Temporal analysis of type 1 interferon activation in tumor cells following external beam radiotherapy or targeted radionuclide therapy.

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies.

Extracellular Vesicles Shedding Promotes Melanoma Growth in Response to Chemotherapy.

Extracellular vesicles (EVs) are emerging as key players in intercellular communication. EVs can transfer biological macromolecules to recipient cells, modulating various physiological and pathological processes. It has been shown that tumor cells secrete large amounts of EVs that can be taken up by malignant and stromal cells, dictating tumor progression. In this study, we investigated whether EVs secreted by melanoma cells in response to chemotherapy modulate tumor response to alkylating drugs. Our findings showed that human and murine melanoma cells secrete more EVs after treatment with temozolomide and cisplatin. We observed that EVs shed by melanoma cells after temozolomide treatment modify macrophage phenotype by skewing macrophage activation towards the M2 phenotype through upregulation of M2-marker genes. Moreover, these EVs were able to favor melanoma re-growth in vivo, which was accompanied by an increase in Arginase 1 and IL10 gene expression levels by stromal cells and an increase in genes related to DNA repair, cell survival and stemness in tumor cells. Taken together, this study suggests that EVs shed by tumor cells in response to chemotherapy promote tumor repopulation and treatment failure through cellular reprogramming in melanoma cells.

Flower extracts from Paeonia decomposita and Paeonia ostii inhibit melanin synthesis via cAMP-CREB-associated melanogenesis signaling pathways in murine B16 melanoma cells.

This investigation seeks to identify the effects of the EtOAc fractions of different flower parts of Paeonia decomposita (Pd) and Paeonia ostii (Po) on melanogenesis and their mechanisms of action in B16 melanoma cells. Cell viability assay showed that Pd-1, Po-1 (the petals of Pd or Po), Pd-3 and Po-3 (the stamens of Pd or Po) at 25 µg/ml produced lower toxic activities in B16 cells. Pd-1 and Po-1 extracts considerably reduced the melanin content and inhibited tyrosinase and DOPA oxidase activity. Moreover, Pd-1 and Po-1 down-regulated the expressions of MC1R, MITF, TRP-1, TRP-2, and tyrosinase. These extracts also reduce cAMP levels and inhibited the phosphorylation of CREB, which might be due to the presence of high concentrations of phenolic compounds and flavonoids. Our results suggested that Pd-1 and Po-1 are able to modulate the cAMP-CREB signaling pathway and down-regulate the melanogenesis-related proteins resulting in the observed anti-melanogenic effects. PRACTICAL APPLICATIONS: In China, the flower of Paeonia is often consumed as a dietary supplement and as an additive in skin whitening products. In November 2013, the National Health and Family Planning Commission of the People's Republic of China has approved the flower of Paeonia ostii as a novel food resource. The current study firstly demonstrated that the effects of EtOAc fractions of the petals of Paeonia decomposita (Pd) and Paeonia ostii (Po) considerably reduced the melanin content in B16 cells, which is due to the modulation of the cAMP-CREB signaling pathway followed by the down-regulation of melanogenesis-related proteins. Pd and Po extracts, as natural tyrosinase inhibitors, may serve as good candidates in food additives, cosmetic materials, or even in treating hyperpigmentation diseases.

Factors Affecting Growth Kinetics and Spontaneous Metastasis in the B16F10 Syngeneic Murine Melanoma Model.

Melanoma is an immunogenic tumor that can metastasize quickly to proximal and distal sites, thus complicating the application of therapeutic modalities. Numerous mouse model systems have been used to gain understanding of the immunobiology and metastatic potential of melanoma. Here, we report the optimization of a syngeneic mouse melanoma model protocol using the mouse B16-derived melanoma cell line B16F10 that ensures the production of tumors on mice pinnae that are similar in size between animals and that enlarge in a time-dependent manner. In this model, B16F10 cells are first allowed to develop tumors after injection in the interscapular area or flank of C57BL/6J mice. Subsequently, the tumors are harvested, cells dissociated and injected into mouse pinnae. Dose-dependent studies revealed that injection of 2 × 105 cells allowed for slow tumor enlargement, producing tumors averaging 100 mm³ within 2 to 3 wk with a metastatic frequency of 100%. This experimental protocol will be useful in dissecting the immunobiology of melanoma tumor development and metastasis and the evaluation of immunotherapeutic antimelanoma therapies.

