulate the activated fibroblast phenotype in benign disease. We investigated the potential for CAF modulation in EAC using PDE5i to enhance the efficacy of chemotherapy.MethodsEAC fibroblasts were treated with PDE5i and phenotypic effects examined using immunoblotting, immunohistochemistry, gel contraction, transwell invasion, organotypics, single cell RNAseq and shotgun proteomics. The combination of PDE5i with standard-of-care chemotherapy (Epirubicin, 5-Fluorouracil and Cisplatin) was tested for safety and efficacy in validated near-patient model systems (3D tumor growth assays (3D-TGAs) and patient derived xenograft (PDX) mouse models).ResultsPDE5i treatment reduced α–SMA expression in CAFs by 50% (pConclusionPDE5 is a candidate for clinical trials to alter the fibroblast phenotype in esophageal cancer. We demonstrate the specificity of PDE5i for fibroblasts to prevent transdifferentiation and revert the CAF phenotype. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patientin vitroandin vivoPDX-based model systems.

Background Glioblastoma (GBM) is a highly aggressive brain tumor with rapid subclonal diversification, harboring molecular abnormalities that vary temporospatially, a contributor to therapy resistance. Fluorescence-guided neurosurgical resection utilizes the administration of 5-aminolevulinic acid (5-ALA) generating individually fluorescent tumor cells within a background population of non-neoplastic cells in the invasive tumor region. The aim of the study was to specifically isolate and interrogate the invasive GBM cell population using a novel 5-ALA-based method. Methods We have isolated the critical invasive GBM cell population by developing 5-ALA-based metabolic fluorescence-activated cell sorting. This allows purification and study of invasive cells from GBM without an overwhelming background “normal brain” signal to confound data. The population was studied using RNAseq, real-time PCR, and immunohistochemistry, with gene targets functionally interrogated on proliferation and migration assays using siRNA knockdown and known drug inhibitors. Results RNAseq analysis identifies specific genes such as SERPINE1 which is highly expressed in invasive GBM cells but at low levels in the surrounding normal brain parenchyma. siRNA knockdown and pharmacological inhibition with specific inhibitors of SERPINE1 reduced the capacity of GBM cells to invade in an in vitro assay. Rodent xenografts of 5-ALA-positive cells were established and serially transplanted, confirming tumorigenicity of the fluorescent patient-derived cells but not the 5-ALA-negative cells. Conclusions Identification of unique molecular features in the invasive GBM population offers hope for developing more efficacious targeted therapies compared to targeting the tumor core and for isolating tumor subpopulations based upon intrinsic metabolic properties.

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF-A), which consists of both the pro- and anti-angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF-A variants in IH progression and its spontaneous involution is unknown. Using patient-derived cells and surgical specimens, we showed that the relative level of VEGF-A165 b was increased in the involuting phase of IH and the relative change in VEGF-A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF-A165 b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF-A165 b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta-like ligand 4 (DLL4) expression. These results indicate that VEGF-A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte-derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF-A isoform-dependent manner, and in perivascular cells in a VEGF-independent manner. The contribution of VEGF-A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The versatility of the Passerini three component reaction (Passerini-3CR) is herein exploited for the synthesis of an amphiphilic diblock copolymer, which self-assembles into polymersomes. Carboxy-functionalized poly(ethylene glycol) methyl ether is reacted with AB-type bifunctional monomers and tert-butyl isocyanide in a single process via Passerini-3CR. The resultant diblock copolymer (P1) is obtained in good yield and molar mass dispersity and is well tolerated in model cell lines. The Passerini-3CR versatility and reproducibility are shown by the synthesis of P2, P3, and P4 copolymers. The ability of the Passerini P1 polymersomes to incorporate hydrophilic molecules is verified by loading doxorubicin hydrochloride in P1DOX polymersomes. The flexibility of the synthesis is further demonstrated by simple post-functionalization with a dye, Cyanine-5 (Cy5). The obtained P1-Cy5 polymersomes rapidly internalize in 2D cell monolayers and penetrate deep into 3D spheroids of MDA-MB-231 triple-negative breast cancer cells. P1-Cy5 polymersomes injected systemically in healthy mice are well tolerated and no visible adverse effects are seen under the conditions tested. These data demonstrate that new, biodegradable, biocompatible polymersomes having properties suitable for future use in drug delivery can be easily synthesized by the Passerini-3CR.

Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-&beta, ) signalling, and of &beta, catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the &beta, catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-&beta, and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients.

Design and implementation of innovative local drug delivery systems (DDS) may overcome current limitations in GBM treatment, such as the lack of therapeutic drug concentrations reaching residual GBM cells following surgery. Here we describe a novel DDS which utilises a bespoke mechanically engineered spray device, designed for safe surgical use, to deliver a mucoadhesive hydrogel containing chemotherapeutic nanoparticles (NPs) into the tumour resection margins. The overall aim is to spray a NP and polymer solution onto the resection cavity and potentially increase penetration of anti-cancer drugs within the 2 cm reoccurrence zone beyond the infiltrative margin. The mucoadhesive gel of choice, pectin, is currently used in other in vivo applications; however we have repurposed this for the brain. Pectin is biocompatible with GBM and human astrocyte cells in vitro and showed neither toxicity nor inflammation for up to 2 weeks upon orthotopic brain injection. Pectin is biodegradable in artificial CSF and is capable of being sprayed from the engineered device. A panel of polymeric, oil-based and polymer-coated NPs have been developed and optimised to maximise drug encapsulation of etoposide and olaparib as proof-of-concept for combination drug delivery. Etoposide/olaparib was chosen due to cytotoxicity from 5 GBM cell lines, including primary lines isolated from the invasive tumour margin (Mean IC50 of 1.1 µM and 8.3 µM respectively). The optimal NP/drug formulation (based on drug encapsulation, spray capability and bio-adhesiveness) will ultimately be assessed for tolerability and efficacy using orthotopic allograft and xenograft high-grade glioma models, including measurement of penetration of drug/nanoparticle in ex vivo murine and porcine brain using novel hybrid time-of-flight/Orbitrap TM secondary ion mass spectrometer (orbiSIMS) technology.

INTRODUCTION: The blood brain barrier is a critical limiting step to achieving therapeutic CNS drug concentrations. We have developed a poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG)-based Platform Technology to interstitially deliver multiple chemotherapy agents from a single thermo-setting paste, potentially filling the 3-week therapy gap that currently exists between surgery and commencement of radiotherapy in the treatment of high grade gliomas. Here we evaluate mouldable PLGA/PEG paste for combined temozolomide (TMZ) and etoposide (ETOP) delivery in an orthotopic high grade glioma model. METHODS: Drug release and preserved stability of the active TMZ molecule (AIC) was evaluated by fluoroscopic and LC-mass spectrometer based methods. In vitro cytotoxicity of released TMZ/ETOP was evaluated against high grade glioma cell lines and patient-derived primary cultures using metabolic assays. In vivo efficacy and overall survival were evaluated in the syngenic orthotopic 9L glioma rat model of intra cavity chemotherapy. RESULTS: TMZ and ETOP were released from PLGA/PEG alone or in combination over 2 weeks in vitro. Cytotoxicity of released drugs in vitro is comparable to directly applied agents, demonstrating the retained molecular integrity of TMZ/ETOP upon loading and releasing from PLGA/PEG. Both high (20% w/w TMZ / 50% w/w ETOP) and low (10% w/w TMZ / 25% w/w ETOP) doses were well tolerated in vivo, with no observable weight loss nor neurological deficits. Significant survival benefits from PLGA/PEG/TMZ/ETOP therapy were observed compared to surgery alone (49 vs. 14 days; p < 0.001), surgery with blank paste (49 vs. 14 days; p < 0.001) or surgery with daily oral TMZ (49 vs. 33 days; p < 0.004). CONCLUSIONS: This study demonstrates that interstitial delivery of TMZ/ETOP achieves significant glioma survival benefits. As such, PLGA/PEG paste applied to the post-surgical resection cavity offers a realistic opportunity for localised control of residual disease in pediatric and adult high grade gliomas.

