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CARATTERIZZAZIONE FUNZIONALE E FENOTIPICA DELLE CELLULE NATURAL KILLER CIRCOLANTI E TUMORALI IN PAZIENTI CON CANCRO DELLA PROSTATA
No full textLe cellule Natural Killer (NK) sono cellule linfoidi innate (ILC) coinvolte nel riconoscimento/eliminazione del tumore e sono state trovate alterate in diversi tumori, incluso il cancro alla prostata (PCa). Inoltre, è stato scoperto che le cellule NK acquisiscono fenotipo/funzioni pro-angiogenici nel cancro solido.
Ho caratterizzato le cellule NK circolanti (TANK) e infiltranti il tumore (TINK) in pazienti con cancro alla prostata, iperplasia prostatica benigna (BPH), rispetto a quelle di individui sani, mediante citometria a flusso multiparametrica. Ho inoltre caratterizzato funzionalmente l'interazione di PCa-TANKs, esercitata tramite fattori solubili, con cellule endoteliali e monociti/macrofagi mediante qPCR, test di formazione di strutture endoteliali, migrazione sulle camere di Boyden. Ho testato l'attivazione di STAT3 e la sua riduzione, da parte dell'agente antipsicotico Pimozide (approccio farmacologico), sulle TANKs di PCa. Ho profilato il secretoma delle TANKs rispetto alle cellule NK dei controlli sani, nonché il secretoma delle TANKs trattati con Pimozide, mediante array proteici di membrana. Ho profilato i sieri dei pazienti con PCa, rispetto ai controlli, mediante l'array Bioplex. Infine, utilizzando un modello di polarizzazione in vitro, ho testato la capacità di linee cellulari di PCa (PC-3, DU-145, LNCaP) o di citochine trovate upregolate nei sieri di pazienti con PCa, di polarizzare le cellule NK verso il fenotipo simil-deciduale. Ho scoperto che sia le TANKs che le TINKs acquisiscono il fenotipo delle cellule NK di tipo deciduo CD9+CD49a+, la cui frequenza è fortemente aumentata, rispetto alle cellule NK circolanti da soggetti con BPH e controlli sani. Inoltre, ho scoperto che le TANKs hanno un fenotipo esaurito e capacità ridotte di degranulazione. I mezzi condizionati delle TANKs sono arricchiti in fattori correlati all'angiogenesi infiammatoria (CXCL8, uPAR, MMP-1 e MMP9), al reclutamento di macrofagi (GM-CSF, CXCL1/GRO, CXCL11/I-TAC, CCL1/I-309, CCL2 /MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL13/MCP-4) e alla polarizzazione in simil-M2 (IL-10). I CMs delle TANKs inducono la formazione di strutture endoteliali simil-tubulari, attivano il programma trascrizionale pro-angiogenico/pro-infiammatorio nelle cellule endoteliali, nonché reclutano sia i monociti THP-1 che CD14+ umani inducendo una polarizzazione simile a M2 nei macrofagi derivati da monociti CD14+. La Pimozide è stata in grado di diminuire l'attivazione di STAT3 nelle TANKs, insieme alla rieducazione delle TANKs che hanno ridotto il rilascio di IL-6, CXCL8/IL-8, IL-10, mentre hanno aumentato la produzione di IFN e TNF. Ho visto che i sieri dei pazienti con PCa sono arricchiti in citochine coinvolte nell'attivazione di STAT3, come IL-4, IL-6, CXCL8/IL-8 e IL-10. In vitro, ho scoperto che i CMs delle linee cellulari di PCa PC-3, DU-145 e LNCaP, inducono il fenotipo CD9+CD49a+ simil-deciduale, aumentano la produzione di fattori pro-angiogenici (angiogenina, angiopoietina-1, CXCL8), mentre riducono la produzione di citochine antitumorali (granzima B, TNFα, IFNγ). Inoltre, le cellule NK esposte ai CMs di cellule PC-3 e DU-145 aumentano l'espressione di PD-1 e TIM-3, insieme a ridotte capacità di degranulazione. Infine, tra tutte le citochine attivanti STAT3 trovate aumentate nei sieri di PCa, solo CXCL8/IL-8 era efficace nell'aumentare l'espressione di CD9 su cellule NK sane derivate dal controllo.
Presi insieme, i risultati del mio progetto hanno mostrato che PCa TANKs e TINKs acquisiscono il fenotipo pro-infiammatorio/pro-angiogenico simil-deciduale e possono indurre angiogenesi ex vivo, tramite fattori solubili, interagendo direttamente con le cellule endoteliali e i macrofagi simil-M2 come cellule astanti pro-angiogeniche. Inoltre, le TANKs sembrano rispecchiare l'alterazione delle TINKs nel PCa, evidenziando così un possibile ruolo per le cellule NK circolanti come marker da tracciare/monitorare nel PCa.Natural killer (NK) cells are innate lymphoid cells (ILC) involved in tumour recognition and/elimination and have been found to be altered in diverse tumours, including prostate cancer (PCa). Also, NK cells have been found to acquire pro-angiogenic phenotype/functions in solid cancer.
