34 research outputs found

    Identification and Preliminary Validation in Mouse Models of Circulating Biomarkers of Pancreatic Cancer

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    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal oncological malignancies in humans. Not-specific symptoms and lack of early diagnostic strategies, frequently lead to late diagnosis which limited therapeutic possibilities (Korc, 2007). The present study aimed at identifying novel potential serum biomarkers for early detection of PDAC.In the first phase, two different mouse models of PDAC were characterized: genetically engineered mice (GEMs) (Hingorani et al., 2003) which developed PanIN (pancreatic intraepithelial neoplasia) lesions and three PDAC patient-derived xenograft.In the second phase, the two mouse models were used to evaluate the reliability of 3 circulating molecules as early diagnostic biomarkers of PDAC. The plasma levels of matrix metalloproteinase-7 (MMP-7), tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) and thrombospondin-2 (THBS-2) were tested on GEMs and PDAC-PDXs bearing mice by ELISA tests, during tumor development, and at sacrifice by immunohistochemistry performed on pancreatic tissue.The three established PDAC-PDXs were found to better reproduce the tumor of origin after intra-pancreas transplantation compared to the subcutaneous ones, and to maintain molecular and morphological features over different passages.At sacrifice, histopathological analysis demonstrated different stages of PanIN lesions in GEMs and the presence of a well-developed pancreatic tumor in all the mice orthotopically inoculated with the PDAC-PDXs.Plasma levels of MMP-7, TIMP-1 and THBS-2 were progressively upregulated, over the time, in GEMs and in PDAC-PDX bearing mice.In both animal models, immunohistochemistry revealed stromal immunoreactivity for TIMP-1 and THBS-2, while MMP-7 expression was mainly localized on epithelial cells. All the markers showed progressive increase of staining intensity along with PanIN progression.In conclusion, the investigated circulating molecules represent promising biomarkers for early diagnosis of PDAC and to monitor the response to treatment in human patients. Both tumoral cells and associated stroma play a role in the production and release of such biomarkers

    Tumor Microenvironment In Experimental Models Of Human Cancer: Morphological Investigational Approaches

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    Introduction. Tumor microenvironment (TME) is defined as the non-tumoral part of tumors. It is composed of different cell populations and structures (such as tumor-associated vasculature, immune-inflammatory cells, fibroblasts…) (Hanahan and Coussens, 2012). TME could either promote or antagonize tumor growth and has a great potential as target for novel therapeutic strategies. Along with several methods (i.e. molecular assays), morphological techniques allow to evaluate the components of TME in the setting of their action. The aim of this work was to set up and define valuable morphological approaches useful in the investigation of the TME.Materials and methods. Histological and immunohistochemical techniques, along with digital image analysis, were tested on experimental mouse models (both xenograft and genetically engineered mice) of four different human tumors (ovarian cancer, pancreatic ductal adenocarcinoma (PDAC), colon adenocarcinoma, thyroid carcinoma).Results. Concerning the vascular compartment, CD31 immunostaining and double-immunofluorescence with CD31 and a-SMA (pericytes marker) allowed to respectively quantify vessels and evaluate their maturation degree. Immunohistochemical detection of previously administrated Pimonidazole, revealed variable extended areas of hypoxia within tumoral masses in a consistent pattern between frozen and formalin-fixed paraffin-embedded samples.Concerning the stromal component, anti-human MHC I and specie-specific markers for Vimentin demonstrated the host-derivation of stroma in xenotumors, while Sirius Red histochemical staining allowed the quantification of desmoplasia in models of PDAC.Concerning immune-inflammatory cells, an immunohistochemical panel with CD3 (T lymphocytes), B220 (B lymphocytes), MPO (neutrophils) and Iba-1 (macrophages), showed high reliability in characterizing the tumoral infiltrate. Moreover, the application of markers specific for different macrophage subsets confirmed the higher prevalence of M2 (Arginase I positive) on M1 (iNOS positive) macrophages. YM1 demonstrated a low performance in detecting the M2 population (Fig. 1).Discussion and conclusions. Due to the microenvironmental heterogeneity which influence tumor development and biological behavior, a sole quantification is unreliable for characterizing the TME. Considering that, morphological techniques proved to be a valuable approach, allowing the evaluation of the spatial distribution and mutual interaction between the different elements. Additional studies are needed for further investigate the biological significance of spatial distribution of the components of the TME

    POT1 mutations are frequent and associated with Ki-67 index in canine diffuse large B-cell lymphoma

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    Diffuse large B-cell lymphoma (DLBCL) represents one of the most frequent and deadliest neoplasia in dogs worldwide and is characterized by a remarkable degree of clinical heterogeneity, with poor chances to anticipate the outcome. Even if in the last years some recurrently mutated genes have been identified, the genetic origin of canine DLBCL (cDLBCL) is not yet completely understood. The aim of the present study was to assess the prevalence of POT1 mutations in cDLBCL and to elucidate the role of such gene in the pathogenesis of this tumor. Mutations in POT1 were retrieved in 34% of cases, in line with previous reports, but no significant associations with any clinico-pathological variable were identified. Likewise, POT1 mutations are not predictive of worse prognosis. Interestingly, Ki-67 index was significantly higher in dogs harboring POT1 mutations compared to wild-type ones. These results suggest that POT1 mutations may exert their pathogenic role in cDLBCL by promoting cellular proliferation

    Selective proliferative response of microglia to alternative polarization signals