The modulating effects of the orally active epidermal growth factor receptor-specific tyrosine kinase inhibitor ZD 1839 (‘Iressa’) on cell growth and signalling were evaluated in four ovarian cancer cell lines (PE01, PE04, SKOV-3, OVCAR-5) that express the epidermal growth factor receptor, and in A2780, which is epidermal growth factor receptor-negative. Transforming growth factor-α stimulated growth was completely inhibited by concentrations of ZD 1839 ⩾0.3 μM in the epidermal growth factor receptor-expressing cell lines, as were transforming growth factor-α stimulated phosphorylation of the epidermal growth factor receptor and downstream components of the MAP kinase and PI-3 kinase signalling cascades. Growth inhibition in the absence of added transforming growth factor-α was also observed which could be consistent with suppression of action of autocrine epidermal growth factor receptor-activating ligands by ZD 1839. In support of this, transforming growth factor-α, EGF and amphiregulin mRNAs were detected by RT–PCR in the epidermal growth factor receptor-expressing cell lines. ZD 1839 inhibited growth of the PE04 ovarian cancer xenograft at 200 mg kg−1 day−1. These data lend further support to the view that targeting the epidermal growth factor receptor in ovarian cancer could have therapeutic benefit. British Journal of Cancer (2002) 86, 456–462. DOI: 10.1038/sj/bjc/6600058 www.bjcancer.com © 2002 The Cancer Research Campaign

Introduction The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma. Methods Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model. Results Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour. Conclusions Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.

c-Raf is an essential component of the extracellular related kinase (ERK) signal transduction pathway. Immunohistochemical staining indicated that c-Raf was present in 49/53 ovarian adenocarcinomas investigated and high c-Raf expression correlated significantly with poor survival (P = 0.002). c-Raf protein was detected in 15 ovarian cancer cell lines. Antisense oligodeoxynucleotides (ODNs) (ISIS 5132 and ISIS 13650) reduced c-Raf protein levels and inhibited cell proliferation in vitro. Selectivity was demonstrated by the lack of effect of ISIS 5132 on A-Raf or ERK, while a random ODN produced only minor effects on growth and did not influence c-Raf expression. ISIS 5132 produced enhanced apoptosis and cells accumulated in S and G 2/M phases of the cell cycle. In vivo, ISIS 5132 inhibited growth of the s.c. SKOV-3 xenograft while a mismatch ODN had no effect. These data indicate that high levels of c-Raf expression may be important in ovarian cancer and use of antisense ODNs targeted to c-Raf could provide a strategy for the treatment of this disease. © 2001 Cancer Research Campaign http://www.bjcancer.com

Changes in tumour volume depend on the relative balance between cell proliferation and cell loss. However, these processes are not independent, and it remains unclear which is more important in tumour progression and regression. For example, the anti-oestrogen tamoxifen, a mainstay in the therapy of breast cancer, has both antiproliferative and pro-apoptotic actions, and their relative importance in clinical efficacy is unknown. Thus, using a model system based on oestrogen receptor (ER)-positive ZR-75-1 breast cancer xenografts, both increased apoptosis and reduced proliferation have been previously shown to occur within 7 days of tamoxifen therapy (Cameron DA, Ritchie AA, Langdon S, Anderson TJ, Miller WR. Tamoxifen induced apoptosis in ZR-75 breast cancer xenografts antedates tumour regression. Breast Cancer Res Treat 1997, 45, 99–107). In the present study, Gompertzian growth curves have been fitted to individual breast cancer xenografts. This demonstrates that the growth rate of the untreated tumours is directly dependent only on the mitotic rate (P

