Timo L M ten Hagen

Erasmus MC, Rotterdam, South Holland, Netherlands

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Publications (161)749.7 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: To develop RGD-targeted thermosensitive liposomes with increased tumor retention, improving drug release efficiency upon mild hyperthermia (HT) in both tumor and angiogenic endothelial cells. Standard termosensitive liposomes (TSL) and TSL containing a cyclic Arg-Gly-Asp (cRGD) pentapeptide with the sequence Arg-Cys-D-Phe-Asp-Gly (RGDf[N-Met]C) were synthetized, loaded with Dox and characterized. Temperature- and time-dependent drug release profiles were assessed by fluorometry. Intracellular Dox delivery was studied by flow cytometry and confocal microscopy. Cytotoxic effect of TSL and RGD-TSL was studied on B16Bl6 melanoma, B16F10 melanoma and HUVEC. Intravital microscopy was performed on B16Bl6 tumors implanted in dorsal-skin fold window-bearing mice. Pharmacokinetic and biodistribution of Dox-TSL and Dox-RGD-TSL were followed in B16Bl6 tumor bearing mice upon normothermia or initial hyperthermia conditions. DLS and cryo-TEM revealed particle homogeneity and size of around 85 nm. Doxorubicin loading efficiency was >95%as assessed by spectrofluorometry. Flow cytometry and confocal microscopy showed a specific uptake of RGD-TSL by melanoma and endothelial cells when compared to TSL and an increased doxorubicin delivery. High resolution intravital microscopy demonstrated specific accumulation of RGD-TSL to the tumor vasculature. Moreover, application of hyperthermia resulted in massive drug release from RGD-TSL. Biodistribution studies showed that initial hyperthermia increases Dox uptake in tumors from TSL and RGD-TSL. RGD-TSL have potency to increase drug efficacy due to higher uptake by tumor and angiogenic endothelial cells in combination with heat-triggered drug release.
    Pharmaceutical Research 07/2015; DOI:10.1007/s11095-015-1746-7 · 3.42 Impact Factor
  • Asha M Das · Alexander M M Eggermont · Timo L M Ten Hagen
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    ABSTRACT: Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier-based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cells of interest are seeded around this barrier, and after the formation of a peripheral monolayer the barrier is removed and migration into the cell-free area is monitored. This assay is highly reproducible and convenient to perform, and it allows the deduction of several parameters of migration, including total and effective migration, velocity and cell polarization. An advantage of this assay over the conventional scratch assay is that the cells move over an unaltered and virgin surface, and thus the effect of matrix components on cell migration can be studied. In addition, the cells are not harmed at the onset of the assay. Through computer automation, four individual barrier assays can be monitored at the same time. The procedure can be used in a 12-well standard plate allowing higher throughput, or it can be modified to perform invasion assays. The basic procedure takes 2-3 d to complete.
    Nature Protocol 06/2015; 10(6):904-915. DOI:10.1038/nprot.2015.056 · 9.67 Impact Factor
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    ABSTRACT: Oxaliplatin (L-OH), a platinum derivative with good tolerability is currently combined with Cetuximab (CTX), a monoclonal antibody (mAb), for the treatment of certain (wild-type KRAS) metastatic colorectal cancer (CRC) expressing epidermal growth factor receptor (EGFR). Improvement of L-OH pharmacokinetics (PK) can be provided by its encapsulation into liposomes, allowing a more selective accumulation and delivery to the tumor. Here, we aim to associate both agents in a novel liposomal targeted therapy by linking CTX to the drug-loaded liposomes. These EGFR-targeted liposomes potentially combine the therapeutic activity and selectivity of CTX with tumor-cell delivery of L-OH in a single therapeutic approach. L-OH liposomes carrying whole CTX or CTX-Fab' fragments on their surface were designed and characterized. Their functionality was tested in vitro using four human CRC cell lines, expressing different levels of EGFR to investigate the role of CTX-EGFR interactions in the cellular binding and uptake of the nanocarriers and encapsulated drug. Next, those formulations were evaluated in vivo in a colorectal cancer xenograft model with regard to tumor drug accumulation, toxicity and therapeutic activity. In EGFR-overexpressing cell lines, intracellular drug delivery by targeted liposomes increased with receptor density reaching up to 3-fold higher levels than with non-targeted liposomes. Receptor specific uptake was demonstrated by competition with free CTX, which reduced internalization to levels similar to non-targeted liposomes. In a CRC xenograft model, drug delivery was strongly enhanced upon treatment with targeted formulations. Liposomes conjugated with monovalent CTX-Fab' fragments showed superior drug accumulation in tumor tissue (2916.0 ± 507.84 ng/g) compared to CTX liposomes (1546.02 ± 362.41 ng/g) or non-targeted liposomes (891.06 ± 155.1 ng/g). Concomitantly, CTX-Fab' targeted L-OH liposomes outperformed CTX-liposomes, which on its turn was still more efficacious than non-targeted liposomes and free drug treatment in CRC bearing mice. These results show that site-directed conjugation of monovalent CTX-Fab' provides targeted L-OH liposomes that display an increased tumor drug delivery and efficacy over a formulation with CTX and non-targeted liposomes. Copyright © 2015. Published by Elsevier B.V.
