Timo L M ten Hagen

Erasmus MC, Rotterdam, South Holland, Netherlands

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Publications (161)712.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Phosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influences the phosphorylation of signaling pathway mediators involved in cancer and is thus postulated to be a tumor-promoting enzyme, but neither unequivocal clinical evidence nor convincing mechanistic actions for a role of LMWPTP have been identified. In the present study, we show that LMWPTP expression is not only significantly increased in colorectal cancer (CRC), but also follows a step-wise increase in different levels of dysplasia. Chemical inhibition of LMWPTP significantly reduces CRC growth. Furthermore, downregulation of LMWPTP in CRC leads to a reduced migration ability in both 2D- and 3D-migration assays, and sensitizes tumor cells to the chemotherapeutic agent 5-FU. In conclusion, this study shows that LMWPTP is not only overexpressed in colorectal cancer, but it is correlated with the malignant potential of this cancer, suggesting that this phosphatase may act as a predictive biomaker of CRC stage and represents a rational novel target in the treatment of this disease.
    Oncotarget 03/2015; · 6.63 Impact Factor
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    ABSTRACT: Further understanding of the molecular biology and pathogenesis of hepatocellular carcinoma (HCC) is crucial for future therapeutic development. SMAD4, recognized as an important tumor suppressor, is a central mediator of transforming growth factor beta (TGFB) and bone morphogenetic protein (BMP) signaling. This study investigated the role of SMAD4 in HCC. Nuclear localization of SMAD4 was observed in a cohort of 140 HCC patients using tissue microarray. HCC cell lines were used for functional assay in vitro and in immune-deficient mice. Nuclear SMAD4 levels were significantly increased in patient HCC tumors as compared with adjacent tissues. Knockdown of SMAD4 significantly reduced the efficiency of colony formation and migratory capacity of HCC cells in vitro and was incompatible with HCC tumor initiation and growth in mice. Knockdown of SMAD4 partially conferred resistance to the anti-growth effects of BMP ligand in HCC cells. Importantly, simultaneous elevation of SMAD4 and phosphorylated SMAD2/3 is significantly associated with poor patient outcome after surgery. Although high levels of SMAD4 can also mediate an antitumor function by coupling with phosphorylated SMAD1/5/8, this signaling, however, is absent in majority of our HCC patients. In conclusion, this study revealed a highly non-canonical tumor-promoting function of SMAD4 in HCC. The drastic elevation of nuclear SMAD4 in sub-population of HCC tumors highlights its potential as an outcome predictor for patient stratification and a target for personalized therapeutic development.Oncogene advance online publication, 22 December 2014; doi:10.1038/onc.2014.425.
    Oncogene 12/2014; DOI:10.1038/onc.2014.425 · 8.56 Impact Factor
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    ABSTRACT: Purpose To improve therapeutic activity of mitoxantrone (MTO)-based chemotherapy by reducing toxicity through encapsulation in nanoliposomes and enhancing intracellular drug delivery using short-chain sphingolipid (SCS) mediated tumor cell membrane permeabilization. Methods Standard (MTOL) and nanoliposomes enriched with the SCS, C8-Glucosylceramide or C8-Galactosylceramide (SCS-MTOL) were loaded by a transmembrane ammonium sulphate gradient and characterized by DLS and cryo-TEM. Intracellular MTO delivery was measured by flow cytometry and imaged by fluorescence microscopy. In vitro cytotoxicity was studied in breast carcinoma cell lines. Additionally, live cell confocal microscopy addressed the drug delivery mechanism by following the intracellular fate of the nanoliposomes, the SCS and MTO. Intratumoral MTO localization in relation to CD31-positive tumor vessels and CD11b positive cells was studied in an orthotopic MCF-7 breast cancer xenograft. Results Stable SCS-MTOL were developed increasing MTO delivery and cytotoxicity to tumor cells compared to standard MTOL. This effect was much less pronounced in normal cells. The drug delivery mechanism involved a transfer of SCS to the cell membrane, independently of drug transfer and not involving nanoliposome internalization. MTO was detected intratumorally upon MTOL and SCS-MTOL treatment, but not after free MTO, suggesting an important improvement in tumor drug delivery by nanoliposomal formulation. Nanoliposomal MTO delivery and cellular uptake was heterogeneous throughout the tumor and clearly correlated with CD31-positive tumor vessels. Yet, MTO uptake by CD11b positive cells in tumor stroma was minor. Conclusions Nanoliposomal encapsulation improves intratumoral MTO delivery over free drug. Liposome bilayer-incorporated SCS preferentially permeabilize tumor cell membranes enhancing intracellular MTO delivery.
