Erratum: Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging: Erratum.

[This corrects the article DOI: 10.7150/thno.37949.].

An In Vivo Model to Study Cell Migration in XYZ-T Dimension Followed by Whole-Mount Re-evaluation.

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.

Liposomal Drug Delivery Systems for Cancer Therapy: The Rotterdam Experience.

At the Nanomedicine Innovation Center (NICE) at the Erasmus MC in Rotterdam, we have approached the treatment of cancer by starting with a vision of first establishing a platform that enables us to overcome the low levels of drugs delivered to tumors and the issue of dose-limiting toxicity. Showing that a reduction of the volume of distribution, and a lowering of toxicity and side-effects, accompanied by augmented intratumoral drug delivery, could change outcomes in patients, paved the way to target, not only localized disease, but also systemic and metastasized cancers. In particular, the detailed studies with intravital microscopy we performed at NICE provided us with the necessary insights and affected to a large extent our program on liposome-based cancer therapy. Together with our experience with the loco-regional treatment of cancer, this helped us to develop a program that focused on the subsequent aspects discussed here. We recognized that passive accumulation of nanoparticles was not as effective as previously believed and undertook to improve the local accumulation by changing the tumor pathophysiology and, in particular, the vascular permeability. We added the targeting of liposomes using vascular and tumor directed moieties, to improve cellular drug delivery. To improve payload delivery, we studied the modification of liposomes with phospholipids that help passive drug release and augment cellular accumulation. Second, and importantly, modification of liposomes was undertaken, to enable triggered drug release. The capability for modifying liposomes to respond to a trigger, and the ability to now apply an external trigger (e.g., hyperthermia) and specifically reach the tumor volume, resulted in the current smart drug delivery systems. Our experience at NICE, after a few decades of research on lipid-based nanoparticles, shows that, after the first liposomal formulation registered for clinical application in cancer therapy, further developments quickly followed, while further clinical applications lagged behind. Now we need to focus on and make the next steps towards the clinic, to fulfil the promise that is found there.

Assessment of the In Vivo Response to Nanobody-Targeted PDT Through Intravital Microscopy.

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.

An adapted dorsal skinfold model used for 4D intravital followed by whole-mount imaging to reveal endothelial cell-pericyte association.

Endothelial cells and pericytes are highly dynamic vascular cells and several subtypes, based on their spatiotemporal dynamics or molecular expression, are believed to exist. The interaction between endothelial cells and pericytes is of importance in many aspects ranging from basic development to diseases like cancer. Identification of spatiotemporal dynamics is particularly interesting and methods to studies these are in demand. Here we describe the technical details of a method combining the benefits of high resolution intravital imaging and whole-mount histology. With intravital imaging using an adapted light weight dorsal skinfold chamber we identified blood flow patterns and spatiotemporal subtypes of endothelial cells and pericytes in a 4D (XYZ, spatial+T, time dimension) manner as representative examples for this model. Thereafter the tissue was extracted and stained as a whole-mount, by which the position and volumetric space of endothelial cells as well as pericytes were maintained, to identify molecular subtypes. Integration of the two imaging methods enabled 4D dissection of endothelial cell-pericyte association at the molecular level.

Preclinical Studies in Small Animals for Advanced Drug Delivery Using Hyperthermia and Intravital Microscopy.

This paper presents three devices suitable for the preclinical application of hyperthermia via the simultaneous high-resolution imaging of intratumoral events. (Pre)clinical studies have confirmed that the tumor micro-environment is sensitive to the application of local mild hyperthermia. Therefore, heating is a promising adjuvant to aid the efficacy of radiotherapy or chemotherapy. More so, the application of mild hyperthermia is a useful stimulus for triggered drug release from heat-sensitive nanocarriers. The response of thermosensitive nanoparticles to hyperthermia and ensuing intratumoral kinetics are considerably complex in both space and time. To obtain better insight into intratumoral processes, longitudinal imaging (preferable in high spatial and temporal resolution) is highly informative. Our devices are based on (i) an external electric heating adaptor for the dorsal skinfold model, (ii) targeted radiofrequency application, and (iii) a microwave antenna for heating of internal tumors. These models, while of some technical complexity, significantly add to the understanding of effects of mild hyperthermia warranting implementation in research on hyperthermia.

Drug transport kinetics of intravascular triggered drug delivery systems.

