PTX3 shapes profibrotic immune cells and epithelial/fibroblast repair and regeneration in a murine model of pulmonary fibrosis.

The long pentraxin 3 (PTX3) is protective in different pathologies but was not analyzed in-depth in Idiopathic Pulmonary Fibrosis (IPF). Here, we have explored the influence of PTX3 in the bleomycin (BLM)-induced murine model of IPF by looking at immune cells (macrophages, mast cells, T cells) and stemness/regenerative markers of lung epithelium (SOX2) and fibro-blasts/myofibroblasts (CD44) at different time points that retrace the progression of the disease from onset at day 14, to full-blown disease at day 21, to incomplete regression at day 28. We took advantage of transgenic PTX3 overexpressing mice (Tie2-PTX3) and Ptx3 null ones (PTX3-KO) in which pulmonary fibrosis was induced. Our data have shown that PTX3 overexpression in Tie2-PTX3 compared to WT or PTX3-KO: reduced CD68+ and CD163+ macrophages and the Tryptase+ mast cells during the whole experimental time; on the contrary, CD4+ T cells are consistently present on day 14 and dramatically decreased on day 21; CD8+ T cells do not show significant differences on day 14, but are significantly reduced on day 21; SOX2 is reduced on days 14 and 21; CD44 is reduced on day 21. Therefore, PTX3 could act on the proimmune and fibrogenic microenvironment to prevent fibrosis in BLM-treated mice.

Double Immunohistochemical Staining on Formalin-Fixed Paraffin-Embedded Tissue Samples to Study Vascular Co-option.

Vascular co-option is a non-angiogenic mechanism whereby tumor growth and progression move on by hijacking the pre-existing and nonmalignant blood vessels and is employed by various tumors to grow and metastasize.The histopathological identification of co-opted blood vessels is complex, and no specific markers were defined, but it is critical to develop new and possibly more effective therapeutic strategies. Here, in glioblastoma, we show that the co-opted blood vessels can be identified, by double immunohistochemical staining, as weak CD31+ vessels with reduced P-gp expression and proliferation and surrounded by highly proliferating and P-gp- or S100A10-expressing tumor cells. Results can be quantified by the Aperio Colocalization algorithm, which is a valid and robust method to handle and investigate large data sets.

Differential P-Glycoprotein/CD31 Expression as Markers of Vascular Co-Option in Primary Central Nervous System Tumors.

BACKGROUND: Vascular co-option is one of the main features of brain tumor progression. It is identified using histopathological analysis, but no antibody-specific markers were found, and no universally accepted histological features were defined. METHODS: We employed double immunohistochemical stainings for CD31, P-gp, S100A10, and mitochondria on formalin-fixed, paraffin-embedded human samples of IDH-WT glioblastoma, IDH-mutant astrocytoma, and meningioma to study vascular co-option across different brain tumors and across normal, peritumoral, and intratumoral areas using the Aperio colocalization algorithm, which is a valid and robust method to handle and investigate large data sets. RESULTS: The results have shown that (i) co-opted vessels could be recognized by the presence of metabolically overactive (evaluated as mitochondria expression) and P-gp+ or S100A10+ tumor cells surrounding CD31+ endothelial cells; (ii) vascular co-option occurs in the intratumoral area of meningioma and astrocytoma; and (iii) vascular co-option is prevalent in peritumoral glioblastoma area. CONCLUSIONS: The described approach identifies new markers for cellular components of the vessel wall and techniques that uncover the order and localization of vascularization mechanisms, which may contribute to developing new and possibly more effective therapeutic strategies.

microRNAs Biogenesis, Functions and Role in Tumor Angiogenesis.

