Endothelial Cells Of Tumor Vessels Research Articles

Abstract PO4-13-05: Spotlight on PSMA as a new theranostic biomarker for breast cancer

Abstract Breast cancer patients are risk-stratified on the basis of the subtype classification. However, this parameter is not completely accurate in discriminating between high- and low-risk disease, creating a need for a reliable marker to determine aggressiveness. Prostate-specific membrane antigen (PSMA) could fulfill this need given that it has been reported to be expressed by breast cancer tumor cells and the endothelial cells of tumor vessels. We analyzed 68 breast cancers (BC) of whom 14 luminal A, 28 Luminal B, 15 HER2 positive and 11 triple negative BC to assess whether PSMA expression by immunohistochemistry was different in the several tumor subtypes and was related to ki67 expression and stromal TILs. PSMA positivity was calculated as the number of positive vessels on 10 fields at 40X magnifications. Kruskal Wallis’s test was used to assess difference in PSMA positivity among BC subgroups and Dunn’s test was performed for post-hoc comparisons; Spearman’s rho coefficient was calculated to analyze the correlation among PSMA, KI67 and TILs. Our results show that median PSMA was higher both considering the number of positive vessels and the staining intensity in TNBC compared to Luminal A and B tumors (p< 0.001). Ki67 was higher in TNBC compared to Luminal A tumors (p< 0.001). We saw a correlation between PSMA and ki67, especially in HER2+ tumors (p=0.002), while a correlation between PSMA and TILs was observed in TNBC (p=0.014). The analysis of PSMA expression on the lymph nodes showed the same trend observed for the primary tumors given that it was higher in TNBC compared to HER2 positive and luminal cancers. Our results suggest that PSMA could be used as theranostic biomarker in BC considering that it is highly expressed in more aggressive tumors and the possibility to treat PSMA-expressing patients for anti-angiogenesis and/or radionuclide treatment. Citation Format: Sara Bravaccini, Stefania Cortecchia, Flavia Foca, Giovanni Martinelli, Sara Ravaioli, Maria Maddalena Tumedei, Federica Matteucci, Roberta Maltoni, Giovanni Paganelli, Francesca Poli, Maurizio Puccetti. Spotlight on PSMA as a new theranostic biomarker for breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-05.

Transvascular delivery of talaporfin sodium to subcutaneous tumors in mice by nanosecond pulsed laser-induced photomechanical waves

BackgroundWe previously developed a site-specific transvascular drug delivery system (DDS) based on photomechanical waves (PMWs) or laser-induced stress/shock waves (LISWs). In this study, we investigated the validity of this method to deliver a clinical photosensitizer, talaporfin sodium (TS), to subcutaneous tumors in mice and to enhance the efficacy of photodynamic therapy (PDT). MethodsTS solution (2.5 mg/kg) was intravenously injected into mice. Immediately thereafter, PMWs were applied to the tumor by irradiating a laser target with a Q-switched ruby laser pulse (0.8 J/cm2). Five hours after TS administration, some tumors were excised to evaluate the depth distribution of the delivered TS under a fluorescence microscope. Other tumors were subjected to PDT by irradiating the tissues with a 665 nm continuous-wave laser diode (75 mW/cm2, 667 s) at this timepoint. The effects of PDT were evaluated on the basis of the two primary therapeutic mechanisms of TS-mediated PDT: i) damage to tumor cells and ii) damage to endothelial cells of tumor vessels, i.e., the vascular shutdown effect on tumors. ResultsPMW application significantly increased the accumulation of TS in the tumor parenchyma but not in the tumor vessel walls; the endothelial cell junctions of tumor vessels should be the route of TS delivery enhanced by PMWs. Thus, as a result of PMW application followed by PDT, while the vascular shutdown effect on the tumors was not enhanced, direct damage to the tumor cells was increased, resulting in significant tumor growth retardation without body weight loss for 7 days after treatment.