Intravascular triggered drug delivery systems (IV-DDS) for local drug delivery include various stimuli-responsive nanoparticles that release the associated agent in response to internal (e.g., pH, enzymes) or external stimuli (e.g., temperature, light, ultrasound, electromagnetic fields, X-rays). We developed a computational model to simulate IV-DDS drug delivery, for which we quantified all model parameters in vivo in rodent tumors. The model was validated via quantitative intravital microscopy studies with unencapsulated fluorescent dye, and with two formulations of temperature-sensitive liposomes (slow, and fast release) encapsulating a fluorescent dye as example IV-DDS. Tumor intra- and extravascular dye concentration dynamics were extracted from the intravital microscopy data by quantitative image processing, and were compared to computer model results. Via this computer model we explain IV-DDS delivery kinetics and identify parameters of IV-DDS, of drug, and of target tissue for optimal delivery. Two parameter ratios were identified that exclusively dictate how much drug can be delivered with IV-DDS, indicating the importance of IV-DDS with fast drug release (~sec) and choice of a drug with rapid tissue uptake (i.e., high first-pass extraction fraction). The computational model thus enables engineering of improved future IV-DDS based on tissue parameters that can be quantified by imaging.

Externally triggered smart drug delivery system encapsulating idarubicin shows superior kinetics and enhances tumoral drug uptake and response.

Rationale: Increasing the bioavailable drug level in a tumor is the key to enhance efficacy of chemotherapy. Thermosensitive smart drug delivery systems (SDDS) in combination with local hyperthermia facilitate high local drug levels, thus improving uptake in the tumor. However, inability to rapidly and efficiently absorb the locally released drug results in reduced efficacy, as well as undesired redistribution of the drug away from the tumor to the system. Methods: Based on this paradigm we propose a novel approach in which we replaced doxorubicin (DXR), one of the classic drugs for nanocarrier-based delivery, with idarubicin (IDA), a hydrophobic anthracycline used solely in the free form for treatment hematologic cancers. We established a series of in vitro and in vivo experiments to in depth study the kinetics of SDDS-based delivery, drug release, intratumor biodistribution and subsequent cell uptake. Results: We demonstrate that IDA is taken up over 10 times more rapidly by cancer cells than DXR in vitro. Similar trend is observed in in vivo online imaging and less drug redistribution is shown for IDA, together resulting in 4-times higher whole tumor drug uptake for IDA vs. DXR. Together his yielded an improved intratumoral drug distribution for IDA-SDDS, translating into superior tumor response compared to DXR-SDDS treatment at the same dose. Thus, IDA - a drug that is not used for treatment of solid cancers - shows superior therapeutic index and better outcome when administered in externally triggered SDDS. Conclusions: We show that a shift in selection of chemotherapeutics is urgently needed, away from the classic drugs towards selection based on properties of a chemotherapeutic in context of the nanoparticle and delivery mode, to maximize the therapeutic efficacy.

Hyperthermia and Temperature-Sensitive Nanomaterials for Spatiotemporal Drug Delivery to Solid Tumors.

Nanotechnology has great capability in formulation, reduction of side effects, and enhancing pharmacokinetics of chemotherapeutics by designing stable or long circulating nano-carriers. However, effective drug delivery at the cellular level by means of such carriers is still unsatisfactory. One promising approach is using spatiotemporal drug release by means of nanoparticles with the capacity for content release triggered by internal or external stimuli. Among different stimuli, interests for application of external heat, hyperthermia, is growing. Advanced technology, ease of application and most importantly high level of control over applied heat, and as a result triggered release, and the adjuvant effect of hyperthermia in enhancing therapeutic response of chemotherapeutics, i.e., thermochemotherapy, make hyperthermia a great stimulus for triggered drug release. Therefore, a variety of temperature sensitive nano-carriers, lipid or/and polymeric based, have been fabricated and studied. Importantly, in order to achieve an efficient therapeutic outcome, and taking the advantages of thermochemotherapy into consideration, release characteristics from nano-carriers should fit with applicable clinical thermal setting. Here we introduce and discuss the application of the three most studied temperature sensitive nanoparticles with emphasis on release behavior and its importance regarding applicability and therapeutic potentials.

Sensitization of drug resistant sarcoma tumors by membrane modulation via short chain sphingolipid-containing nanoparticles.

