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.
Le 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.
CARATTERIZZAZIONE FUNZIONALE E FENOTIPICA DELLE CELLULE NATURAL KILLER CIRCOLANTI E TUMORALI IN PAZIENTI CON CANCRO DELLA PROSTATA / Matteo Gallazzi , 2022 Dec 19. 35. ciclo, Anno Accademico 2021/2022.
CARATTERIZZAZIONE FUNZIONALE E FENOTIPICA DELLE CELLULE NATURAL KILLER CIRCOLANTI E TUMORALI IN PAZIENTI CON CANCRO DELLA PROSTATA
GALLAZZI, MATTEO
2022-12-19
Abstract
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.File | Dimensione | Formato | |
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Tesi_FINAL_MG.pdf
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Descrizione: PHENOTYPE AND FUNCTIONAL CHARACTERIZATION OF CIRCULATING AND TUMOR INFILTRATING NATURAL KILLER CELLS IN PATIENTS WITH PROSTATE CANCER
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