Manipulating Neovasculature-Targeting Capability of Biomimetic Nanodiscs for Synergistic Photoactivatable Tumor Infarction and Chemotherapy.

Tumor infarction therapy is a promising antitumor strategy with the advantages of taking a short therapy duration, less risk of resistance, and effectiveness against a wide range of tumor types. However, its clinical application is largely hindered by tumor recurrence in the surviving rim and the potential risk of thromboembolic events due to nonspecific vasculature targeting. Herein, a neovasculature-targeting synthetic high-density lipoprotein (sHDL) nanodisc loaded with pyropheophorbide-a and camptothecin (CPN) was fabricated for photoactivatable tumor infarction and synergistic chemotherapy. By manipulating the anisotropy in ligand modification of sHDL nanodiscs, CPN modified with neovaculature-targeting peptide on the planes (PCPN) shows up to 7-fold higher cellular uptake compared with that around the edge (ECPN). PCPN can efficiently bind to endothelial cells of tumor vessels, and upon laser irradiation, massive local thrombus can be induced by the photodynamic reaction to deprive nutrition supply. Meanwhile, CPT could be released in response to the tumor reductive environment, thus killing residual tumor cells in the surviving rim to inhibit recurrence. These findings not only offer a powerful approach of synergistic cancer therapy but also suggest the potential of plane-modified sHDL nanodiscs as a versatile drug delivery nanocarrier.

Abstract 3802: Micromilieu-dependent transcriptional memory of tumor vessel endothelial cells in colorectal cancer

Abstract Purpose of the study: Tumor microenvironment (TME)-induced plasticity of fibroblasts and immune cells are known to contribute to cancer pathogenesis. In contrast, only little is known about the existence, stability, functions and prognostic potential of different plastic phenotypes of tumor vessel endothelial cells (TECs) in cancer. Recently, transcriptional memory effects have been observed in different cell lines after cytokine stimulation in vitro. From this we developed the hypothesis that isolated TEC may exhibit TME-dependent transcriptional memories, which may provide novel insights into the intratumoral effects on TEC plasticity in cancer. To investigate this, we isolated ultrapure TECs from colorectal cancer (CRC) with prognostically favorable (Th-1-like) or worse (non-Th1-like) immune-micro-environments and analyzed the prescence of TME-associated transcriptional memory patterns. Methods: TECs and as controls normal colon endothelial cells (NECs), PBMCs and tumor cells (isolated by laser-microdissection) from the same CRC patients (n=75) were isolated by FACS-sorting. Ultra high purity (98%) of TECs and NECs was validated by qPCR and immunocytochemistry. Quality validated complete sample sets from 12 patients (Th-1-like, n=6, non-Th1-like, n=6) were compared using exome sequencing, Clariome S chips and EPICmethylation. Integrative bioinformatics was used to identify differential gene sets and to predict their association with prognosis. Results: Differential transcriptional memory patterns were detected in TECs derived from CRC with different prognostic TMEs. Of note, the aggressiveness of the originating tumors was reflected by the functions of the vascular transcriptional memory genes. The transcriptional memory was imprinted by epigenetic DNA methylation and not due to acquired somatic variants in the genome. These results could be successfully confirmed by alternative methods. Most importantly, integrative bioinformatics analyses retrieved seven vascular memory genes (VMG), which were expressed in the TECs in a TME-dependent manner and predicted the outcome of patients in an independent cohort of CRC patients. Conclusion: Our findings demonstrate for the first time a TME-dependent transcriptional memory of TECs, providing a clinically relevant indicator of intra-tumoral TME-dependent vascular plasticity with perspectives for patient stratification in vessel-directed tumor therapies. Citation Format: Michael Stürzl, Maximilian Fuchs, Meik Kunz, Nicholas Dickel, Bernt Popp, Steffen Uebe, Arif B. Ekici, Carol I. Geppert, Susanne Merkel, Vera S. Schellerer, Elisabeth Naschberger. Micromilieu-dependent transcriptional memory of tumor vessel endothelial cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3802.