Multiparametric High-Resolution MRI as a Tool for Mapping of Hypoxic Level in Tumors.

Hypoxia is a condition, common to most malignant tumors, where oxygen tension in the tissue is below the physiological level. Among consequences of tumor hypoxia is also altered cancer cell metabolism that contributes to cancer therapy resistance. Therefore, precise assessment of tumor hypoxia is important for monitoring the tumor treatment progression. In this study, we propose a simple model for prediction of hypoxic level in tumors based on multiparametric magnetic resonance imaging. The study was performed on B16F1 murine melanoma tumors ex vivo that were first magnetic resonance scanned and then analyzed for hypoxic level using hypoxia-inducable factor 1-alpha antibody staining. Each tumor was analyzed in identical sections and in identical regions of interest for pairs of hypoxic level and magnetic resonance values (apparent diffusion coefficient and T2). This was followed by correlation analysis between hypoxic level and respective magnetic resonance values. A moderate correlation was found between hypoxic level and apparent diffusion coefficient (ρ = 0.56, P < .00001) and lower between hypoxic level and T2 (ρ = 0.38, P < .00001). The data were analyzed further to obtain simple predictive models based on the multiple linear regression analysis of the measured hypoxic level (dependent variable) and apparent diffusion coefficient and T2 (independent variables). Among the hypoxic level models, the most efficient was the 3-parameter model given by relation ( HL = kADC ADC + kT2 T2 + b), where kADC = 26%/µm2/ms, kT2 = 0.8%/ms, and b = -32%. The model can be used for calculation of the predicted hypoxic level map based on magnetic resonance-measured apparent diffusion coefficient and T2 maps. Similar prediction models, based on tumor apparent diffusion coefficient and T2 maps, can be done also for other tumor types in vivo and can therefore help in assessment of tumor treatment as well as to better understand the role of hypoxia in cancer progression.

Effects of volatile oil from ginger on the murine B16 melanoma cells and its mechanism.

In this study, the inhibitory effects of volatile oil from ginger on melanogenesis and its antioxidant characteristics were investigated. The effects of volatile oil from ginger on cell proliferation, melanin content and tyrosinase activity were determined using a microplate reader. In addition, the expression of melanogenesis-related protein was determined by western blotting. The results indicate that the tested concentrates of volatile oil from ginger showed inhibitory effects on cell proliferation and melanogenesis. This revealed that volatile oil from ginger exhibited apparent capacities for scavenging ROS and lipid peroxidation. Furthermore, volatile oil from ginger improved the activities of GSH, SOD and CAT in B16 melanoma cells. These results demonstrated that volatile oil from ginger suppressed melanin synthesis through its antioxidant properties and the inhibitory effects on tyrosinase activity and melanogensis-related proteins. Hence, volatile oil from ginger could be used as an effective skin-whitening agent applied in food industry.

EFFECT OF FUCOIDAN ON B16 MURINE MELANOMA CELL MELANIN FORMATION AND APOPTOSIS.