Nanoparticles such as liposomes are able to overcome cancer treatment challenges such as multidrug resistance by increasing the bioavailability of the encapsulated drug, bypassing drug pumps or through targeting resistant cells. Here, we merge enhanced drug delivery by nanotechnology with tumor cell membrane modulation combined in a single formulation. This is achieved through the incorporation of Short chain sphingolipids (SCSs) in the liposomal composition, which permeabilizes cell membranes to amphiphilic drugs such as Doxorubicin (Dxr). To study the mechanism and capability of SCS-containing nanodevices to overcome Dxr resistance, a sensitive uterine sarcoma cell line, MES-SA, and a resistant derived cell line, MES-SA/MX2, were used. The mechanism of resistance was explored by lipidomics and flow cytometry, revealing significant differences in lipid composition and in P glycoprotein (Pgp) expression. In vitro assays show that SCS liposomes were able to reverse cell resistance, and importantly, display a higher net effect on resistant than sensitive cells. SCS lipids modulated the cell membrane of MES-SA/MX2 drug resistant cells, while Pgp expression was not affected. Furthermore, SCS-modified liposomes were evaluated in a sarcoma xenograft model on drug accumulation, pharmacokinetics and efficacy. SCS liposomes improved Dxr levels in tumor nuclei of MES-SA/MX2 tumor cells, which was accompanied by a delay in tumor growth of the resistant model. Here we show that Dxr accumulation in tumor cells by SCS-modified liposomes was especially improved in Dxr resistant cells, rendering Dxr as effective as in sensitive cells. Moreover, this phenomenon translated to improved efficacy when Dxr liposomes where modified with SCSs in the drug resistant tumor model, while no benefit was seen in the sensitive tumors.

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging.

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h compared to 1 h after administration. Discussion: Although differences were observed in distribution of the two NB-PS conjugates, both led to similar necrosis. Clearly, the response to PDT using NB-PS conjugates is the result of a complex mixture of tumor cell responses and vascular effects, which is likely to be necessary for a maximally effective treatment.

A microcarrier-based spheroid 3D invasion assay to monitor dynamic cell movement in extracellular matrix.

BACKGROUND: Cell invasion through extracellular matrix (ECM) is a critical step in tumor metastasis. To study cell invasion in vitro, the internal microenvironment can be simulated via the application of 3D models. RESULTS: This study presents a method for 3D invasion examination using microcarrier-based spheroids. Cell invasiveness can be evaluated by quantifying cell dispersion in matrices or tracking cell movement through time-lapse imaging. It allows measuring of cell invasion and monitoring of dynamic cell behavior in three dimensions. Here we show different invasive capacities of several cell types using this method. The content and concentration of matrices can influence cell invasion, which should be optimized before large scale experiments. We also introduce further analysis methods of this 3D invasion assay, including manual measurements and homemade semi-automatic quantification. Finally, our results indicate that the position of spheroids in a matrix has a strong impact on cell moving paths, which may be easily overlooked by researchers and may generate false invasion results. CONCLUSIONS: In all, the microcarrier-based spheroids 3D model allows exploration of adherent cell invasion in a fast and highly reproducible way, and provides informative results on dynamic cell behavior in vitro.

CREPT Promotes Melanoma Progression Through Accelerated Proliferation and Enhanced Migration by RhoA-Mediated Actin Filaments and Focal Adhesion Formation.

Melanoma is one of the most aggressive cancers, and patients with distant metastases have dire outcomes. We observed previously that melanoma progression is driven by a high migratory potential of melanoma cells, which survive and proliferate under harsh environmental conditions. In this study, we report that CREPT (cell-cycle related and expression-elevated protein in tumor), an oncoprotein highly expressed in other cancers, is overexpressed in melanoma cells but not melanocytes. Overexpression of CREPT stimulates cell proliferation, migration, and invasion in several melanoma cell lines. Further, we show that CREPT enhances melanoma progression through upregulating and activating Ras homolog family member A (RhoA)-induced actin organization and focal adhesion assembly. Our study reveals a novel role of CREPT in promoting melanoma progression. Targeting CREPT may be a promising strategy for melanoma treatment.

Liposomes targeted to MHC-restricted antigen improve drug delivery and antimelanoma response.

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.

Spatiotemporal endothelial cell - pericyte association in tumors as shown by high resolution 4D intravital imaging.

