Adipocytes WNT5a mediated dedifferentiation: a possible target in pancreatic cancer microenvironment

A significant epidemiological association between obesity and pancreatic ductal adenocarcinoma (PDAC) has previously been described, as well as a correlation between the degree of pancreatic steatosis, PDAC risk and prognosis. The underlying mechanisms are still not completely known.After co-culture of 3T3-L1 adipocytes and MiaPaCa2 with an in vitro transwell system we observed the appearance of fibroblast-like cells, along with a decrease in number and size of remaining adipocytes. RT-PCR analyses of 3T3-L1 adipocytes in co-culture showed a decrease in gene expression of typical markers of mature adipocytes, in parallel with an increased expression of fibroblast-specific and reprogramming genes. We found an increased WNT5a gene and protein expression early in MiaPaCa2 cells in co-culture. Additionally, EMSA of c-Jun and AP1 in 3T3-L1 demonstrated an increased activation in adipocytes after co-culture. Treatment with WNT5a neutralizing antibody completely reverted the activation of c-Jun and AP1 observed in co-cultured adipocytes.Increasing doses of recombinant SFRP-5, a competitive inhibitor for WNT5a receptor, added to the co-culture medium, were able to block the dedifferentiation of adipocytes in co-culture.These data support a WNT5a-mediated dedifferentiation process with adipocytes reprogramming toward fibroblast-like cells that might profoundly influence cancer microenvironment