microRNAs (miRNAs) are small non-coding RNA molecules, evolutionary conserved. They target more than one mRNAs, thus influencing multiple molecular pathways, but also mRNAs may bind to a variety of miRNAs, either simultaneously or in a context-dependent manner. miRNAs biogenesis, including miRNA transcription, processing by Drosha and Dicer, transportation, RISC biding, and miRNA decay, are finely controlled in space and time. miRNAs are critical regulators in various biological processes, such as differentiation, proliferation, apoptosis, and development in both health and disease. Their dysregulation is involved in tumor initiation and progression. In tumors, they can act as onco-miRNAs or oncosuppressor-miRNA participating in distinct cellular pathways, and the same miRNA can perform both activities depending on the context. In tumor progression, the angiogenic switch is fundamental. miRNAs derived from tumor cells, endothelial cells, and cells of the surrounding microenvironment regulate tumor angiogenesis, acting as pro-angiomiR or anti-angiomiR. In this review, we described miRNA biogenesis and function, and we update the non-classical aspects of them. The most recent role in the nucleus, as transcriptional gene regulators and the different mechanisms by which they could be dysregulated, in tumor initiation and progression, are treated. In particular, we describe the role of miRNAs in sprouting angiogenesis, vessel co-option, and vasculogenic mimicry. The role of miRNAs in lymphoma angiogenesis is also discussed despite the scarcity of data. The information presented in this review reveals the need to do much more to discover the complete miRNA network regulating angiogenesis, not only using high-throughput computational analysis approaches but also morphological ones.

Inflammatory Infiltrate and Angiogenesis in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive and rare B-cell non-Hodgkin lymphoma classified in two clinicopathological subtypes according to SOX11 expression and mutation state of immunoglobulin variable region heavy chain (IgVH) gene. The transcription factor SOX11, overexpressed in 78%-93% of MCL patients, plays a central role in modulating tumor microenvironment prosurvival signals and angiogenic genes. In this work, we have explored the lymph node microenvironment of three subgroups of MCL patients classified according to SOX11 expression as negative, light, and strong. CD34+ microvessels, CD4+ and CD8+ T-lymphocytes, CD68+ and CD163+ macrophages, and the oncogene p53 expression were evaluated by immunohistochemistry. Moreover, STAT3 mRNA expression was analyzed by RNA-scope assay. Our results confirmed increased angiogenesis in the sample of patients positive to SOX11 compared to the negative ones and demonstrated that angiogenesis and SOX11 expression positively correlate to a higher T-lymphocytes inflammatory infiltrate. On the contrary, angiogenesis and SOX11 expression negatively correlate with macrophage's inflammatory infiltrate and p53 expression. STAT3 mRNA expression level was not relevant concerning angiogenesis or SOX11 expression. Overall, our data indicate that, in MCL, SOX11 expression is associated with increased angiogenesis and a high CD4+ and CD8+ T-cell infiltration, which are not sustained by CD163+ macrophages infiltrate and p53 expression.

RNAscope dual ISH-IHC technology to study angiogenesis in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) are the most common types of Non-Hodgkin's lymphomas and are highly heterogeneous in terms of phenotype and treatment response. The natural course of DLBCLs tumor progression is featured by a flow of events leading to the enhancement of proliferative and invasive capabilities and, therefore, towards the establishment of a more aggressive phenotype. Angiogenesis is a constant hallmark of DLBCLs progression, has prognostic potential and promote DLBCLs dissemination. The study of DLBCLs angiogenesis mechanisms, and the tumor endothelium characterization, will allow us to identify new prognostic/predictive biomarkers to proper patient selection to antiangiogenic treatment. In our previous work, by RNAscope technology, we have demonstrated that Janus kinase (Jak) and signal transducer activator of transcription pathway (STAT) is one of the proangiogenic pathways activated in DLBCLs and it drives neoangiogenesis occurred by vasculogenesis mechanism. Here, we describe a detailed protocol to perform RNAscope technology alone and in combination with immunohistochemistry (called dual RNAscope ISH-IHC) in DLBCLs formalin-fixed, paraffin-embedded sections. We propose dual ISH-IHC as an extremely powerful method to study angiogenesis in DLBCLs, because it allows one to answer important biological questions that are difficult to address using other single methods.

Dp71 Expression in Human Glioblastoma.

BACKGROUND: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). METHODS: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. RESULTS: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. CONCLUSION: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.