Wiltrud Lederle

Passive versus Active Tumor Targeting Using RGD- and NGR-Modified Polymeric Nanomedicines

Enhanced permeability and retention (EPR) and the (over-) expression of angiogenesis-related surface receptors are key features of tumor blood vessels. As a consequence, EPR-mediated passive and Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) based active tumor targeting have received considerable attention in the last couple of years. Using several different in vivo and ex vivo optical imaging techniques, we here visualized and quantified the benefit of RGD- and NGR-based vascular vs EPR-mediated passive tumor targeting. This was done using ∼ 10 nm sized polymeric nanocarriers, which were either labeled with DY-676 (peptide-modified polymers) or with DY-750 (peptide-free polymers). Upon coinjection into mice bearing both highly leaky CT26 and poorly leaky BxPC3 tumors, it was found that vascular targeting did work, resulting in rapid and efficient early binding to tumor blood vessels, but that over time, passive targeting was significantly more efficient, leading to higher overall levels and to more efficient retention within tumors. Although this situation might be different for larger carrier materials, these insights indicate that caution should be taken not to overestimate the potential of active over passive tumor targeting.

Ultrasound Microbubbles for Molecular Diagnosis, Therapy, and Theranostics

Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.

Recent advances in molecular, multimodal and theranostic ultrasound imaging

Ultrasound (US) imaging is an exquisite tool for the non-invasive and real-time diagnosis of many different diseases. In this context, US contrast agents can improve lesion delineation, characterization and therapy response evaluation. US contrast agents are usually micrometer-sized gas bubbles, stabilized with soft or hard shells. By conjugating antibodies to the microbubble (MB) surface, and by incorporating diagnostic agents, drugs or nucleic acids into or onto the MB shell, molecular, multimodal and theranostic MBs can be generated. We here summarize recent advances in molecular, multimodal and theranostic US imaging, and introduce concepts how such advanced MB can be generated, applied and imaged. Examples are given for their use to image and treat oncological, cardiovascular and neurological diseases. Furthermore, we discuss for which therapeutic entities incorporation into (or conjugation to) MB is meaningful, and how US-mediated MB destruction can increase their extravasation, penetration, internalization and efficacy.

Vascular endothelial growth factor-induced skin carcinogenesis depends on recruitment and alternative activation of macrophages

Inflammation contributes to tumour growth, invasion and angiogenesis. We investigated the contribution of macrophages and their polarization to tumour progression in a model of VEGF‐A‐induced skin carcinogenesis. Transfection of the human non‐tumourigenic keratinocyte cell line HaCaT with murine VEGF‐A leads to malignant tumour growth in vivo. The resulting tumours are characterized by extensive vascularization, invasive growth and high numbers of M2‐polarized macrophages that crucially contribute to the establishment of the malignant phenotype. Accordingly, macrophage depletion from tumour‐bearing animals resulted in reduced tumour growth, inhibition of invasion, decreased proliferation and reduced angiogenesis. In vitro, VEGF‐A exerted a chemo‐attracting effect on macrophages, but did not induce M2 polarization. We identified IL‐4 and IL‐10 as the factors involved in M2 polarization. These factors were produced by tumour cells (IL‐10) and macrophages (IL‐4) in vivo. Addition of recombinant IL‐4 and IL‐10 in vitro induced a pro‐invasive M2 macrophage phenotype and inhibition of the IL‐4 receptor in vivo blocked M2 polarization of macrophages, resulting in a less aggressive tumour phenotype. Thus, we provide evidence that M2 macrophages are crucial for the development of VEGF‐A‐induced skin tumours and that VEGF‐A contributes to malignant tumour growth, not only by enhancing angiogenesis but also by establishing an anti‐inflammatory microenvironment. However, VEGF‐A alone is not sufficient to create a tumour‐promoting microenvironment and requires the presence of IL‐4 and IL‐10 to induce M2 polarization of macrophages. Copyright

MMP13 as a stromal mediator in controlling persistent angiogenesis in skin carcinoma

Matrix metalloproteinases (MMPs) such as MMP13 promote tumour growth and progression by mediating extracellular matrix (ECM) reorganization and regulating the biological activity of cytokines. Using Mmp13-/- mice, we demonstrate an essential role of this single collagenase for highly malignant and invasive growth in skin squamous cell carcinoma (SCC). Lack of host MMP13 strongly impaired tumour growth of malignant SCC cells, leading to small, mostly avascular cysts. While initial stromal activation in tumour transplants of Mmp13+/+ and Mmp13-/- animals was similar, MMP13 was essential for maintenance of angiogenesis and for invasion. MMP13 was induced in fibroblasts of the wild-type animals at the onset of invasion and correlated with a strong increase in vascular endothelial growth factor (VEGF) protein and its association with vascular endothelial growth factor receptor-2 on endothelial cells in invasive areas. In contrast, VEGF protein in the stroma was barely detectable and tumour invasion was downregulated in Mmp13-/- animals, despite ongoing VEGF messenger RNA expression. Taken together with in vitro data showing the release of VEGF from the ECM by MMP13 expressing fibroblasts, these data strongly suggest a crucial role of MMP13 in promoting angiogenesis via releasing VEGF from the ECM and thus allowing the invasive growth of the SCC cells.

