Abnormalization of Tumor Vessels to Improve the Efficacy of Chemotherapy

Achieving an adequate drug concentration at the tumor site is a major challenge in systemic therapy. Besides massive dilution in the blood and uptake by other organs, the pathophysiology of the tumor hampers drug uptake. Vasoactive compounds, like tumor necrosis factor-alpha (TNF) or Cilengitide manipulates the tumor-associated vasculature improving the permeability of these vessels. In this process, we have called tumor vessel abnormalization, the co-administered chemotherapeutic drug, melphalan or liposomal doxorubicin (Doxil), can traverse more easily from the blood circulation into the surrounding tumor tissue. This increased intratumoral drug accumulation ultimately results in an improved tumor response

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

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-carrie

Hyperthermia and smart drug delivery systems for solid tumor therapy

Chemotherapy is a cornerstone of cancer therapy. Irrespective of the administered drug, it is crucial that adequate drug amounts reach all cancer cells. To achieve this, drugs first need to be absorbed, then enter the blood circulation, diffuse into the tumor interstitial space and finally reach the tumor cells. Next to chemoresistance, one of the most important factors for effective chemotherapy is adequate tumor drug uptake and penetration. Unfortunately, most chemotherapeutic agents do not have favorable properties. These compounds are cleared rapidly, distribute throughout all tissues in the body, with only low tumor drug uptake that is heterogeneously distributed within the tumor. Moreover, the typical microenvironment of solid cancers provides additional hurdles for drug delivery, such as heterogeneous vascular density and perfusion, high interstitial fluid pressure, and abundant stroma. The hope was that nanotechnology will solve most, if not all, of these drug delivery barriers. However, in spite of advances and decades of nanoparticle development, results are unsatisfactory. One promising recent development are nanoparticles which can be steered, and release content triggered by internal or external signals. Here we discuss these so-called smart drug delivery systems in cancer therapy with emphasis on mild hyperthermia as a trigger signal for drug delivery

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

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

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-induc

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

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 i

Enhanced Specificity and Drug Delivery in Tumors by cRGD - Anchoring Thermosensitive Liposomes

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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