Expression of Prostate Specific Membrane Antigen (PSMA) in Breast Cancer.

Background Prostate specific membrane antigen (PSMA) is a promising protein for breast cancer patients. It has not only been detected in prostate cancer but is also expressed by tumor cells and the endothelial cells of tumor vessels in breast cancer patients. PSMA plays a role in tumor progression and tumor angiogenesis. For this reason, a number of diagnostic and therapeutic methods to target PSMA have been developed. Method This paper provides a general structured overview of PSMA and its oncogenic potential, with a special focus on its role in breast cancer. This narrative review is based on a selective literature search carried out in PubMed and the library of Freiburg University Clinical Center. The following key words were used for the search: “PSMA”, “PSMA and breast cancer”, “PSMA PET/CT”, “PSMA tumor progression”. Relevant articles were explicitly read through, processed, and summarized. Conclusion PSMA could be a new diagnostic and therapeutic alternative, particularly for triple-negative breast cancer. It appears to be a potential predictive and prognostic marker.

Tumor Vessel Targeted Self-Assemble Nanoparticles for Amplification and Prediction of the Embolization Effect in Hepatocellular Carcinoma.

Vessel embolization is recommended as the first line treatment for unresectable hepatocellular carcinoma (HCC). However, owing to the imprecise vessel embolization and heterogeneous response performance among patients, its survival benefits are often compromised. Herein, we reported an innovative strategy to extensively embolize the tumor by triggering the coagulation cascade, and predict the embolization effect with vessel density assessment. We synthesized manganese dioxide (MnO2)/verteporfin (BPD) nanocomposites, in which BPD bound to the tumor vessel endothelial cells (TVECs) and MnO2 nanosheets served as the carrier. MnO2 was reduced to Mn2+ ions and self-assembled with BPD to produce nanoBPD, resulting in enhanced TVECs apoptosis and coagulation cascade compared to that with free BPD. Furthermore, multimodal imaging was used to visualize tumor vessel density, which can be used as a predictor to identify the patients who would benefit from embolization. Our findings describe a promising strategy for both tumor eradication and effect prediction to improve survival benefits in unresectable HCC patients.

Abstract 3444: Intratumor regulatory T cells prevent IFN-gamma-dependent tumor vessel regression and can be selectively targeted by anti-CD25 near-infrared photoimmunotherapy

Abstract Regulatory T cells (Tregs) play pivotal roles in inhibiting anti-tumor immunity at different phases and locations (priming phase in lymph node and effector phase in tumor tissue) through their ability to suppress the function of T, NK, and antigen-presenting cells. However, our knowledge about the roles of Tregs in tumor immunity has largely derived from analyses after systemic depletion of Tregs or systemic manipulation of Treg functions in murine tumor models. The specific roles that intra- and extra-tumor Tregs play remain unclear due to technical difficulties to deplete or manipulate Tregs in a spatially specific manner. In order to achieve spatially specific depletion of Tregs in a clinically applicable manner, we have previously developed an anti-CD25 F(ab')2 near-infrared photoimmunotherapy (NIR-PIT), in which an intravenous injection of IR700-conjugated anti-CD25 F(ab')2 followed by irradiation of near-infrared light on tumors achieves selective depletion of intratumor Tregs and suppresses tumor growth. The suppression of tumor growth by the anti-CD25 F(ab')2 NIR-PIT depends on CD8 T and NK cells and their production of IFN-γ; however, the exact target of IFN-γ and the mechanisms of tumor regression remain to be elucidated. In this study, we aimed to dissect the mechanisms of the anti-tumor effect of intratumor Treg depletion by anti-CD25 F(ab')2 NIR-PIT using a series of Ifngr1 knockout mice models including bone-marrow chimera and conditional knockout models. Unexpectedly, we found that the target of IFN-γ in anti-CD25 F(ab')2 NIR-PIT was endothelial cells of tumor vessels. In both MC38 and EO771 tumor models (murine colon and breast cancer, respectively), CD8 T cells, NK cells, and Tregs were mainly located along the tumor vessels, and depletion of intratumor Tregs by anti-CD25 F(ab')2 NIR-PIT induced IFN-γ expression from these perivascular CD8 T and NK cells for up to 6 hours. This transient but synchronized induction of IFN-γ/STAT1 signaling along tumor vessels caused rapid vessel regression, intratumor ischemia, and apoptotic death of tumor cells. Combination with IL-15 treatment further eradicated residual tumor cells to achieve complete remission of MC38 and EO771 tumors. Citation Format: Yutaka Kurebayashi, Peter L. Choyke, Hisataka Kobayashi, Noriko Sato. Intratumor regulatory T cells prevent IFN-gamma-dependent tumor vessel regression and can be selectively targeted by anti-CD25 near-infrared photoimmunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3444.