    Pharmaceutical Research 10/2014; 32(4). DOI:10.1007/s11095-014-1539-4 · 3.95 Impact Factor
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    ABSTRACT: Liposomal nanoparticles can circumvent toxicity of encapsulated chemotherapeutic drugs, but fall short in tumor-specific and efficient intracellular drug delivery. To overcome these shortcomings, we designed a multifunctional dual targeted, heat-responsive nanocarrier encapsulating doxorubicin (Dox) as a chemotherapeutic content. Dox-loaded cationic thermosensitive liposomes (Dox-CTSL) carry targeting functions addressing tumor cells and tumor vasculature and have a heat-responsive lipid bilayer. Targeted Dox-CTSL demonstrated superior uptake by and toxicity to different tumor cell lines and endothelial cells compared to non-targeted TSL. Heat triggered intracellular Dox release in acidic cell compartments was visualized as fluorescent Dox nanobursts by live cell confocal microscopy. In vivo, using high resolution intravital microscopy, we demonstrated that Dox-CTSL upon an external heat-trigger delivered 3-fold higher Dox quantity to tumors than TSL. Dox-CTSL bound specifically to tumor vasculature, which in combination with the heat-triggered drug release caused significant tumor vessel damage, which was not observed when non-targeted TSL were administered. Therefore, Dox-CTSL have strong potency to increase drug efficacy due to targeted delivery and heat-triggered drug release in tumors.
    Journal of Controlled Release 08/2014; 195. DOI:10.1016/j.jconrel.2014.07.058 · 7.26 Impact Factor
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    ABSTRACT: Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells. We synthesized two conjugates containing the somatostatin analogue [Tyr3]-octreotate and Chlorin e6 (Ce6): Ce6-K3-[Tyr3]-octreotate (1) and Ce6-[Tyr3]-octreotate-K3-[Tyr3]-octreotate (2). Investigation of the uptake and photodynamic activity of conjugates in-vitro in human erythroleukemic K562 cells showed that conjugation of [Tyr3]-octreotate with Ce6 in conjugate 1 enhances uptake (by a factor 2) in cells over-expressing sst2 compared to wild-type cells. Co-treatment with excess free Octreotide abrogated the phototoxicity of conjugate 1 indicative of a specific sst2-mediated effect. In contrast conjugate 2 showed no receptor-mediated effect due to its high hydrophobicity. When compared with un-conjugated Ce6, the PDT activity of conjugate 1 was lower. However, it showed higher photostability which may compensate for its lower phototoxicity. Intra-vital fluorescence pharmacokinetic studies of conjugate 1 in rat skin-fold observation chambers transplanted with sst2+ AR42J acinar pancreas tumors showed significantly different uptake profiles compared to free Ce6. Co-treatment with free Octreotide significantly reduced conjugate uptake in tumor tissue (by a factor 4) as well as in the chamber neo-vasculature. These results show that conjugate 1 might have potential as an in-vivo sst2 targeting photosensitizer conjugate.
    PLoS ONE 08/2014; 9(8):e104448. DOI:10.1371/journal.pone.0104448 · 3.53 Impact Factor
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    ABSTRACT: Abstract Aims: With the aim to improve peptide receptor radionuclide therapy effects in patients with gastroenteropancreatic neuroendocrine tumor (GEPNET) liver metastases we explored the effect of intra-arterial (IA) administration of [(111)In-DTPA]octreotide ((111)In-DTPAOC) on tumor uptake in an animal model and in a patient study. Methods: Preclinical study: After administering (111)In-DTPAOC intra-venously (IV) or IA, biodistribution studies were performed in rats with a hepatic somatostatin receptor subtype 2 (sst2)-positive tumor. Clinical study: 3 patients with neuroendocrine liver metastases were injected twice with (111)In-DTPAOC. The first injection was given IV, and 2 weeks later, the second was injected IA (hepatic artery). Planar images of the abdomen were made up to 72 hours after injection. Blood samples were taken and urine was collected. Pharmacokinetic modeling was performed on the IV and IA data of the same patient. Based on this model, additional (177)Lu dosimetry calculations for IV and IA administrations were performed. Results: The preclinical study showed a two-fold higher (111)In-DTPAOC tumor uptake after IA administration than after IV injection. Patient data showed a large variability in radioactivity increment in liver metastases after IA administration compared with IV administration. Renal radioactivity was not significantly lower after IA administration; (177)Lu dosimetry simulations in 1 patient using a maximum kidney radiation dose of 23 Gy showed IA administration resulted in a mean increase in tumor radiation dose of 2.9-fold. Conclusion: Preclinical and clinical data both indicate that IA administration of radiolabeled somatostatin analogs via the hepatic artery can significantly increase radionuclide uptake in GEPNET, sst2-positive, liver metastases up to 72 hours postinjection, although the effect of IA administration can differ between patients.