BACKGROUND: Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. It not only inhibits cancer cell growth but also inhibits tyrosinase in vitro. Therefore, it is of interest to investigate the effect of fucoidan on B16 murine melanoma cells as the findings may provide new insights into the underlying mechanism regarding the inhibition of melanin formation by fucoidan. In the present study, we aimed to investigate the anti-melanogenic effect of fucoidan and its inhibitory effect on B16 cells. MATERIALS AND METHODS: The influence of fucoidan on B16 melanoma cells and cellular tyrosinase was examined. Cell viability was examined by the cell counting kit-8 assay. Cellular tyrosinase activity and melanin content were determined using spectrophotometric methods and protein expression was analyzed by immunoblotting. Morphological changes in B16 melanoma cells were examined by phase contrast microscopy and apoptosis was analyzed by flow cytometry. RESULTS: In vitro studies were performed using cell viability analysis and showed that fucoidan significantly decreased viable cell number in a dose-response manner with an IC50 of 550 ±4.3 µg/mL. Cell morphology was altered and significant apoptosis was induced when cells were exposed to 550 µg/mL fucoidan for 48 h. CONCLUSION: This study provides substantial evidence to show that fucoidan inhibits B16 melanoma cell proliferation and cellular tyrosinase activity. Fucoidan may be useful in the treatment of hyperpigmentation and as a skin-whitening agent in the cosmetics industry.

The spider toxin Phα1ß recombinant possesses strong analgesic activity.

The native Phα1ß - a Voltage-Gated Calcium Channel (VGCC) blocker - and its Recombinant Version - were both tested in rodent pain models with an intraplantar injections of capsaicin or formalin, a chronic constriction injury, and melanoma cancer related pain. The formalin nociceptive behaviour in the neurogenic phase was not affected by the toxin pre-treatments, while in the inflammatory phase, Phα1ß and the Recombinant form caused a significant reduction. The nociception that was triggered by capsaicin, an agonist of the TRPV1 vanilloid receptor, was totally blocked by 100 pmol/site, i.t. of Phα1ß or the recombinant version. For the neuropathic pain that was induced by a chronic constriction injury of the sciatic nerve, Phα1ß and its Recombinant reduced the allodynia that was induced by the CCI procedure in the rats and the hypersensitivity lasted for 4 h. Fourteen days after the inoculation of the B16-F10 melanoma cells in the mice, a marked hyperalgesia was induced in the melanoma cancer pain model. Phα1ß and the Recombinant form reduced the hyperalgesia with a full reversion at 100 pmol/site i.t. The inhibitory effects of the nociception that was induced by native Phα1ß and the Recombinant in the studied pain models were not statistically different and they developed with no side effects.

Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential.

AIM: To evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice. METHODS: Impact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer. RESULTS: Selenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals. CONCLUSIONS: Our results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.

Mesenchymal stem cells induce epithelial mesenchymal transition in melanoma by paracrine secretion of transforming growth factor-ß.

Mesenchymal stem cells (MSCs) are considered for potential use as an ideal vehicle to efficiently deliver therapeutic agents in treatment against cancers including melanoma. However, emerging evidence indicates that MSCs promote tumor growth and progression. Therefore, a comprehensive understanding of the role of MSCs is very important to evaluate the MSCs-based therapy in melanoma. B16 melanoma cells treated by MSC conditioned medium (CM), showed significantly enhanced migration and invasion, which was also confirmed in a lung metastasis mice model in vivo. Later, it was found that MSC CM induced an epithelial mesenchymal transition (EMT) in B16 cells. The ELISA assay showed that transforming growth factor-ß (TGF-ß) was secreted by MSCs and EMT was also induced by recombinant TGF-ß in B16 melanoma cells, which suggests the important role of TGF-ß in mediating EMT caused by MSC CM. When TGF-ß signaling was inhibited by SB431542, the EMT process was significantly eliminated in vitro and in xenograft tumors in vivo. Snail, as a downstream target of TGF-ß signaling and an EMT regulator, was upregulated by MSC CM and inhibited by SB431542, which confirms the key role of TGF-ß signaling in EMT induced by MSC CM in B16 cells. Taken together, this study shows that MSC induces EMT in melanoma cells in a paracrine manner, which might be mediated by the TGF-ß/Snail signaling pathway. Thus, caution should be exercised when considering MSCs-based therapy in melanoma and also in other cancers. Targeting TGF-ß signaling and Snail could be further investigated as potential therapeutic approaches for melanoma.