Twenty-four nude mice bearing MCF-7 breast cancer cells grown as xenografts and treated with tamoxifen (2.5 mg slow-release pellet) were studied for up to 35 days. Tumour size was measured in 2 dimensions at regular time-intervals and tumours were harvested on each of days 2, 4, 7, 14, 28 and 35 after the start of treatment. Control animals (8) received no treatment and the tumours were harvested after 0 or 35 days. Tumour sections were assessed for prevalence of apoptosis and mitosis and examined immunocytochemically for Ki(67)(MIB-1) and bcl-2 expression. Tumours increased in size during tamoxifen-treatment, but at a significantly slower rate (max. 2.6-fold) than in the untreated control animals; thus tumours not actually regressing may, nevertheless, be responding significantly to tamoxifen. MIB-1 and bcl-2 immunostaining and mitosis failed to show any consistent change over the period of study. Apoptosis, however, increased progressively and significantly to day-28 in tamoxifen-treated tumours, reaching approximately a 5-fold increase over day-0 values, then decreasing again to nearly 3-fold by day-35 (P= 0.0002). The apoptosis: mitosis ratio in treated tumours also increased to approximately 10-fold on day-28 over day-0 values, decreasing to nearly 4-fold by day-35 (P= 0.037). Within the treated group, apoptosis was significantly inversely correlated with both mitosis (R = -0.38, P= 0.03) and expression of bcl-2 (R = -0.48, P= 0.0056) and strongly positively correlated with both time on tamoxifen (R = +0.63, P= 0.0003) and the % inhibition of growth by tamoxifen (R = +0.58,P = 0.0012) in the 28 individual, treated tumours (estimated relative to the mean growth rate in the controls). The apoptosis: mitosis ratio was also inversely correlated with bcl-2 expression (R = -0.56, P= 0.0021) and positively correlated with both time on tamoxifen (R = +0.50, P= 0.0068) and % inhibition of growth (R = +0.56, P= 0.0019). In this hormone-sensitive tumour model for breast cancer, in which tamoxifen caused inhibition rather than regression, it was not possible to detect significant changes in the marker proteins Ki(67)and bcl-2, or in the prevalence of mitosis in relation to treatment; these factors may therefore not be accurate indices of response to tamoxifen in all situations. By contrast, however, tamoxifen induced a significant, early increase in the prevalence of apoptosis associated with inhibition of tumour growth and an inverse relationship in both mitosis and bcl-2 expression, suggesting that apoptosis may be an accurate and sensitive early marker of even a moderate response to tamoxifen.

ABSTRACT. EO9 is a novel bioreductive drug which has recently undergone extensive clinical evaluation. Its mechanism of action remains to be clearly defined. Antitumour activity of EO9 has been determined in 2 human colon cancer xenografts (HT-29 and BE) and 2 murine colon adenocarcinomas (MAC 16 and 26) after intratumoural injection of 250 μg of drug. Levels of the major bioreductive enzymes (DT-diaphorase, cytochrome P-450 reductase and cytochrome b5 reductase) were measured in tumours using cytochrome c reduction and menadione as the intermediate electron acceptor. There was no correlation between chemosensitivity (T/C: HT-29, 15%; BE, 27%; MAC 16, 33% and MAC 26, 60%) and enzyme activity (r2 = 0.47 for DT-diaphorase, r2 = 0.1 for cytochrome P-450 reductase and r2 = 0.52 for cytochrome b5 reductase). Drug metabolism was followed in vitro using tumour homogenates incubated under aerobic and anaerobic conditions. Four metabolites were identified by HPLC and characterised by UV-visible spectroscopy. With the exception of the hydrolysis product EO5A, all other metabolites appeared to be drug adducts. No correlation was observed between the kinetics of metabolite formation and antitumour activity. A good correlation (r2 = 0.86) was found with the rate of disappearance of parent drug and antitumour activity. These data show that the overall capacity of a tumour to metabolise EO9 is the most important determinant of antitumour activity rather than the expression of the major bioreductive enzymes and that the parent drug rather than a metabolite leads to the active form of the drug.

JONES, D. A., A. J. MacLELLAN, J. CUMMINGS, A. A. RITCHIE, S. P. LANGDON AND J. F. SMYTH. Processing of [ d-arg1,d -Phe5, d -Trp7,9,Leu11]substance P in xenograft bearing Nu/Nu mice. PEPTIDES 18(7) 1073–1077, 1997.—[ d -Arg1, d -Phe5, d -Trp7,9,Leu11]Substance P is a broad-spectrum neuropeptide growth factor antagonist that has exhibited in vitro activity against a range of human cancer cell lines. The fate of this compound in vivo following IP administration at 12 μg/g to nu/nu mice bearing the H69 small-cell lung cancer xenograft has been studied. Metabolism was confined to the C-terminus producing [ d -Arg1, d -Phe5, d -Trp7,9,Leu11]substance P acid and [ d -Arg1, d -Phe5, d -Trp7,9]substance P(1–10). The peptide had a long half-life in plasma (45.9 min) and became widely distributed among the tissues studied with the highest accumulation observed in the liver (AUC 1102 μg/g × min) and the lowest in the brain (5 μg/g × min). Uptake into the tumor xenograft was poor (AUC 189 μg/g × min); however, uptake into the lungs was much greater (AUC 507 μg/g × min), offering encouragement that therapeutic concentrations may be targeted to primary lung tumors.