Improving the antitumor activity of R-CHOP with NGR-hTNF in primary CNS lymphoma: final results of a phase 2 trial

AbstractRituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is the standard treatment of diffuse large B-cell lymphoma (DLBCL). Primary DLBCL of the central nervous system (CNS) (primary central nervous system lymphoma [PCNSL]) is an exception because of the low CNS bioavailability of related drugs. NGR–human tumor necrosis factor (NGR-hTNF) targets CD13+ vessels, enhances vascular permeability and CNS access of anticancer drugs, and provides the rationale for the treatment of PCNSL with R-CHOP. Herein, we report activity and safety of R-CHOP preceded by NGR-hTNF in patients with PCNSL relapsed/refractory to high-dose methotrexate-based chemotherapy enrolled in a phase 2 trial. Overall response rate (ORR) was the primary endpoint. A sample size of 28 patients was considered necessary to demonstrate improvement from 30% to 50% ORR. NGR-hTNF/R-CHOP would be declared active if ≥12 responses were recorded. Treatment was well tolerated; there were no cases of unexpected toxicities, dose reductions or interruptions. NGR-hTNF/R-CHOP was active, with confirmed tumor response in 21 patients (75%; 95% confidence interval, 59%-91%), which was complete in 11. Seventeen of the 21 patients with response to treatment received consolidation (ASCT, WBRT, and/or lenalidomide maintenance). At a median follow-up of 21 (range, 14-31) months, 5 patients remained relapse-free and 6 were alive. The activity of NGR-hTNF/R-CHOP is in line with the expression of CD13 in both pericytes and endothelial cells of tumor vessels. High plasma levels of chromogranin A, an NGR-hTNF inhibitor, were associated with proton pump inhibitor use and a lower remission rate, suggesting that these drugs should be avoided during TNF-based therapy. Further research on this innovative approach to CNS lymphomas is warranted. The trial was registered as EudraCT: 2014-001532-11.

Species-, organ- and cell-type-dependent expression of SPARCL1 in human and mouse tissues.

SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ- and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice.

Tumor vasculature remolding by thalidomide increases delivery and efficacy of cisplatin

BackgroundA promising strategy to overcome the chemoresistance is the tumor blood vessel normalization, which restores the physiological perfusion and oxygenation of tumor vasculature. Thalidomide (Thal) has been shown to increase the anti-tumor effect of chemotherapy agents in solid tumors. However, it is not yet known whether the synergistic effect of Thal combined with other cytotoxic drugs is attributable to tumor vascular normalization.MethodsWe used two homograft mice models (4 T1 breast tumor model and CT26 colorectal tumor model) to investigate the effect of Thal on tumor growth, microvessel density, vascular physiology, vascular maturity and function, drug delivery and chemosensitivity. Immunofluorescence, immunohistochemistry and scanning electron microscopy were performed to determine the vessel changes. Protein array assay, qPCR and western blotting were used to detect the molecular mechanism by which Thal regulates tumor vascular.ResultsHere we report that Thal potently suppressed tumor growth, angiogenesis, hypoxia, and vascular permeability in animal models. Thal also induced a regular monolayer of endothelial cells in tumor vessels, inhibiting vascular instability, and normalized tumor vessels by increasing vascular maturity, pericyte coverage and endothelial junctions. The tumor vessel stabilization effect of Thal resulted in a decrease in tumor vessel tortuosity and leakage, and increased vessel thickness and tumor perfusion. Eventually, the delivery of cisplatin was highly enhanced through the normalized tumor vasculature, thus resulting in profound anti-tumor and anti-metastatic effects. Mechanistically, the effects of Thal on tumor vessels were caused in part by its capability to correct the imbalance between pro-angiogenic factors and anti-angiogenic factors.ConclusionsOur findings provide direct evidence that Thal remodels the abnormal tumor vessel system into a normalized vasculature. Our results may lay solid foundation for the development of Thal as a novel candidate agent to maximize the therapeutic efficacy of chemotherapeutic drugs for solid tumors.