    Cancer Biotherapy & Radiopharmaceuticals 05/2014; 29(4):179-87. DOI:10.1089/cbr.2013.1552 · 1.44 Impact Factor
  • Journal of Hepatology 04/2014; 60(1):S87-S88. DOI:10.1016/S0168-8278(14)60226-9 · 10.40 Impact Factor
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    ABSTRACT: The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (FcγRI) and the low-affinity IgG-binding Fc receptor (FcγRIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity.
    The Journal of clinical investigation 01/2014; DOI:10.1172/JCI66776 · 15.39 Impact Factor
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    ABSTRACT: Liposomal chemotherapy brings the advantage of minimizing systemic toxicity towards healthy organs and tissues, while has the drawbacks of limited nanoparticle accumulation and low drug bioavailability at targeted tumors. The aim of our study is to apply a clinically available mild hyperthermia (HT) treatment with thermosensitive liposomes (TSL) to tackle both issues A two-step HT approach was combined with systemic administration of doxorubicin (Dox) TSL, in a first step to maximize nanoparticle accumulation in tumors and second step to actively trigger Dox release. The therapeutic activity of the two-step approach was compared to a one-step HT triggering intravascular Dox release from circulating TSL. Whereas the intravascular drug release approach requires fast releasing Dox-TSL (Dox-fTSL), the TSL formulation used in the two-step approach is fine-tuned to prolong Dox retention at physiological temperature in circulation, while releasing their drug content at mild HT at a slower rate (Dox-sTSL). Cytotoxicity assays show that a first-step HT at 41°C for 1hour causes no drug resistance on murine BFS-1 sarcoma, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) towards subsequent exposure to Dox. However, HT sensitizes HUVEC towards Dox at higher concentrations (10-100μM). After 2hours of intratumoral Dox-TSL accumulation, HT at 42°C for 1hour was applied to trigger Dox release from Dox-sTSL. Quantification of intratumoral Dox accumulation revealed that the two-step HT approach increased TSL accumulation and Dox bioavailability reaching levels comparable to the intravascular release approach. The two-step HT in combination with Dox-sTSL delayed tumor growth for 12days compared to PBS group, however, was less effective compared to intravascular Dox release from Dox-fTSL using one-step HT. The two-step approach focuses on interstitial drug release upon mild HT, instead of intravascular drug release. This novel two-step approach represents an attractive alternative for the treatment of large and deep seated tumors, which are difficult to heat precisely and require loco-regional HT of the tumor area and accumulated Dox-sTSL therein to obtain a precise intratumoral drug delivery.