Forsythiae Fructus Inhibits B16 Melanoma Growth Involving MAPKs/Nrf2/HO-1 Mediated Anti-Oxidation and Anti-Inflammation.

Forsythiae Fructus, the fruits of Forsythia suspensa (Thunb.) Vahl, Lianqiao in Chinese, is one of the most fundamental herbs in traditional Chinese medicine (TCM). It is a typical heat-clearing and detoxicating herb, according to TCM theory. In this study, we investigated the antitumor effect of Forsythiae Fructus aqueous extract (FAE) on B16-F10 melanoma cells in vivo. The transplanted B16-F10 melanoma in C57BL/6 mice was established and used for the evaluation of the in vivo antitumor effect of FAE. FAE strongly inhibited the growth of B16-F10 cells in vitro and the tumor in vivo. The survival time of tumor-bearing mice was significantly prolonged by FAE. FAE inhibited cancer cell proliferation and angiogenesis in the tumor, as indicated by the decreased expressions of Ki67 and CD31. The levels of ROS, MDA, TNF-[Formula: see text] and IL-6 decreased, while GSH increased in the FAE treatment group, indicating FAE possesses strong anti-oxidative and anti-inflammatory activity. The expression of anti-oxidant proteins Nrf-2 and HO-1, tumor suppressors P53 and p-PTEN, and the MAPK pathways in tumor tissues were upregulated by FAE treatment. These data demonstrated that FAE exhibited strong antitumor activity against B16-F10 murine melanoma both in vitro and in vivo. The antitumor effect of FAE involved decreases in oxidative stress and inflammation in the tumor, which is closely related to the heat-clearing and detoxicating properties of FAE.

Primary melanoma tumor inhibits metastasis through alterations in systemic hemostasis.

Progression from a primary tumor to distant metastases requires extensive interactions between tumor cells and their microenvironment. The primary tumor is not only the source of metastatic cells but also can also modulate host responses to these cells, leading to an enhancement or inhibition of metastasis. Tumor-mediated stimulation of bone marrow can result in pre-metastatic niche formation and increased metastasis. However, a primary tumor can also inhibit metastasis through concomitant tumor resistance-inhibition of metastatic growth by existing tumor mass. Here, we report that the presence of a B16F10 primary tumor significantly restricted numbers and sizes of experimental lung metastases through reduction of circulating platelets and reduced formation of metastatic tumor cell-associated thrombi. Tumor-bearing mice displayed splenomegaly, correlated with primary tumor size and platelet count. Reduction in platelet numbers in tumor-bearing animals was responsible for metastatic inhibition, as restoration of platelet numbers using isolated platelets re-established both tumor cell-associated thrombus formation and experimental metastasis. Consumption of platelets due to a B16F10 primary tumor is a form of concomitant tumor resistance and demonstrates the systemic impact of a growing tumor. Understanding the interplay between primary tumors and metastases is essential, as clarification of concomitant tumor resistance mechanisms may allow inhibition of metastatic growth following tumor resection. Key messages Mice with a primary B16F10 tumor had reduced metastasis vs. mice without a primary tumor. Tumor-bearing mice had splenomegaly and fewer platelets and tumor-associated thrombi. Restoring platelets restored tumor-associated thrombi and increased metastasis. This work shows the impact that a primary tumor can have on systemic metastasis. Understanding these interactions may lead to improved ways to inhibit metastasis.

Application of vasoactive and matrix-modifying drugs can improve polyplex delivery to tumors upon intravenous administration.

