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Cell migration is a very dynamic process involving several chemical as well as biological interactions with other cells and the environment. Several models exist to study cell migration ranging from simple 2D in vitro cultures to more demanding 3D multicellular assays, to complex evaluation in animals. High-resolution 4D (XYZ, spatial + T, time dimension) intravital imaging using transgenic animals with a fluorescent label in cells of interest is a powerful tool to study cell migration in the correct environment. Here we describe an advanced dorsal skinfold chamber model to study endothelial cell and pericyte migration and association.

Methods that allow real-time, longitudinal, intravital detection of the fluorescence distribution and the cellular and vascular responses within tumor and normal tissue are important tools to obtain valuable information when investigating new photosensitizers and photodynamic therapy (PDT) responses. Intravital confocal microscopy using the dorsal skinfold chamber model gives the opportunity to visualize and determine the distribution of photosensitizers within tumor and normal tissue. Next to that, it also allows the visualization of the effect of treatment with respect to changes in vascular diameter and blood flow, vascular leakage, and tissue necrosis, in the first days post-illumination. Here, we describe the preparation of the skinfold chamber model and the intravital microscopy techniques involved, for a strategy we recently introduced, that is, the nanobody-targeted PDT. In this particular approach, photosensitizers are conjugated to nanobodies to target these specifically to cancer cells.

Purpose Melanoma is the most aggressive form of skin cancer. Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy. Materials and methods To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1). With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro. In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft. Results We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes. Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes. In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively. Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1−/A1+ tumors. Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo. Conclusion Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma. We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response. Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy. Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Real-time evaluation of vascular effects in an animal skinfold window model by intravital microscopy (IVM) provides a powerful tool to improve insight into vascular development and vascular therapy. The potential of IVM to examine processes in tissues (e.g., tumors, inflammatory sites), in a noninvasive way, enables determination of the kinetics of processes under study at any given time point. The introduction of sensitive digital cameras, confocal and multiphoton microscopy, and powerful imaging software greatly improved the quality of the images acquired. Together with the introduction of better fluorescent probes, with a shift towards red and near-infrared fluorescence, confocal and multiphoton microscopy enables deeper imaging with less (photo)toxicity. IVM is particularly useful for examination of processes in time, which span seconds up to days or even weeks, such as tumor vascular development. Here we describe an advanced dorsal skinfold window chamber for high-resolution intravital microscopy of tumor angiogenesis.

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60% of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90% of the pa...

The application of tumor necrosis factor-alpha (TNF) for the treatment of solid tumors is limited by its severe, life-threatening, toxicity. Therefore, only low dosages of this cytokine can be applied systemically, which results in poor tumor response. It has been demonstrated previously that administration of high-dose TNF in a so-called isolated perfusion system markedly improved tumor response when combined with chemotherapy. It appeared that TNF had a major impact specifically on the tumor-associated vasculature. At these high concentrations, endothelial cell death is induced by TNF, resulting in complete collapse of the tumor vascular bed. Strikingly, this effect alone is not enough to induce a tumor response, but addition of a chemotherapeutic drug is mandatory to obtain an anti-tumor effect. We showed that TNF has no anti-tumor effect by itself but augmented drug accumulation mainly in the tumor, most likely by enhancing vascular leakage. It seems that enhanced vascular leakage, but not endothelial cell death, explains the interaction between TNF and the co-administered drug. We hypothesized that in a low-dose setting TNF could induce tumor accumulation of chemotherapeutic drugs and consequently improve tumor response. We demonstrate that free TNF has a strong effect on the pharmacokinetics of co-administered Doxil in B16BL6 melanoma-bearing mice, resulting in strongly augmented drug accumulation in the tumor and improved tumor response. Co-injection of Stealth liposomal TNF with Doxil resulted in comparable or less pronounced tumor responses as compared to free TNF. These results imply that systemic application of clinically tolerable doses of TNF may improve drug distribution and tumor response and could be useful in a number of anti-cancer therapies.