    Journal of Controlled Release 11/2013; 174. DOI:10.1016/j.jconrel.2013.11.012 · 7.63 Impact Factor
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    ABSTRACT: We demonstrated previously that the administration of tumor necrosis factor alpha (TNF-α) for the treatment of solid tumors enhanced the response to chemotherapy by augmenting intratumoral drug accumulation. TNF-α changes the integrity of the endothelial cell monolayer in combination with interferon gamma (IFN-γ), which is further enhanced by the addition of peripheral blood mononuclear cells (PBMCs). The improved effect of PBMCs was mostly induced by the endogenous production of interleukin-1beta (IL-1ß) after TNF-α stimulation. In the current study, we demonstrate that exposing endothelial cells to TNF-α and PBMCs mediates the loss of vascular endothelial (VE)-cadherin, an important adherens junction protein for maintaining endothelial integrity, through endogenous IL-1ß. This loss increases permeability of the endothelial layer, thereby explaining the augmented passage of chemotherapeutics into the tumor. Human umbilical vein endothelial cells were exposed to TNF-α, IFN-γ, PBMCs, or IL-1ß, and the effects on the endothelial integrity were assessed by morphological changes and permeability changes with the use of fluorescein isothiocyanate-labeled bovine serum albumin flux. The loss of VE-cadherin was assessed using immunofluorescence, western blotting, and polymerase chain reaction. Incubating endothelial cells with TNF-α, IFN-γ, and PBMCs increased cell elongation, gap formation, and subsequently the permeability of fluorescein isothiocyanate-labeled bovine serum albumin compared with control or TNF-α and IFN-γ-treated cells (P < .05). When PBMCs were replaced with IL-1ß, identical changes were observed. These changes in integrity were associated with a loss of VE-cadherin at the membrane. We conclude that VE-cadherin is lost at the membrane when endothelial cells are exposed to TNF-α, IFN-γ, and PBMCs, which results in loss of integrity. IL-1ß can mimic the effects of PBMCs, indicating a dominant role of endogenously produced IL-1ß in this process.
    Surgery 10/2013; 155(3). DOI:10.1016/j.surg.2013.10.019 · 3.37 Impact Factor
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    ABSTRACT: The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.
    Angiogenesis 09/2013; DOI:10.1007/s10456-013-9385-2 · 4.41 Impact Factor
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    ABSTRACT: Doxil, also known as Caelyx, is an established liposomal formulation of doxorubicin used for the treatment of ovarian cancer, sarcoma and multiple myeloma. While showing reduced doxorubicin related toxicity, Doxil does not greatly improve clinical outcome. To become biologically active, doxorubicin needs to be released from its carrier. Uptake and breakdown of the liposomal carrier and subsequent doxorubicin release is not fully understood and in this study we explored the hypothesis that Doxil is taken up by tumor cells and slowly degraded intracellularly. We investigated the kinetics of liposomal doxorubicin (Doxil) in vitro as well as in vivo by measuring cytotoxic effect, intracellular bioavailability and fate of the carrier and its content. To prevent fixation artifacts we applied live cell imaging in vitro and intravital microscopy in vivo. Within 8h after administration of free doxorubicin, 26% of the drug translocated to the nucleus and when reaching a specific concentration killed the cell. Unlike free doxorubicin, only 0.4% of the doxorubicin added as liposomal formulation entered the nucleus. Looking at the kinetics, we observed a build-up of nuclear doxorubicin within minutes of adding free doxorubicin. This was in contrast to Doxil showing slow translocation of doxorubicin to the nucleus and apparent accumulation in the cytoplasm. Observations made with time-lapse live cell imaging as well as in vivo intravital microscopy revealed the liposomal carrier colocalizing with doxorubicin in the cytoplasm. We also demonstrated the sequestering of liposomal doxorubicin in the lysosomal compartment resulting in limited delivery to the nucleus. This entrapment makes the bioavailable concentration of Doxil-delivered doxorubicin significantly lower and therefore ineffective as compared to free doxorubicin in killing tumor cells.
    Journal of Controlled Release 09/2013; DOI:10.1016/j.jconrel.2013.08.034 · 7.63 Impact Factor
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    ABSTRACT: Experimental evidence supports an association between heterogeneity in tumor perfusion and response to chemotherapy/radiotherapy, disease progression and malignancy. Therefore, changes in tumor perfusion may be used to assess early effects of tumor treatment. However, evaluating changes in tumor perfusion during treatment is complicated by extensive changes in tumor type, size, shape and appearance. Therefore, this study assesses the regional heterogeneity of tumors by dynamic contrast-enhanced MRI (DCE-MRI) and evaluates changes in response to isolated limb perfusion (ILP) with tumor necrosis factor alpha and melphalan. Data were acquired in an experimental cancer model, using a macromolecular contrast medium, albumin-(Gd-DTPA)45. Small fragments of BN 175 (a soft-tissue sarcoma) were implanted in eight brown Norway rats. MRI of five drug-treated and three sham-treated rats was performed at baseline and 1 h after ILP intervention. Properly co-registered baseline and follow-up DCE-MRI were used to estimate the volume transfer constant (K(trans) ) pharmacokinetic maps. The regional heterogeneity was estimated in 16 tumor sectors and presented in cumulative map-volume histograms. On average, ILP-treated tumors showed a decrease in regional heterogeneity on the histograms. This study shows that heterogenic changes in regional tumor perfusion, estimated using DCE-MRI pharmacokinetic maps, can be measured and used to assess the short-term effects of a potentially curative treatment on the tumor microvasculature in an experimental soft-tissue sarcoma model. Copyright © 2013 John Wiley & Sons, Ltd.