Low efficacy of cationic polymer-based formulations (polyplexes) for systemic gene delivery to tumors remains the crucial concern for their clinical translation. Here we show that modulating the physiological state of a tumor using clinically approved pharmaceuticals can improve delivery of intravenously injected polyplexes to murine melanoma tumors with different characteristics. Direct comparison of drugs with different mechanisms of action has shown that application of nitroglycerin or losartan improved extravasation and tumor uptake of polyplex nanoparticles, whereas angiotensin II had almost no effect on polyplex accumulation and microdistribution in the tumor tissue. Application of nitroglycerin and losartan caused from 2- to 6-fold enhanced efficacy of polyplex-mediated gene delivery depending on the tumor model. The results obtained on polyplex behavior in tumor tissues depending on physiological state of the tumor can be relevant to optimize delivery of polyplexes and other nanomedicines with similar physicochemical properties.

The protein kinase IKKepsilon contributes to tumour growth and tumour pain in a melanoma model.

Inhibitor-kappaB kinase epsilon (IKKε) constitutes a non-canonical I-κB kinase, which amongst others modulates NF-κB activity. IKKε and NF-κB have both been described for their role in cell proliferation and their dysregulation has been associated with tumourigenesis and metastasis in multiple cancer types. Accordingly, overexpression and constitutive activation of NF-κB have also been shown in melanoma, however, the role of IKKε in this cancer type has not been investigated so far. Thus, we determined IKKε expression in malignant melanoma cells and we were able to show a significant overexpression of IKKε in tumour cells in comparison to melanocytes. Inhibition of IKKε either by shRNA or the pharmacological inhibitor amlexanox resulted in reduced cell proliferation associated with a cell cycle block in the G1-phase. Functional analysis indicated that NF-κB, Akt1 and MAPK pathways might be involved in the IKKε-mediated effects. In vivo, we applied a mouse melanoma skin cancer model to assess tumour growth and melanoma-associated pain in IKKε knockout mice as well as C57BL/6 mice after inoculation with IKKε-negative cells. In IKKε knockout mice, tumour growth was not altered as compared to IKKε wild type mice. However, melanoma associated pain was strongly suppressed accompanied by a reduced mRNA expression of a number of pain-relevant genes. In contrast, after inoculation of IKKε-depleted tumour cells, the development of melanoma was almost completely prevented. In conclusion, our data suggest that IKKε in the tumour plays an essential role in tumour initiation and progression while IKKε expression in tumour surrounding tissues contributes to melanoma-associated pain.

Butein inhibits metastatic behavior in mouse melanoma cells through VEGF expression and translation-dependent signaling pathway regulation.

BACKGROUND: Melanoma is an aggressive skin cancer and a predominant cause of skin cancer-related deaths. A previous study has demonstrated the ability of butein to inhibit tumor proliferation and invasion. However, the anti-metastatic mechanisms and in vivo effects of butein have not been fully elucidated. METHODS: MTT cell viability assays were used to evaluate the antitumor effects of butein in vitro. Cytotoxic effects of butein were measured by lactate dehydrogenase assay. Anti-migratory effects of butein were evaluated by two-dimensional scratch and transwell migration assays. Signaling transduction and VEGF-releasing assays were measured by Western blotting and ELISA. We also conducted an experimental analysis of the metastatic potential of tumor cells injected into the tail vein of C57BL/6 mice. RESULTS: We first demonstrated the effect of butein on cell viability at non-cytotoxic concentrations (1, 3, and 10 µM). In vitro, butein was found to inhibit the migration of B16F10 cells in a concentration-dependent manner using transwell and scratch assays. Butein had a dose-dependent effect on focal adhesion kinase, Akt, and ERK phosphorylation in B16F10 cells. Butein efficiently inhibited the mTOR/p70S6K translational inhibition machinery and decreased the production of VEGF in B16F10 cells. Furthermore, the in vivo antitumor effects of butein were demonstrated using a pulmonary metastasis model. CONCLUSION: The results of the present study indicate the potential utility of butein in the treatment of melanoma.

Oncogenic properties of apoptotic tumor cells in aggressive B cell lymphoma.

BACKGROUND: Cells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects. RESULTS: Here, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased "in situ transcriptomics" analysis-gene expression profiling of laser-captured TAMs to establish their activation signature in situ-we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma. CONCLUSIONS: In addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis.