Introduction\ud Polymer-based delivery systems offer innovative intra-cavity administration of drugs, with the potential to better target micro-deposits of cancer cells in brain parenchyma beyond the resected cavity. Here we evaluate clinical utility, toxicity and sustained drug release capability of a novel formulation of poly(lactic-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) microparticles.\ud \ud Methods\ud PLGA/PEG microparticle-based matrices were molded around an ex vivo brain pseudo-resection cavity and analyzed using magnetic resonance imaging and computerized tomography. In vitro toxicity of the polymer was assessed using tumor and endothelial cells and drug release from trichostatin A-, etoposide- and methotrexate-loaded matrices was determined. To verify activity of released agents, tumor cells were seeded onto drug-loaded matrices and viability assessed.\ud \ud Results\ud PLGA/PEG matrices can be molded around a pseudo-resection cavity wall with no polymer-related artifact on clinical scans. The polymer withstands fractionated radiotherapy, with no disruption of microparticle structure. No toxicity was evident when tumor or endothelial cells were grown on control matrices in vitro. Trichostatin A, etoposide and methotrexate were released from the matrices over a 3-4 week period in vitro and etoposide released over 3 days in vivo, with released agents retaining cytotoxic capabilities. PLGA/PEG microparticle-based matrices molded around a resection cavity wall are distinguishable in clinical scanning modalities. Matrices are non-toxic in vitro suggesting good biocompatibility in vivo. Active trichostatin A, etoposide and methotrexate can be incorporated and released gradually from matrices, with radiotherapy unlikely to interfere with release.\ud \ud Conclusion\ud The PLGA/PEG delivery system offers an innovative intra-cavity approach to administer chemotherapeutics for improved local control of malignant brain tumors.

The effect of Matrigel, a solubilised tissue basement membrane extract, has been investigated on the tumorigenicity of 3 breast (MCF-7, T47D and MDA.MB.231) and 5 ovarian [PEO1, PEO1 cDDPr, PEO4, PEO14 and OV(hyg)CAR3] carcinoma cell lines. In the absence of Matrigel, the PEO14 and MDA.MB.231 cell lines produced take rates of 30% and 50%, respectively, while the other cell lines either did not develop or only occasionally developed as tumours. With Matrigel, 100% take rates were achieved for 7 of the 8 cell lines (MCF-7, T47D, MDA.MB.231, PEO1, PEO1 cDDPr, PEO4 and PEO14); in the remaining cell line [OV(hyg)CAR3] 2/6 (33%) tumours grew. Xenografts established with Matrigel could be transferred into recipient animals and grown in the absence of Matrigel, suggesting that Matrigel is necessary only for initial establishment of tumours. Furthermore, cells which had been re-established from a T47D xenograft and then inoculated into mice without Matrigel showed a take rate greater than that of the original cell line but less than that of the xenograft. In conclusion, Matrigel has proven to be extremely useful in establishing a variety of cell lines as xenografts.

BACKGROUND 8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is undergoing phase I clinical trials as an anticancer drug. However, there is debate as to whether it is a prodrug for its 8-Cl-adenosine metabolite. DESIGN Pharmacokinetics, metabolism and tumour disposition studies have been performed in 7 breast cancer patients receiving continuous infusion (28 day) 8-Cl-cAMP (0.54 or 1.08 mg/kg/day) and tumour biopsies were obtained before and on the last day of infusion. Parallel studies were performed in nude mice bearing the HT29 human colon cancer xenograft after continuous infusion (7 day) of active drug doses (50 or 100 mg/kg/day). RESULTS Steady state plasma levels (Css) of 8-Cl-cAMP in patients ranged from 0.15-0.72 microM but 8-Cl-adenosine was not detected in plasma. In contrast, 8-Cl-cAMP was not detectable in 3 tumour biopsies but 8-Cl-adenosine was present in 2 samples at high concentrations (1.33 and 2.02 microM). In mice, Css of 8-Cl-cAMP ranged from 3.2-4.6 microM and 8-Cl adenosine was present in plasma only at the higher dose (100 mg/kg/day, peak concentration of 2.3 microM). In the HT29 xenograft, 8-Cl-cAMP levels were considerably lower than in plasma (0.37-1.22 microM) while 8-Cl-adenosine was present at 5.3-21.0 microM and 8-Cl-AMP was found at 11.3-35.7 microM. CONCLUSIONS The fate of 8-Cl-cAMP in human tumours is characterised by extensive metabolism to products which are not generally observed in plasma. These data raise the possibility that 8-Cl-cAMP is a prodrug for a product of its metabolism in human tumours.