Extragonadal FSHR Expression and Function-Is It Real?

Expression of the follicle-stimulating hormone receptor (FSHR), besides gonadal tissues, has recently been detected in several extragonadal normal and tumorous tissues, including different types of primary and metastatic cancer and tumor vessel endothelial cells (TVEC). The suggested FSH actions in extragonadal tissues include promotion of angiogenesis, myometrial contractility, skeletal integrity, and adipose tissue accumulation. Non-malignant cells within cancer tissue have been shown to be devoid of FSHR expression, which implies a potential role of FSHR as a diagnostic, prognostic, or even a therapeutic tool. There are shared issues between several of the published reports questioning the validity of some of the conclusion. Firstly, protein expression of FSHR was performed solely with immunohistochemistry (IHC) using either an unavailable “in house” FSHR323 monoclonal antibody or poorly validated polyclonal antibodies, usually without additional methodological quality control and confirmations. Secondly, there is discrepancy between the hardly traceable or absent FSHR gene amplification/transcript data and non-reciprocal strong FSHR protein immunoreactivity. Thirdly, the pharmacological high doses of recombinant FSH used in in vitro studies also jeopardizes the physiological or pathophysiological meaning of the findings. We performed in this review a critical analysis of the results presenting extragonadal expression of FSHR and FSH action, and provide a rationale for the validation of the reported results using additional more accurate and sensitive supplemental methods, including in vivo models and proper positive and negative controls.

Specific tissue factor delivery using a tumor-homing peptide for inducing tumor infarction

Targeting the human blood coagulation-inducing protein tissue factor (TF) to the tumor vasculature to induce infarction and disrupt the blood vessels has proven to be an effective approach for tumor therapy. In this study, we investigated the thrombogenic activity and anti-tumor potential of a novel fusion protein (tTF-CREKA) comprising the extracellular domain of human tissue factor (truncated TF, tTF) and a tumor targeting pentapeptide, Cys-Arg-Glu-Lys-Ala (CREKA). tTF is soluble and inactive in its free state, but when it is targeted to the plasma membrane of both tumor vessel endothelial cells and stromal cells by the CREKA peptide, its native coagulation-inducing activity is restored. Systemic administration of the tTF-CREKA fusion protein into tumor-bearing mice induced tumor-selective intravascular thrombosis and reduced tumor blood perfusion, consequently inhibiting tumor growth. The development of tTF-CREKA introduces a new method for treating a wide spectrum of solid tumors by selectively blocking tumor blood supply.

Roles of Albumin-Binding Proteins in Cancer Progression and Biomimetic Targeted Drug Delivery.

Nutrient transporters have attracted significant attention for their promising application in biomimetic delivery. Due to the active consumption of nutrients, cancer cells generally overexpress nutrient transporters to meet their increased need for energy and materials. For example, albumin-binding proteins (ABPs) are highly overexpressed in malignant cells, stromal cells, and tumor vessel endothelial cells responsible for albumin uptake. ABP (e.g., SPARC) is a promising target for tumor-specific drug delivery, and albumin has been widely used as a biomimetic delivery carrier. Apart from the transportation function, ABPs are closely associated with neoplasia, invasion, and metastasis. Herein, a summary of the roles of ABP in cancer progression and the application of albumin-based biomimetic tumor-targeted delivery through the ABP pathway is presented.