    Contrast Media & Molecular Imaging 07/2013; 8(4):340-9. DOI:10.1002/cmmi.1528 · 2.87 Impact Factor
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    ABSTRACT: Whereas aberrant activation of canonical Wnt/β-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling datasets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed pro-tumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion, and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.
    Carcinogenesis 06/2013; DOI:10.1093/carcin/bgt215 · 5.27 Impact Factor
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    ABSTRACT: Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor-α (TNF) is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline two hours before and three hours after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a 3- to 7- fold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting. © 2012 Wiley Periodicals, Inc.
    International Journal of Cancer 06/2013; 132(11). DOI:10.1002/ijc.27940 · 6.20 Impact Factor
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    ABSTRACT: PURPOSE: To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C8-glucosylceramide (C8-GluCer), C8-galactosylceramide (C8-GalCer) or C8-lactosylceramide (C8-LacCer). METHODS: DoxNL enriched with C8-GluCer or C8-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells. RESULTS: C8-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C8-GluCer or C8-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox. CONCLUSION: C8-GluCer or C8-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.
    Pharmaceutical Research 05/2013; 30(7). DOI:10.1007/s11095-013-1031-6 · 4.74 Impact Factor
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    ABSTRACT: Liposome mediated anticancer drug delivery has the advantage of reducing cytotoxicity in healthy tissues. However, undesired slow drug release impedes the therapeutic efficacy of clinically applied PEG-liposomal doxorubicin (Dox). The aim of this study is to combine stealth thermosensitive liposomes (TSL) and local mild hyperthermia (HT) to increase bioavailable Dox levels in tumors. Dox was encapsulated in stealth TSL (~80 nm) with optimized PEG concentration in the membrane, and compared with lysolipid-based Dox-LTSL for in vitro stability, release kinetics, and in vivo tumor growth control. In vitro cytotoxicity of Dox-TSL against murine BFS-1 sarcoma, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) under normothermia (37°C) and HT (42°C) was compared with non-encapsulated Dox. In vitro Dox uptake in nuclei was imaged in BLM and HUVEC. In vivo intravascular Dox release from TSL in BFS-1 tumors under local mild HT in dorsal skin flap window chamber models was captured by intravital confocal microscopy. Intravascular Dox-TSL release kinetics, penetration depth and interstitial Dox density were subjected to quantitative image analysis. Systemic Dox-TSL administration in combination with local mild HT on subcutaneous tumor growth control was compared to Dox-LTSL plus local mild HT. Dox-TSL were stable at 37°C, while released over 95% Dox within 1 minute in 90% serum at 42°C. Dox-TSL demonstrated efficient in vivo intratumoral Dox release under local mild HT, followed by significant Dox uptake by tumor and tumor vascular endothelial cells. Dox-TSL plus mild HT showed improved tumor growth control over Dox-LTSL plus mild HT. Survival after a single treatment of Dox-TSL plus mild HT was 67%, while survival after Dox-LTSL plus mild HT was 22%. This combination of Dox-TSL and local mild HT offers promising clinical opportunities to improve liposomal Dox delivery to solid tumors.