Summary Background [Arg6, D-Trp7-9, NmePhe8)-Substance P (6–11) (codenamed antagonist G) represents the first broad spectrum antagonist of a number of neuropeptides shown to act as growth factors in small-cell lung cancer (SCLC) and is shortly to enter clinical trials. Design Pharmacokinetics, metabolism, tissue and tumour disposition have been studied in mice (nu/nu) bearing the NCI-H69 human SCLC xenograft after systemic drug administration at an active dose (45 mg/kg i.p.). Results The peptide exhibited a relatively long half life (28.9 min; clearance 45.6 ml/min/kg) and distributed widely (volume of distribution 1490 ml/kg). Marked accumulation of antagonist G (and its metabolites) was noted in the liver (AUC 5278 μg/g × min) and to a lesser extent the spleen (AUC 930 μg/g × min) but only low levels appeared to cross the blood brain barrier (AUC in brain, 20 μg/g × min) or be taken up into the heart (AUC 101 ng/g × min). Tumour uptake was intermediate in value out of the 7 tissues studied (AUC 195 (μg/g × min). Metabolism was restricted almost exclusively to the C terminal of the peptide producing 4 major products: M1, deamidated antagonist G; M2, H-Arg-DTrp-NmePhe-DTrp-Leu-OH, both of which retain growth factor antagonist activity; M3, a combination of oxidised antagonist G [Met11(O)] and oxidised deamidated antagonist G; and M4, a combination of H-Arg-DTrp-NmePhe-DTrp-OH and H-DTrp-NmePhe-DTrp-Leu-OH. Extensive bio-transformation to predominately Ml and M2 occurred in most tissues including the tumour where the parent peptide accounted for only 48.5% of the total. Conclusions Levels of antagonist G required to produce a small but significant effect on the growth of SCLC cell lines in vitro are in the region of 4–7 μM. Taking into account metabolites, a peak concentration of 4.1 μg/g (4.3 uM) was achieved in the H69 xenograft. These studies reveal a favourable preclinical pharmacology profile for antagonist G and offer hope that anticancer activity may be achievable in man.

Interferons (IFNs) augment the effect of some antitumor agents, including cis-diamminedichloroplatinum(II) (cDDP), in experimental systems. The effect of human recombinant interferon-alpha 2b (rIFN alpha) on the cDDP-dependent growth delay of a human non-small cell lung cancer established as a xenograft in nude mice (NX002) has been investigated. IFN (10(5) IU/mouse, s.c.) as a single agent had no effect on the growth of the xenograft. cDDP (4.2 mg/kg, i.p.) caused a specific growth delay of 0.42, and this delay was significantly enhanced (to 1.08) by concomitant dosing with the otherwise inactive IFN. Possible mechanisms for this supra-additive relationship between IFN and cDDP have been investigated: increased intratumoral accumulation of platinum was seen at late time points (maximally at 36 hr) during the pharmacokinetic beta-phase of cDDP elimination from the plasma of the nude mice. Tumor:plasma platinum concentration ratios at 36-48 hr indicated significantly increased accumulation of platinum in tumors from IFN-treated mice compared to controls (p0.05). Scheduling experiments suggest that this IFN-mediated effect can persist for 4 hr. These differences may account for the enhanced antitumor activity of cDDP when coadministered with IFN.

This paper presents results of the in vitro and in vivo effects of anti-oestrogens on the growth of human ovarian cancer cells. Tamoxifen and the "pure" anti-oestrogens, ICI 164,384 and ICI 182,780, inhibited the oestrogen-stimulated growth of the oestrogen receptor (ER)-positive PE04 and PE01 cell lines grown in culture, the latter two compounds being more potent than tamoxifen. In the absence of 17 beta-oestradiol (E2), tamoxifen, but not the pure anti-oestrogens, produced a small degree of growth stimulation in the PE01 and PE04 lines at concentrations between 10((7) and 10(-9) M. In contrast, growth of the ER-negative PE014 line was unaffected by E2 and all three anti-oestrogens. The effects of tamoxifen and ICI 182,780 on PE04 cells grown as xenografts in nude mice were also studied. Both anti-oestrogens produce significant growth inhibitory effects. These results indicate that ovarian carcinoma cells may be sensitive to anti-oestrogens in vitro and in vivo, and support the view that anti-oestrogens merit further clinical studies in patients with ER-positive tumours.