L-type amino acid transporter 1 (LAT1) in endothelial cells of tumor vessels contributes to tumor angiogenesis

L-type amino acid transporter 1 (LAT1) in endothelial cells of tumor vessels contributes to tumor angiogenesis

Pericyte-targeting prodrug overcomes tumor resistance to vascular disrupting agents.

Blood vessels in the tumor periphery have high pericyte coverage and are resistant to vascular disrupting agents (VDAs). VDA treatment resistance leads to a viable peripheral tumor rim that contributes to treatment failure and disease recurrence. Here, we provide evidence to support a hypothesis that shifting the target of VDAs from tumor vessel endothelial cells to pericytes disrupts tumor peripheral vessels and the viable rim, circumventing VDA treatment resistance. Through chemical engineering, we developed Z-GP-DAVLBH (from the tubulin-binding VDA desacetylvinblastine monohydrazide [DAVLBH]) as a prodrug that can be selectively activated by fibroblast activation protein α (FAPα) in tumor pericytes. Z-GP-DAVLBH selectively destroys the cytoskeleton of FAPα-expressing tumor pericytes, disrupting blood vessels both within the core and around the periphery of tumors. As a result, Z-GP-DAVLBH treatment eradicated the otherwise VDA-resistant tumor rim and led to complete regression of tumors in multiple lines of xenografts without producing the drug-related toxicity that is associated with similar doses of DAVLBH. This study demonstrates that targeting tumor pericytes with an FAPα-activated VDA prodrug represents a potential vascular disruption strategy in overcoming tumor resistance to VDA treatments.

Distinctive role of vasohibin-1A and its splicing variant vasohibin-1B in tumor angiogenesis.

Vasohibin-1 (VASH1) was isolated as a negative-feedback regulator of angiogenesis expressed in endothelial cells (ECs). There are two transcripts of VASH1, that is, the full-length VASH1A consisting of seven exons and the splicing variant VASH1B consisting of four exons. Here, we compared the effects of VASH1A and VASH1B on tumor angiogenesis. When ECs were transfected with VASH1A or VASH1B cDNAs, VASH1B transfectants, but not VASH1A ones, induced autophagic cell death of ECs. With sonoporation, the VASH1A or VASH1B gene were transfected specifically in ECs of tumor vessels in mice. Both VASH1A and VASH1B decreased tumor vessel density and inhibited tumor growth. VASH1A normalized the remaining tumor vessels, increased their rate of perfusion, decreased tumor hypoxia and enhanced the efficacy of anticancer chemotherapy, whereas VASH1B pruned tumor vessels without causing normalization, increased tumor hypoxia and tumor necrosis and did not enhance the efficacy of anticancer chemotherapy. The alternate transfection of mice with the VASH1A and VASH1B gene showed the highest effects on antitumor activity and normalization of tumor vessels. Our present findings on VASH1A and VASH1B should provide an innovative approach that would improve the efficacy of antiangiogenic cancer therapy by balancing vascular normalization and pruning.

Activation of multiple angiogenic signaling pathways in hemangiopericytoma.

Hemangiopericytoma (HPC) is a highly vascularized mesenchymal tumor. Local recurrence and distant metastasis are common features of HPC. Considering the remarkable hyper-vasculature phenotype of HPC, we assumed that dysregulated angiogenic signaling pathways were involved in HPC. The key components of angiogenic signaling pathways including VEGF-VEGF-R2, EphrinB2-EphB4 and DLL4-Notch were examined by real-time RT-PCR, Western blotting and immunostaining in 17 surgical specimens of HPC patients and in 6 controls. A significant upregulation of VEGF and VEGF-R2 associated with elevated levels of p-Akt and proliferating cell nuclear antigen (PCNA) was detected in HPC. Moreover, a dramatic increase in the mRNA and protein expression of EphB4 and its downstream factor p-Erk1/2 was found in HPC. A massive activation of core-components of DLL4-Notch signaling was detected in HPC. Double-immunofluorescent staining confirmed the expression of these upregulated key factors in the endothelial cells of tumor vessels. The present study identified the activation of multiple and crucial angiogenic signaling pathways, which could function individually and/or synergistically to stimulate angiogenesis in HPC and eventually contribute to tumor growth and progression. Our findings emphasize the importance to target multiple angiogenic signaling pathways when an anti-angiogenic therapy is considered for this highly vascularized tumor.