Endothelial cells and pericytes are integral cellular components of the vasculature with distinct interactive functionalities. To study dynamic interactions between these two cells we created two transgenic animal lines. A truncated eNOS (endothelial nitric oxide synthase) construct was used as a GFP tag for endothelial cell evaluation and an inducible Cre-lox recombination, under control of the Pdgfrb (platelet derived growth factor receptor beta) promoter, was created for pericyte assessment. Also, eNOStag-GFP animals were crossed with the already established Cspg4-DsRed mice expressing DsRed fluorescent protein in pericytes. For intravital imaging we used tumors implanted in the dorsal skinfold of these transgenic animals. This setup allowed us to study time and space dependent complexities, such as distribution, morphology, motility, and association between both vascular cell types in all angiogenetic stages, without the need for additional labeling. Moreover, as fluorescence was still clearly detectable after fixation, it is possible to perform comparative histology following intravital evaluation. These transgenic mouse lines form an excellent model to capture collective and individual cellular and subcellular endothelial cell - pericyte dynamics and will help answer key questions on the cellular and molecular relationship between these two cells.

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

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.

Melanomas prevent endothelial cell death under restrictive culture conditions by signaling through AKT and p38 MAPK/ ERK-1/2 cascades.

Although melanoma progression and staging is clinically well characterized, a large variation is observed in pathogenesis, progression, and therapeutic responses. Clearly, intrinsic characteristics of melanoma cells contribute to this variety. An important factor, in both progression of the disease and response to therapy, is the tumor-associated vasculature. We postulate that melanoma cells communicate with endothelial cells (ECs) in order to establish a functional and supportive blood supply. We investigated the angiogenic potential of human melanoma cell lines by monitoring the survival of ECs upon exposure to melanoma conditioned medium (CM), under restrictive conditions. We observed long-term (up to 72 h) EC survival under hypoxic conditions upon treatment with all melanoma CMs. No such survival effect was observed with the CM of melanocytes. The CM of pancreatic and breast tumor cell lines did not show a long-term survival effect, suggesting that the survival factor is specific to melanoma cells. Furthermore, all size fractions (up to < 1 kDa) of the melanoma CM induced long-term survival of ECs. The survival effect observed by the < 1 kDa fraction excludes known pro-angiogenic factors. Heat inactivation and enzymatic digestion of the CM did not inactivate the survival factor. Global gene expression and pathway analysis suggest that this effect is mediated in part via the AKT and p38 MAPK/ ERK-1/2 signaling axis. Taken together, these data indicate the production of (a) survival factor/s (< 1 kDa) by melanoma cell lines, which enables long-term survival of ECs and promotes melanoma-induced angiogenesis.

High-Resolution Intravital Microscopy of Tumor Angiogenesis.

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.

Pharmacokinetics, Tissue Distribution and Therapeutic Effect of Cationic Thermosensitive Liposomal Doxorubicin Upon Mild Hyperthermia.

PURPOSE: To evaluate pharmacokinetic profile, biodistribution and therapeutic effect of cationic thermosensitive liposomes (CTSL) encapsulating doxorubicin (Dox) upon mild hyperthermia (HT). METHODS: Non-targeted thermosensitive liposomes (TSL) and CTSL were developed, loaded with Dox and characterized. Blood kinetics and biodistribution of Dox-TSL and Dox-CTSL were followed in B16BL6 tumor bearing mice upon normothermia (NT) or initial hyperthermia conditions. Efficacy study in B16BL6 tumor bearing mice was followed with Dox-TSL or Dox-CTSL upon NT or HT. Efficacy study in LLC tumor bearing mice was performed upon two HT conditions. Intravital microscopy was performed on B16BL6 tumors implanted in dorsal-skin fold window-bearing mice. RESULTS: Targeting did not cause faster blood clearance of CTSL compared to TSL. Highest uptake of liposomes was observed in spleen, kidneys and liver. Applying HT prior to CTSL administration increased drug delivery to the tumor and CTSL delivered ~1.7 fold higher Dox concentration compared to TSL. Efficacy in B16BL6 murine melanoma showed that HT had a significant effect on CTSL in tumor suppression and prolonged survival. Efficacy in LLC Lewis lung carcinoma tumor model demonstrates that two HT treatments hold promises for a successful treatment option. CONCLUSION: CTSL have potency to increase drug efficacy in tumors due to their targeted and drug release functions.