The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6ß1. In contrast, the binding of α6ß1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6ß1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.

Spatio-temporal changes of lymphatic contractility and drainage patterns following lymphadenectomy in mice.

OBJECTIVE: To investigate the redirection of lymphatic drainage post-lymphadenectomy using non-invasive near-infrared fluorescence (NIRF) imaging, and to subsequently assess impact on metastasis. BACKGROUND: Cancer-acquired lymphedema arises from dysfunctional fluid transport after lymphadenectomy performed for staging and to disrupt drainage pathways for regional control of disease. However, little is known about the normal regenerative processes of the lymphatics in response to lymphadenectomy and how these responses can be accelerated, delayed, or can impact metastasis. METHODS: Changes in lymphatic "pumping" function and drainage patterns were non-invasively and longitudinally imaged using NIRF lymphatic imaging after popliteal lymphadenectomy in mice. In a cohort of mice, B16F10 melanoma was inoculated on the dorsal aspect of the paw 27 days after lymphadenectomy to assess how drainage patterns affect metastasis. RESULTS: NIRF imaging demonstrates that, although lymphatic function and drainage patterns change significantly in early response to popliteal lymph node (PLN) removal in mice, these changes are transient and regress dramatically due to a high regenerative capacity of the lymphatics and co-opting of collateral lymphatic pathways around the site of obstruction. Metastases followed the pattern of collateral pathways and could be detected proximal to the site of lymphadenectomy. CONCLUSIONS: Both lymphatic vessel regeneration and co-opting of contralateral vessels occur following lymphadenectomy, with contractile function restored within 13 days, providing a basis for preclinical and clinical investigations to hasten lymphatic repair and restore contractile lymphatic function after surgery to prevent cancer-acquired lymphedema. Patterns of cancer metastasis after lymphadenectomy were altered, consistent with patterns of re-directed lymphatic drainage.

Pathways of tumor development and progression in drug-induced nonmelanoma skin cancer: a new hope or the next great confusion?

The factors that lead to the clinical manifestation of the nonmelanocytic skin tumors are different. Ultraviolet radiation, infections with human papillomaviruses, and inherited or iatrogenic-induced immunosuppression (in cases of autoimmune diseases and organ transplant recipients) are considered to be some of the most important generators and/or costimulating factors supporting the appearance of "de-novo" mutations and obstruct, in one or another way, the cell cycle arrest, the programmed cell death (apoptosis), and the immunosurveillance. Preconditions are thus created for the initial persistence and subsequent proliferation of the malignant cell branch in the genome, with the simultaneous increase of the risk of nonmelanocytic skin tumor manifestation.A number of medical drugs that possess a currently well-known selective, targeting, and immunomodulating effect, like the TNF-alpha inhibitors for example, most probably possess an additional blocking action on the death receptors within the framework of the extrinsic apoptotic pathway. In this way, they seem to be one of the major factors for the clinical manifestation not only of nonmelanocytic skin but also of a number of other type of tumors with a dependency on the genetic predisposition of each separate patient.This article focuses the attention on the basic exogenic and endogenic factors that affect the regulatory processes of the cellular cycle, apoptosis, immunosurveillance, and the human inflammasome in patients with nonmelanocytic skin tumors. These processes are interwoven in a complex network and are controlled by (1) the genome regulator p53, (2) its interaction with the proapoptotic acting proteins Bak and Bax, (3) as well as the interaction with the key regulatory protein of the inflammasome-ASC/TMS1.As a process, the malignant transformation is exceptionally dynamic, plastic, and adaptive. The exterior "interferences", on the part of the clinician, in the form of a planned therapy should be targeted at the simultaneous impact on the various pathogenetic chains with the objective of bringing the tumor cells to their total collapse. This can be made possible only after the careful and simultaneous-or parallel-examination of a much greater number of markers that serve to characterize the process of the malignant transformation-a fact, which is currently being disregarded by many researchers.