Temporal bone carcinoma: a first glance beyond the conventional clinical and pathological prognostic factors

Temporal bone squamous cell carcinoma (TBSCC) is an uncommon, aggressive malignancy with a poor prognosis in advanced cases. The dismal outcome is partially related to: the lack of reliable clinical or pathological prognostic factors and the largely unstandardized surgical and integrated treatments adopted. There is an undeniable need for novel diagnostic/therapeutic strategies to improve the prognosis. The purpose of this critical review was to explore the level of available knowledge concerning the molecular markers involved in the biology of TBSCC that have a prognostic potential. The Pub-Med and Scopus electronic databases were searched without publication date limits for studies investigating molecular markers in cohorts of patients with primary TBSCC. The search terms used were: "temporal bone cancer", "temporal bone carcinoma", "temporal bone malignancy", "ear cancer", "ear carcinoma", and "ear malignancy". We decided preliminarily not to consider series with less than five cases. Nine retrospective case series of TBSCC were found in which different analytical techniques had been used to study the role of several biomarkers (HPV, vimentin, transforming growth factor β, CD105, RECK, matrix metalloproteinase-9, MASPIN, EBV, p16, TP53 mutation, pSTAT3, relaxin-2). CD105 expression (in tumor vessel endothelial cells) and MASPIN cytoplasmic expression (in carcinoma cells) were, respectively, found directly and inversely related with the neoplasm's recurrence rate. CD105 expression was also inversely related with disease-free survival in TBSCC. A future goal of such analyses should be to ascertain the radio- and chemo-sensitivity profiles of individual TBSCCs, enabling truly personalized therapies. A further, more ambitious goal will be to find targets for therapeutic agents that might prove crucial in improving the disease-specific survival for patients with advanced TBSCC.

Anginex lipoplexes for delivery of anti-angiogenic siRNA

Angiogenesis is one of the hallmarks of cancer which renders it an attractive target for therapy of malignancies. Tumor growth suppression can be achieved by inhibiting angiogenesis since it would deprive tumor cells of oxygen and vital nutrients. Activation of endothelial cells of tumor vasculature is the first step in angiogenesis which is mediated by various factors. One of the major triggers in this process is vascular endothelial growth factor (VEGF) which binds to VEGF receptors on endothelial cells of tumor vessels. This induces a series of signaling cascades leading to activation of cellular processes involved in angiogenesis, and therefore down-regulation of VEGF receptor-2 (VEGFR-2) expression seems a viable option to inhibit angiogenesis. In our investigations, this aim has been pursued by using siRNA interfering with the expression of VEGFR-2. Since the discovery of RNA interference (RNAi) as a gene regulation process, successful delivery of small non-coding RNA has presented itself as a major challenge. In the current study, we have characterized a galectin-1 targeted anginex-coupled lipoplex (Angiplex) containing siRNA against the gene of VEGFR-2 as an angiostatic therapeutic. Angiplex particles had a size of approximately 120nm with a net negative charge and were stable in vitro. These particles were internalized in a specific manner by HUVECs compared to a non-targeted lipoplex system, and their uptake was higher than Lipofectamine 2000. Gene silencing efficiency of Angiplex was shown to be 61%.

Non-invasive monitoring of tumor-vessel infarction by retargeted truncated tissue factor tTF–NGR using multi-modal imaging

The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmallsuperparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.