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    Background: Microglia are resident myeloid cells of the central nervous system (CNS) that are maintained by self-renewal and actively participate in tissue homeostasis and immune defense. Under the influence of endogenous or pathological signals, microglia undertake biochemical transformations that are schematically classified as the pro-inflammatory M1 phenotype and the alternatively activated M2 state. Dysregulated proliferation of M1-activated microglia has detrimental effects, while an increased number of microglia with the alternative, pro-resolving phenotype might be beneficial in brain pathologies; however, the proliferative response of microglia to M2 signals is not yet known. We thus evaluated the ability of interleukin-4 (IL-4), a typical M2 and proliferative signal for peripheral macrophages, to induce microglia proliferation and compared it with other proliferative and M2 polarizing stimuli for macrophages, namely colony-stimulating factor-1 (CSF-1) and the estrogen hormone, 17β-estradiol (E2).Methods: Recombinant IL-4 was delivered to the brain of adult mice by intracerebroventricular (i.c.v.) injection; whole brain areas or ex vivo-sorted microglia were analyzed by real-time PCR for assessing the mRNA levels of genes related with cell proliferation (Ki67, CDK-1, and CcnB2) and M2 polarization (Arg1, Fizz1, Ym-1) or by FACS analyses of in vivo BrdU incorporation in microglia. Primary cultures of microglia and astrocytes were also tested for proliferative effects.Results: Our results show that IL-4 only slightly modified the expression of cell cycle-related genes in some brain areas but not in microglia, where it strongly enhanced M2 gene expression; on the contrary, brain delivery of CSF-1 triggered proliferation as well as M2 polarization of microglia both in vivo and in vitro. Similar to IL-4, the systemic E2 administration failed to induce microglia proliferation while it increased M2 gene expression.Conclusions: Our data show that, in contrast to the wider responsiveness of peripheral macrophages, microglia proliferation is stimulated by selected M2 polarizing stimuli suggesting a role for the local microenvironment and developmental origin of tissue macrophages in regulating self-renewal following alternative activating stimuli

    The ER stress response mediator ERO1 triggers cancer metastasis by favoring the angiogenic switch in hypoxic conditions

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    : Solid tumors are often characterized by a hypoxic microenvironment which contributes, through the hypoxia-inducible factor HIF-1, to the invasion-metastasis cascade. Endoplasmic reticulum (ER) stress also leads tumor cells to thrive and spread by inducing a transcriptional and translational program, the Unfolded Protein Response (UPR), aimed at restoring ER homeostasis. We studied ERO1 alpha (henceforth ERO1), a protein disulfide oxidase with the tumor-relevant characteristic of being positively regulated by both ER stress and hypoxia. Analysis of the redox secretome indicated that pro-angiogenic HIF-1 targets, were blunted in ERO1-devoid breast cancer cells under hypoxic conditions. ERO1 deficiency reduced tumor cell migration and lung metastases by impinging on tumor angiogenesis, negatively regulating the upstream ATF4/CHOP branch of the UPR and selectively impeding oxidative folding of angiogenic factors, among which VEGF-A. Thus, ERO1 deficiency acted synergistically with the otherwise feeble curative effects of anti-angiogenic therapy in aggressive breast cancer murine models and it might be exploited to treat cancers with pathological HIF-1-dependent angiogenesis. Furthermore, ERO1 levels are higher in the more aggressive basal breast tumors and correlate inversely with the disease- and metastasis-free interval of breast cancer patients. Thus, taking advantage of our in vitro data on ERO1-regulated gene products we identified a gene set associated with ERO1 expression in basal tumors and related to UPR, hypoxia, and angiogenesis, whose levels might be investigated in patients as a hallmark of tumor aggressiveness and orient those with lower levels toward an effective anti-angiogenic therapy

    Development of monoclonal antibodies targeting canine PD-L1 and PD-1 and their clinical relevance in Canine Apocrine Gland Anal Sac Adenocarcinoma

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    Canine apocrine gland anal sac adenocarcinoma (AGASACA) is an aggressive canine tumor originating from the anal sac glands. Surgical resection, with or without adjuvant chemotherapy, represents the standard of care for this tumor, but the outcome is generally poor, particularly for tumors diagnosed at an advanced stage. For this reason, novel treatment options are warranted, and a few recent reports have suggested the activation of the immune checkpoint axis in canine AGASACA. In our study, we developed canine-specific monoclonal antibodies targeting PD-1 and PD-L1. A total of 41 AGASACAs with complete clinical and follow-up information were then analyzed by immunohistochemistry for the expression of the two checkpoint molecules (PD-L1 and PD-1) and the presence of tumor-infiltrating lymphocytes (CD3 and CD20), which were evaluated within the tumor bulk (intratumor) and in the surrounding stroma (peritumor). Seventeen AGASACAs (42%) expressed PD-L1 in a range between 5% and 95%. The intratumor lymphocytes were predominantly CD3+ T-cells and were positively correlated with the number of PD-1+ intratumor lymphocytes (ρ = 0.36; p = 0.02). The peritumor lymphocytes were a mixture of CD3+ and CD20+ cells with variable PD-1 expression (range 0–50%). PD-L1 expression negatively affected survival only in the subgroup of dogs treated with surgery alone (n = 14; 576 vs. 235 days). The presence of a heterogeneous lymphocytic infiltrate and the expression of PD-1 and PD-L1 molecules support the relevance of the immune microenvironment in canine AGASACAs and the potential value of immune checkpoints as promising therapeutic targets

    Chronic cholesterol administration to the brain supports complete and long-lasting cognitive and motor amelioration in Huntington's disease

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    : Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors
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