Regorafenib Inhibits Growth, Angiogenesis, and Metastasis in a Highly Aggressive, Orthotopic Colon Cancer Model

The combination of target-specific drugs like bevacizumab with chemotherapeutics has improved treatment efficacy in advanced colorectal cancer (CRC). However, the clinical prognosis of metastatic CRCs is still poor, and novel drugs are currently assessed with respect to their efficacies in patients with CRCs. In a phase III study, the multikinase inhibitor regorafenib (BAY 73-4506) has recently been shown to prolong survival of patients with CRCs after standard therapies failed. In the present study, the activity of regorafenib was investigated in comparison with the angiogenesis inhibitor DC101 in the highly aggressive, murine CT26 metastatic colon cancer model. While a treatment for 10 days with DC101 given at a dose of 34 mg/kg every third day significantly delayed tumor growth compared with vehicle-treated animals, regorafenib completely suppressed tumor growth at a daily oral dose of 30 mg/kg. Regorafenib also induced a stronger reduction in tumor vascularization, as longitudinally assessed in vivo by dynamic contrast-enhanced MRI (DCE-MRI) and confirmed by immunohistochemistry. In addition, regorafenib inhibited the angiogenic activity more strongly and induced a three times higher apoptosis rate than DC101. Even more important, regorafenib completely prevented the formation of liver metastases, whereas in DC101-treated animals, the metastatic rate was only reduced by 33% compared with the vehicle group. In addition, regorafenib significantly reduced the amount of infiltrating macrophages. These data show that the multikinase inhibitor regorafenib exerts strong antiangiogenic, antitumorigenic, and even antimetastatic effects on highly aggressive colon carcinomas indicative for its high potential in the treatment of advanced CRCs. Mol Cancer Ther; 12(7); 1322–31. ©2013 AACR.

Bioactive Gyroid Scaffolds Formed by Sacrificial Templating of Nanocellulose and Nanochitin Hydrogels as Instructive Platforms for Biomimetic Tissue Engineering

A sacrificial templating process using lithographically printed minimal surface structures allows complex de novo geo-metries of delicate hydrogel materials. The hydrogel scaffolds based on cellulose and chitin nanofibrils show differences in terms of attachment of human mesenchymal stem cells, and allow their differentiation into osteogenic outcomes. The approach here serves as a first example toward designer hydrogel scaffolds viable for biomimetic tissue engineering.

IL-6 promotes malignant growth of skin SCCs by regulating a network of autocrine and paracrine cytokines.

Cytokines play a crucial role in tumor initiation and progression. Here, we demonstrate that interleukin (IL)‐6 is a key factor by driving tumor progression from benign to malignant, invasive tumors in the HaCaT‐model of human skin carcinoma. IL‐6 activates STAT3 and directly stimulates proliferation and migration of the benign noninvasive HaCaT‐ras A‐5 cells in vitro. Furthermore, IL‐6 induces a complex, reciprocally regulated cytokine network in the tumor cells that includes inflammatory and angiogenic factors such as IL‐8, GM‐CSF, VEGF and MCP‐1. These IL‐6 effects lead to tumor cell invasion in organotypic cultures in vitro and to the formation of malignant and invasive s.c. tumors in vivo. Tumor invasion is supported by the IL‐6 induced overexpression of MMP‐1 in vitro and in vivo. These data demonstrate a key function of IL‐6 in the progression of skin SCCs by regulating a complex cytokine and protease network and suggest new therapeutic approaches to target this central player in skin carcinogenesis.

Assessment of vascular remodeling under antiangiogenic therapy using DCE-MRI and vessel size imaging†

To assess vascular remodeling in tumors during two different antiangiogenic therapies with dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI) and vessel size imaging and to evaluate the vessel size index (VSI) as a novel biomarker of therapy response.

Comparison of conventional time-intensity curves vs. maximum intensity over time for post-processing of dynamic contrast-enhanced ultrasound

Our aim was to prospectively compare two post-processing techniques for dynamic contrast-enhanced ultrasound and to evaluate their impact for monitoring antiangiogenic therapy. Thus, mice with epidermoid carcinoma xenografts were examined during administration of polybutylcyanoacrylate-microbubbles using a small animal ultrasound system (40 MHz). Cine loops were acquired and analyzed using time-intensity (TI) and maximum intensity over time (MIOT) curves. Influences of fast (50 microl/2s) vs. slow (50 microl/10s) injection of microbubbles on both types of curves were investigated. Sensitivities of both methods for assessing effects of antiangiogenic treatment (SU11248) were examined. Correlative histological analysis was performed for vessel-density. Mann-Whitney test was used for statistical analysis. Microbubble injection rates significantly influenced upslope, time-to-peak and peak enhancement of conventional TI curves (p<0.05) but had almost no impact on maximum enhancement of MIOT curves (representing relative blood volume). Additionally, maximum enhancement of MIOT curves captured antiangiogenic therapy effects more reliably and earlier (already after 1 day of therapy; p<0.05) than peak enhancement of TI curves. Immunohistochemistry validated the significantly (p<0.01) lower vessel densities in treated tumors and high correlation (R(2)=0.95) between vessel-density and maximum enhancement of MIOT curves was observed. In conclusion, MIOT is less susceptible to variations of the injections speed. It enables to assess changes of the relative blood volume earlier and with lower standard deviations than conventional TI curves. It can easily be translated into clinical practice and thus may provide a promising tool for cancer therapy monitoring.