    Journal of Controlled Release 05/2015; 210. DOI:10.1016/j.jconrel.2015.05.271 · 7.71 Impact Factor
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    ABSTRACT: Mitoxantrone (MTO) is clinically used for treatment of various types of cancers providing an alternative for similarly active, but more toxic chemotherapeutic drugs like anthracyclines. To further decrease its toxicity MTO was encapsulated into liposomes. Although liposomal drugs can accumulate in target tumor tissue, they still face the plasma membrane barrier for effective intracellular delivery. Aiming to improve MTO tumor cell availability, we used short chain lipids to target and modulate the tumor cell membrane, promoting MTO plasma membrane traversal. MTO was encapsulated in liposomes containing the Short Chain Sphingolipid (SCS), C8-Glucosylceramide (C8-GluCer) or C8-Galactosylceramide (C8-GalCer) in their bilayer. These new SCS-liposomes containing MTO (SCS-MTOL) were tested in vivo for tolerability, pharmacokinetics, biodistribution, tumor drug delivery by intravital microscopy and efficacy, and compared to standard MTO liposomes (MTOL) and free MTO. Liposomal encapsulation decreased MTO toxicity and allowed administration of higher drug doses. SCS-MTOL displayed increased clearance and lower skin accumulation compared to standard MTOL. Intratumoral liposomal drug delivery was heterogeneous and rather limited in hypoxic tumor areas, yet SCS-MTOL improved intracellular drug uptake in comparison to MTOL. The increased MTO availability correlated well with the improved antitumor activity of SCS-MTOL in a MDAMB-231 breast carcinoma model. Multiple dosing of liposomal MTO strongly delayed tumor growth compared to free MTO and prolonged mouse survival, whereas among the liposomal MTO treatments, C8-GluCer-MTOL was most effective. Targeting plasma membranes with SCS improved MTO tumor availability and thereby therapeutic activity and represents a promising approach to improve MTO-based chemotherapy. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 05/2015; 94(1). DOI:10.1016/j.ejpb.2015.05.003 · 3.38 Impact Factor
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    ABSTRACT: Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSL) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSL in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing Doxorubicin (Dox) were incubated at 4 °C and 37 °C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox. SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSL. SC-GSL enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4 °C. Our results demonstrate that SC-GSL preferentially insert into tumor cell plasma membranes enhancing cells intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 04/2015; 1848(8). DOI:10.1016/j.bbamem.2015.04.011 · 4.66 Impact Factor
  • Gastroenterology 04/2015; 148(4):S-375. DOI:10.1016/S0016-5085(15)31260-9 · 16.72 Impact Factor
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    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(10). DOI:10.18632/oncotarget.3224 · 6.36 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; 34(39). DOI:10.1038/onc.2014.425 · 8.46 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 · 3.42 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 · 7.71 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 · 3.23 Impact Factor
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    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 · 1.78 Impact Factor
  • Journal of Hepatology 04/2014; 60(1):S87-S88. DOI:10.1016/S0168-8278(14)60226-9 · 11.34 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; 124(2). DOI:10.1172/JCI66776 · 13.22 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(1). DOI:10.1016/j.jconrel.2013.11.012 · 7.71 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 · 3.38 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; 17(1). DOI:10.1007/s10456-013-9385-2 · 4.88 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; 172(1). DOI:10.1016/j.jconrel.2013.08.034 · 7.71 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 · 2.92 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; 34(11). DOI:10.1093/carcin/bgt215 · 5.33 Impact Factor

Publication Stats

3k Citations
749.70 Total Impact Points


  • 1997–2015
    • Erasmus MC
      • • Department of Surgery
      • • Department of Oncological Surgery
      Rotterdam, South Holland, Netherlands
  • 1995–2015
    • Erasmus Universiteit Rotterdam
      • • Department of Surgery
      • • Department of Surgical Oncology
      • • Department of Medical Microbiology and Infectious Diseases
      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
    • University of Groningen
      • Department of Pathology and Laboratory Medicine
      Groningen, Groningen, Netherlands
    • Loma Linda University
      • Department of Radiation Medicine
      Loma Linda, CA, United States
  • 1999
    • University of Antwerp
      Antwerpen, Flemish, Belgium
  • 1994
    • Emory University
      • Department of Pathology and Laboratory Medicine
      Atlanta, GA, United States