Here, I characterize circulating (TANKs) and tumor-infiltrating (TINKs) NK cells in patients with prostate cancer, prostatic benign hyperplasia (BPH), compared to those from healthy individuals, by multicolor flow cytometry. I functionally characterize the interaction of PCa-TANK, exerted via soluble factors, with endothelial cells and monocytes/macrophages by qPCR, tube formation assay, migration on Boyden chambers. I tested the activation of STAT3 and its reduction, by the anti-psychotic agent Pimozide (drug-repurposing approach), in PCa TANKs. I profiled the secretome of PCa TANKs compared to NK cells of healthy controls, as well as PCa TANKs secretome treated with Pymozide, by membrane protein arrays. I profiled the sera of PCa patients, compared to controls, by Bioplex array. Finally, using an in vitro model of polarization, I tested the capability of PCa cells lines (PC-3, DU-145, LNCaP) or cytokines found to be up-regulated in the sera of PCa patients, to polarize NK cells toward the decidual-like phenotype.
I found that PCa TANKs and TINKs acquire the CD9+CD49a+ decidual-like NK cell phenotype, whose frequency was strongly increased, compared to circulating NK cells form BPH subjects and healthy controls. Also, I found that PCa TANKs have an exhausted phenotype and impaired degranulation capabilities. Conditioned media of PCa TANKs are enriched in factors related to inflammatory angiogenesis (CXCL8, uPAR, MMP-1, and MMP9), macrophage recruiting (GM-CSF, CXCL1/GRO, CXCL11/I-TAC, CCL1/I-309, CCL2/MCP-1, CCL5/RANTES, CCL7/MCP-3, CCL13/MCP-4) and M2-like polarization (IL-10). CMs from PCa TANKs induce tube formation, activates a pro-angiogenic/pro-inflammatory transcriptional program in endothelial cells, as well as recruit THP-1 and human CD14+ monocytes and induce an M2-like polarization in CD14+ monocyte-derived macrophages. Pymozide was able to decrease STAT3 activation in PCa TANKs, together with the re-education of PCa TANKs that have reduced release of IL-6, CXCL8/IL-8, IL-10, while increase the production of IFN and TNF. I observed that sera from PCa patients are enriched in cytokines involved in STAT3 activation, such as IL-4, IL-6, CXCL8/IL-8 and IL-10. In vitro, I found that CMs of PC-3, DU-145 and LNCaP prostate cancer cell lines, induce the decidual-like CD9+CD49a+ phenotype, increase the production of pro-angiogenic factors (angiogenin, angiopoietin-1, CXCL8), while reducing the production of anti-tumor cytokines (granzyme B, TNFα, IFNγ). In addition, NK cells exposed to CMs of PC-3 and DU-145 cells increase the expression of PD-1 and TIM-3, together with reduced degranulation capabilities. Finally, between all STAT3-activating cytokines found increased in PCa sera, only CXCL8/IL-8 was effective in increasing CD9 expression on healthy control-derived NK cells.
Taken together, results from my project showed that PCa TANKs and TINKs acquire the pro-inflammatory/ pro-angiogenic decidual-like phenotype and can induce angiogenesis ex vivo, via soluble factors, by directly interacting with endothelial cells of by using M2-like polarized macrophages as pro-angiogenic bystander cells. My results also suggest that PCa TANKs seems to mirror NK cell alteration in PCa TINKs, thus highlighting a possible role for circulating NK cells as a marker to be traced/monitored in PCa
The tumor innate immune microenvironment in prostate cancer: an overview of soluble factors and cellular effectors
Get PDFProstate cancer (PCa) accounts as the most common non-cutaneous disease affecting males, and
as the first cancer, for incidence, in male. With the introduction of the concept of immunoscore,
PCa has been classified as a cold tumor, thus driving the attention in the development of strategies
aimed at blocking the infiltration/activation of immunosuppressive cells, while favoring the
infiltration/activation of anti-tumor immune cells. Even if immunotherapy has revolutionized the approaches to cancer therapy, there is still a window failure, due to the immune cell plasticity within PCa, that can acquire pro-tumor features, subsequent to the tumor microenvironment (TME) capability to polarize them. This review discussed selected relevant soluble factors [transforming growth factor-beta (TGFβ), interleukin-6 (IL-6), IL-10, IL-23] and cellular components of the innate immunity, as drivers of tumor progression, immunosuppression, and angiogenesis within the PCa-TME
Prostate cancer associated natural killer cells show a pro-angiogenic and pro-inflammatory phenotype.
No full textThe Tumor-Endothelium Interaction in Pioneering Studies and the Revisited Concept on the Angiogenesis Process During Tumor Progression and Metastasis
Get PDFThe growth of solid tumors and their dissemination require the continuous formation of new capillary blood vessels. However, the association of uncontrolled growth of tumors and angiogenesis, i.e. the mechanism that allows the formation of new blood vessels starting from pre-existing vessels, dates back to 1971, in relation of Judah Folkman's works. Since then, his group and other researchers added new key results confirming the important role played by angiogenesis in tumor growth and metastasis, and multiple efforts have been made to exploit this knowledge in developing innovative anti-cancer therapies. In this article, we discuss seminal works regarding molecular mechanisms involved in aberrant tumor angiogenesis, biology of endothelial cells within extracellular matrix, function of diverse pro- and anti-angiogenic factors, roles of metalloproteinases and protumor effects played by stromal and immune cells in the tumor microenvironment. Interestingly, growing evidences indicate a key role played by inflammatory and stromal cells in both tumor development and progression. The present article also aim to provide up-to-date information concerning new therapeutic concepts involving tumor vessels normalization and anti-angiogenic agents, among which inhibitors of metalloproteinases and of the main angiogenic factor, vascular endothelial growth factor (VEGF) or its receptors, and the combination of them with immune checkpoint blockade, that seem to be the most promising ones at present
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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