A human ovarian adenocarcinoma cell line (PE04) has been established as a xenograft in nude mice. In vitro, this cell line is estrogen receptor (ER)-positive and its growth is stimulated by 17 beta-estradiol at concentrations between 10(-12) and 10(-6) M. When xenografted, PE04 cells remain ER-positive and also possess progesterone receptors (PR); treatment with 17 beta-estradiol reduces the concentration of ER and increases levels of PR. Growth of the xenograft is reduced in ovariectomized animals while implantation of estrogen pellets also results in growth inhibition. Similar treatment with estrogen does not inhibit the ER-negative HOX 60 ovarian xenograft, and stimulates growth of the ER-positive ZR-75-I breast carcinoma xenograft. Serum measurements of 17 beta-estradiol confirm that ovariectomy reduces the level of 17 beta-estradiol while implantation of estrogen pellets results in raised levels of the hormone. Tamoxifen inhibits growth of the PE04 xenograft but not that of the HOX 60 xenograft, consistent with ER status. These results indicate that ER-positive PE04 ovarian cancer cells are sensitive to 17 beta-estradiol in vivo but that the response may be of a different type from the in vitro response. This lends further support to the concept that ovarian cancer may be hormone-sensitive and potentially responsive to endocrine therapy.

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation. Images Figure 1

The use of human tumor xenografts grown in immunodeficient animals as a model for human cancers is well established and their value depends on the extent to which their characteristics reflect the properties of a particular cancer in the clinical situation. For endocrine-sensitive tumors, the retention of hormone receptors in xenografts and their responsiveness to hormonal stimuli are essential criteria if they are to serve as appropriate models. Provided these criteria are met, such experimental systems allow detailed studies of the effects of hormones on hormone-sensitive tumor cells and the potential of endocrine therapies in an in vivo system (1).

Recent investigations into bioreductive anticancer drugs have focused on profiling reductase enzymes and relating their expression to therapeutic activity in an approach referred to as enzyme directed drug development. However, few studies have attempted to validate this approach in vivo and even less is known about how the expression of reductases relates quantitatively and qualitatively to metabolic activation. In the present study, the antitumour activity, pharmacokinetics and metabolism of mitomycin C (MMC) has been determined in vivo in two murine adenocarcinomas of the colon, MAC 16 (high DT-diaphorase activity) and MAC 26 (low DT-diaphorase activity) after intra-tumoural injection of drug. Over a broad range of drug concentrations (50-250 microg), MAC 16 proved to be consistently the more sensitive tumour (e.g. 75 microg of MMC, T/C 11% for MAC 16 and 31% for MAC 26). Higher levels of parent drug (peak concentration 103 microg/tumour compared to 58 microg/tumour) were maintained over 45 min in MAC 16 after which time clearance was rapid from both tumours. Four metabolites were detected in both tumours characteristic of different pathways of metabolism. However, by far the major metabolite was 2,7-diaminomitosene (2,7-DM), an accurate indicator of metabolic activation of MMC. Despite higher reductase levels and greater sensitivity to the drug, there was 4-fold less production of 2,7-DM in MAC 16. These results indicate a lack of a simple relationship in vivo between reductase expression and metabolic activation and suggest factors other than pharmacological determinants being responsible for the chemosensitivity of the MAC tumours to MMC.