The toxicity associated with tumor necrosis factor-alpha (TNF-alpha) has limited its usefulness as an anticancer agent. However, encapsulation of TNF-alpha in Stealth (SL) liposomes can minimize risk for toxicity and thus increase its potential as an adjuvant treatment. Our recent studies have shown that SL-TNF-alpha plus radiation is more effective at inhibiting LS174T colon tumor growth than either radiation alone or free TNF-alpha plus radiation. This increase in efficacy was coincident with a modulation of immune parameters in blood and spleen. The aim of this study was to determine if infiltration of natural killer (NK) cells, macrophages, and neutrophils into LS174T tumors was altered by SL-TNF-alpha treatment and whether any observed changes could potentially contribute to the enhanced antitumor efficacy seen with SL-TNF-alpha plus radiation treatment. Sections of excised tumors were examined histologically and quantitative analysis was performed using laser scanning cytometry. The data showed that the group receiving multiple treatments with SL-TNF-alpha plus radiation had the smallest tumors, but yet the level of necrosis was similar to that found in groups with much larger tumors. Furthermore, the necrotic areas in the SL-TNF-alpha plus radiation group had signs of recent and/or continuing cell death and the highest levels of NK cell and macrophage infiltrates. In time course experiments, a single injection of SL-TNF-alpha (but not free TNF-alpha) induced fluctuations in leukocyte infiltration into tumors that correlated inversely with our previous findings in blood and spleen. Overall, the data indicate that the mechanisms underlying the increased efficacy of SL-TNF-alpha compared to free TNF-alpha include a rapid and relatively sustained recruitment of NK cells, macrophages, and neutrophils.

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically-stabilized PEGylated STEALTH liposomes (SL) can better and more safely augment the efficacy of other treatment modalities than free TNF-alpha. The aim of this study was to examine the effects of SL-TNF-alpha in the LS174T human colon tumor xenograft model and to correlate its administration with alterations in innate immune system parameters.Nude mice (n = 128) were injected subcutaneously with LSI 74T cells and treated intravenously with SL-TNF-alpha SL-placebo, or free recombinant human TNF-alpha; the animals were euthanized at 6, 18, 36 and 96 hours after injection.Significant increases in leukocyte, granulocyte, monocyte and NK cell numbers were observed early (6 hours) in the blood from both SL-TNF-alpha and free TNF-alpha treated mice compared to the control group. In contrast, during the 18- to 36-hours interval, SL-TNF-alpha induced significantly higher (p0.05) leukoctyte, T cell, and NK cell numbers, basal leukocyte proliferation, and CD25+ activation marker expression; the modulation occurred primarily in the spleen.These data indicate that both SL-TNF-alpha and free TNF-alpha can induce dramatic up-regulation in leukocyte populations early after injection. However, the up-regulation produced by SL-TNF-alpha was more prolonged and pronounced than that of TNF-alpha and had better correlation with cell activation. These findings suggest that sustained leukocyte recruitment and/or activation may be an important factor in the greater than additive or synergistic antitumor effects observed when SL-TNF-alpha is used in combination with other cancer therapies.

Previously we reported that encapsulation of tumor necrosis factor-alpha (TNF) in pegylated (STEALTH) liposomes (TNF-PEGL) dramatically improved circulation times of the protein and augmented accumulation in tumor tissue. We and others have demonstrated enhanced antitumor activity of doxorubicin or melphalan by free TNF when used in high doses in an isolated limb perfusion setting. In the present study the antitumor activity of TNF-PEGL was studied in combination with liposomal chemotherapy. BN rats with subcutaneous BN175 sarcomas (8-12 mm diameter) received no treatment or pegylated liposomal doxorubicin (Doxil) alone or in combination with various doses of TNF-PEGL (15-200 microg/kg). The evaluated endpoints were tumor response and toxicity of the treatment regimens. Here we demonstrate that TNF-PEGL at a dose of 15 microg/kg markedly augments the antitumor activity of liposomal doxorubicin, without resulting in the increased toxic side effects observed with free TNF at doses resulting in a similar enhancement of the antitumor effects. Even at a TNF dose of 200 microg/kg TNF, repeated administration of TNF-PEGL did not result in severe weight loss or cause diarrhea. Repeated dosing of free TNF at this dose resulted in severe, life-threatening weight loss and occurrence of diarrhea in all animals. These results indicate that pegylated liposomal encapsulation may be effective in systemic application of TNF for combined treatment with liposomal chemotherapy of advanced solid tumors.