    Journal of Controlled Release 03/2013; DOI:10.1016/j.jconrel.2013.03.011 · 7.63 Impact Factor
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    ABSTRACT: Accumulation of nanoparticles in solid tumors depends on their extravasation. However, vascular permeability is very heterogeneous within a tumor and among different tumor types, hampering efficient delivery. Local hyperthermia at a tumor can improve nanoparticle delivery by increasing tumor vasculature permeability, perfusion and interstitial fluid flow. The aim of this study is to investigate hyperthermia conditions required to improve tumor vasculature permeability, subsequent liposome extravasation and interstitial penetration in 4 tumor models. Tumors are implanted in dorsal skin flap window chambers and observed for liposome (~85nm) accumulation by intravital confocal microscopy. Local hyperthermia at 41°C for 30minutes initiates liposome extravasation through permeable tumor vasculature in all 4 tumor models. A further increase in nanoparticle extravasation occurs while continuing heating to 1hour, which is a clinically relevant duration. After hyperthermia, tumor vasculature remains permeable for 8hours. We visualize gaps in the endothelial lining of up to 10μm induced by HT. Liposomes extravasate through these gaps and penetrate into the interstitial space to at least 27.5μm in radius from vessel walls. Whole body optical imaging confirms HT induced extravasation while liposome extravasation was absent at normothermia. In conclusion, thermal dose of 41°C for 1hour is effective to induce long-lasting permeable tumor vasculature for liposome extravasation and interstitial penetration. These findings hold promise for improved intratumoral drug delivery upon application of local mild hyperthermia prior to administration of nanoparticle-based drug delivery systems.
    Journal of Controlled Release 02/2013; DOI:10.1016/j.jconrel.2013.01.026 · 7.63 Impact Factor
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    ABSTRACT: Inactivation of the tumor suppressor E-cadherin is an important event during breast tumorigenesis, as its decreased expression is linked to aggressiveness and metastasis. However, the relationship between the different modes of E-cadherin inactivation (mutation versus promotor hypermethylation) and breast cancer cell behavior is incompletely understood. The high correlation between E-cadherin inactivation status and cell morphology in vitro suggests different biological roles for the two inactivation modes during breast tumorigenesis. Because E-cadherin has been linked to cell invasion and metastasis, and cell motility is a crucial prerequisite to form metastases, we here compared the cell motility capacities of breast cancer cell lines with known E-cadherin status. Using barrier migration assays and time-lapse microscopy, we analyzed the migratory capacity of nine well-characterized human breast cancer cell lines (MDA-MB-231, MCF-7, T47D, BT549, MPE600, CAMA-1, SUM159PT, SUM52PE, and SK-BR-3). This subset was chosen based on E-cadherin gene status (wild-type, mutated, and promotor hypermethylated): three cell lines of each group. In addition, cell proliferation assays were performed for all conditions, to dissect migratory from proliferative effects. In this study, we demonstrate an overt association between the mode of E-cadherin inactivation and cell migration. Promotor hypermethylated E-cadherin cell lines showed a higher migration capacity, while cell lines with mutated E-cadherin were less motile compared to wild-type E-cadherin cell lines. Migration induction by fibronectin and basic fibroblast growth factor did not alter the cell motility association differences. Cell proliferation assays showed that the associations found were not caused by proliferation differences. Inhibition and overexpression of E-cadherin as well as DNA demethylation confirmed the relationship between E-cadherin and breast cancer cell motility. Our results demonstrate an association between the mode of E-cadherin inactivation and migration of breast cancer cells, which justifies more detailed research on the role of E-cadherin inactivation in cell migration and metastasis.
    Breast Cancer Research and Treatment 09/2012; 136(2). DOI:10.1007/s10549-012-2261-8 · 4.47 Impact Factor
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    ABSTRACT: Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream. Cancer Res; 72(21); 1-10. ©2012 AACR.
    Cancer Research 09/2012; 72(21). DOI:10.1158/0008-5472.CAN-12-1683 · 9.28 Impact Factor

Publication Stats

3k Citations
712.91 Total Impact Points

Institutions

  • 1997–2014
    • Erasmus MC
      • • Department of Surgery
      • • Department of Oncological Surgery
      Rotterdam, South Holland, Netherlands
  • 1995–2014
    • Erasmus Universiteit Rotterdam
      • • Department of Surgery
      • • Department of Surgical Oncology
      Rotterdam, South Holland, Netherlands
  • 2010
    • Radboud University Nijmegen
      Nymegen, Gelderland, Netherlands
  • 2009
    • Attikon University Hospital
      Athínai, Attica, Greece
  • 2005
    • University of Leuven
      Louvain, Flemish, Belgium
  • 2002
    • Loma Linda University
      • Department of Radiation Medicine
      Loma Linda, CA, United States
  • 2000
    • Hong Kong Red Cross Blood Transfusion Service
      Hong Kong, Hong Kong
  • 1999
    • University of Antwerp
      Antwerpen, Flemish, Belgium