8-Chloroadenosine-3',5'-monophosphate (8-ClcAMP) is a novel antitumour agent currently undergoing phase I clinical trials in several European centres. In this study, its antitumour activity against human tumour xenografts and its dependence on schedule were investigated. When administered by continuous infusion at doses of 100 or 50 mg/kg/day to nude mice bearing human tumour xenografts, 8-ClcAMP inhibited the growth of the HT 29 colorectal, ZR-75-1 breast, HOX 60 and PE04 ovarian and PANC-1 pancreatic carcinoma xenografts. However, these infusion schedules produced hypercalcaemia and severe weight loss. In an attempt to optimise antitumour activity and minimise toxicity, several other schedules were studied. In comparison with continuous administration of 8-ClcAMP at 50 mg/kg/day for 14 days which, although producing complete growth inhibition in the HOX 60 model, was associated with a marked body weight loss, schedules in which the infusion was interrupted (infusion on either days 0-4; 7-11 or days 0-2; 6-8) produced minimal weight loss but also reduced antitumour activity. However, co-administration of salmon calcitonin with continuous infusion of 8-ClcAMP prevented both hypercalcaemia and body weight loss in 3/6 animals while still producing marked inhibition of tumour growth. These data indicate that 8-ClcAMP has broad-spectrum antitumour activity and the major side-effect of hypercalcaemia may at least in part be ameliorated by the use of salmon calcitonin.

The ZR-75-1 ER positive breast cancer cell line, xenografted in female nude mice, has been used to determine the effect of tamoxifen on cell proliferation (as measured by mitosis) and cell death (as evidenced by apoptosis and necrosis). After 2 days treatment, there was a significant rise in apoptosis (p < 0.05), whereas a fall in mitosis was not apparent until 7 days (p < 0.05). Furthermore there was an increase in the apoptotic:mitotic ratio on day 7 (p < 0.05). These changes antedated tumour regression, which did not reach not significance until day 14. Tamoxifen did not increase necrosis (which significantly decreased in treated tumours once they had regressed (p < 0.01). In contrast tamoxifen treatment of xenografted MDA-MB-231 ER-negative breast cancer cells produced no significant effects on growth, apoptosis, or mitosis. This study presents clear evidence for tamoxifen inducing apoptosis in ZR-75-1 xenografts (but not MDA-MB-231 tumours). Since changes in apoptosis and mitosis antedate tumour regression, their assessment may provide the potential by which to predict tumour response to tamoxifen therapy.

Significant dose-related inhibition of growth of HT29 human colorectal cancer xenografts and ZR-75-1 breast cancer xenografts in immune-suppressed mice was induced by the cyclic AMP analogue, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cyclic AMP) when given by alzet mini-pumps over a 7-day period at doses of either 50 or 100 mg/kg/day. Levels and types of cyclic AMP binding proteins were measured by ligand binding and photoaffinity labelling, respectively, in tumours harvested at the end of the treatment period. Compared with levels in tumours from control animals, values of tumour cyclic AMP binding proteins from treated animals were significantly reduced. These effects were associated with an apparent modulation of the types of cyclic AMP binding proteins, 8-Cl-cyclic AMP-treated xenografts displaying a reduced ratio of RI/RII isoforms compared with untreated control tumours.

The novel anticancer compound GR63178A is being evaluated in the clinic, having demonstrated activity against a wide range of experimental tumour systems in animals without significant toxic side-effects being apparent. In this work, we have demonstrated significant antitumour action of this compound against one murine colon cancer model (colon 38 tumour in BDF-1 mice, specific growth delay = 1.2) when given at 10 mg/kg over 21 consecutive days and in contrast shown minimal sensitivity of another similar murine colon adenocarcinoma, MAC 26, in NMRI mice with the same dose regime. We investigated the disposition of both the parent drug and the 9-OH metabolite (GR54374X) in plasma, tissues and tumours, using solid phase extraction followed by reversed-phase high performance liquid chromatography. Although plasma clearance profiles of GR63178A were similar, significant differences were seen in the disposition of the drug to major organs in two mouse strains. Noteably, the liver and kidneys of the sensitive model had higher levels of parent drug and 9-OH metabolite at both 30 min and 4 h post-injection. However, this was not apparent in the tumours themselves, and the levels of 9-OH metabolite were lower in the plasma and higher in the urine of the sensitive mice, indicating possible rapid renal clearance of this compound. Neither GR63178A nor GR54374X proved cytotoxic in in vitro experiments. The data presented here have revealed considerable variation in drug handling by these two mouse strains, but this did not produce different levels of either parent drug or GR54374X in the tumours, which are the presumed targets, suggesting that differences in disposition are probably not responsible for the different sensitivities of the two tumours. Other possible explanations include the production of a hitherto undetected ultimate cytotoxic metabolite in the sensitive, but not in the resistant, mouse/tumour combination, or differences in inherent tumour sensitivity, or in host-mediated effects. These possibilities are discussed.