Adipocita e Flogosi: Ruolo di Adipochine e Vitamina D

L\u2019obesit\ue0 \ue8 una condizione caratterizzata da infiammazione cronica del tessuto adiposo (TA), che si associa ad aumentato rischio non solo metabolico ma anche neoplastico, con prognosi peggiorative. \uc8 stato ipotizzato un ruolo del TA nel crosstalk con le cellule del sistema immunitario e quindi della patogenesi dello stato infiammatorio subclinico correlato all\u2019obesit\ue0. In questa tesi si \ue8 voluto indagare la modulazione della flogosi in diverse condizioni sperimentali. Nel modello sperimentale di cellule murine 3T3-L1, stimolate con lipopolisaccaride (LPS) a diversi dosaggi e tempi, sono state indagate le espressioni di diverse adipochine, sia come mRNA da lisato cellulare, che come proteine secrete nel surnatante, osservando un aumento di espressione di citochine infiammatorie (IL-6, TNF-\u3b1, CCL3, CCL4, CXCL12) e riduzione di proteina antinfiammatoria (IL-10). \uc8 stato quindi valutato il ruolo del calcitriolo (1,25[OH]2D3) nella fisiologia e patofisiologia dell\u2019adipocita, ed in particolare nella modulazione della flogosi del TA stesso. Si sono perci\uf2 indagati gli effetti trascrizionali e morfologici sia di 1,25[OH]2D3 che del precursore colecalciferolo (D3) su adipociti 3T3-L1, SGBS, ottenuti da colture primarie o isolati a fresco, in relazione alle stimolazioni con LPS in diverse [Ca2+]e. L\u2019adipocita presentava enzimi atti al metabolismo della vitamina D3, CYP27A1 e CYP27B1. La stimolazione con D3 ha mostrato andamento simile a quella con 1,25[OH]2D3, ossia antinfiammatorio, con riduzione di IL-6, TNF-\u3b1 ed aumento di IL-10. D3 e 1,25[OH]2D3 infatti, erano in grado di aumentare area e diametro degli adipociti con riduzione di densit\ue0 ottica (optical density = OD), effetti opposti al trattamento con LPS. Tali azioni di D3 e 1,25[OH]2D3 nell\u2019adipocita sembrano accentuati in presenza di aumentate [Ca2+]e. Sono state quindi analizzate le caratteristiche infiammatorie del TA peritumorale (PAT), in pazienti affetti da cancro al colon retto (CCR). Utilizzando campioni bioptici di TA \ue8 stato studiato il livello di infiammazione e infiltrazione macrofagica in PAT, TA viscerale (VAT) e sottocutaneo (SAT). Le indagini istopatologiche hanno evidenziato che gli adipociti del PAT abbiano dimensioni pi\uf9 ridotte rispetto a quelli di VAT e SAT. Inoltre PAT ha rivelato una prevalente infiltrazione di macrofagi M2, mentre in VAT e SAT i macrofagi infiltrati mostrano caratteristiche M1. Il PAT presenta altres\uec una maggiore espressione di adiponectina, paragonata con SAT e VAT. In conclusione nella loro globalit\ue0, questi dati dimostrano come la flogosi nel TA sia finemente regolata. Il TA \ue8 in grado di rispondere a diversi stimoli pro- ed anti-infiammatori, modulandone l\u2019azione. Pertanto, soprattutto nell\u2019ambito di alcune patologie (quali in cancro e la sindrome metabolica), cruciale sembra la possibilit\ue0 di poter interferire con tali processi, allo scopo di ridurre la flogosi del TA, coinvolta nella loro patogenesi. Futuri studi dovranno proseguire nell\u2019identificazione dei potenziali target utili per la modulazione della flogosi del TA.Obesity is bound to a cronic, low-grade inflamed status of adipose tissue (AT), associated not only with a metabolic risk, but also neoplastic, leading to worse prognosis about a broad spectrum of pathologies. It was supposed a leading role for AT as regard crosstalk with immune cells and AT inflamed status. The aim of this study was to investigate the relationship between adipocytes and inflammation. From an experimental model based on 3T3-L1 cells, stimulated with lipopolysaccharide (LPS) in various times and doses, expressions of adipokines was examined, as regards both mRNA from cellular lysate, and secreted proteins in cellular medium (with ELISA). Modulations were observed with general augmented expressions of inflammatory citokines (IL-6, TNF-\u3b1, CCL3, CCL4, CXCL12), while there was a reduction in anti-inflammatory response (IL-10). The role of calcitriol (1,25[OH]2D3) was investigated in adipocyte\u2019s physiology and pathophysiology, in relation to adipogenic and inflammatory functions. For this reasons, transcriptional and morphological effects were investigated, both of 1,25[OH]2D3 and of its parent compound colecalciferol (D3) on adipocytes differentiated from 3T3-L1, SGBS, primary cultures preadipocytes and fresh-isolated adipocytes. Also in conjunction with [Ca2+]e and LPS stimulations. Adipocytes showed enzymes for D3, such as CYP27A1 and CYP27B1. D3 treatment showed a similar antiinflammatory trend compared to 1,25[OH]2D3 one, with a reduction in IL-6, TNF-\u3b1 and an increase in IL-10. As regards morphology, some changes were observed, about lipid droplets\u2019 optical density (OD), area and diameter of adipocytes. D3 and 1,25[OH]2D3, indeed, increased adipocytes area and diameter, reducing OD. LPS showed opposite effects, while pretreatment with D3 and 1,25[OH]2D3 spared variations induced by LPS stimulation. These effects seem to be highlighted by the increase of [Ca2+]e, also in relation with LPS treatment. Using AT bioptic material, the differences in adipocytes\u2019 morphology were studied, inflammatory status and macrophages\u2019 infiltration among peritumoral AT (PAT), visceral AT distant from the tumor (VAT) and subcutaneous AT (SAT). Histopathological analyses revealed that PAT adipocytes were smaller then VAT and SAT adipocytes. Furthermore, the PAT was mainly infiltrated by M2-subtype macrophages, while the macrophages identified in other AT depots presented mainly M1-features. PAT also presented a higher expression of adiponectin, compared to SAT and VAT. In conclusion, these data showed the AT response capabilities to inflammation are strictly regulated. AT seems to be able to react both to inflammatory and anti-inflammatory stimulations. Therefore, AT alterations could involve pathological effects, alike cancer or metabolic syndrome. So, any possibility to interfere with these processes could be pivotal, reducing AT inflammation. Future studies may identify potential targets useful to modulate inflammation in AT

LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

OBJECTIVE: In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times. METHODS: 3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 \u3bcg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed. RESULTS: Cells activated by increasing doses of LPS upregulated TNF-\u3b1 expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1\u3b1. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-\u3b1 and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 \ub0C. The chemokines MIP-1\u3b1/CCL3 and MIP-1\u3b2/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments. CONCLUSION: These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte

Phenotypic Shift of Adipocytes by Cholecalciferol and 1\u3b1,25 Dihydroxycholecalciferol in Relation to Inflammatory Status and Calcium Content

Recent experimental data seem to suggest a relevant role for 1,25[OH]2cholecalciferol (1,25[OH]2D3) in adipocyte physiology and pathophysiology, with some studies showing adipogenic and pro-inflammatory properties, and others lipolytic and anti-inflammatory functions. Moreover, to our knowledge, the role of cholecalciferol (D3) in adipocytes function is still not known. Therefore, the aim of this study was to investigate in vitro the effects of 1,25[OH]2D3, as well as of D3, in 3T3-L1 adipocytes in basal and inflammatory conditions, testing the effects of different calcium concentrations in adipocytes culture medium. In 3T3-L1 adipocytes, CYP27A1 and CYP27B1 mRNA were detected in basal conditions and induced after D3 treatment. Pre-treatment of 3T3-L1 adipocytes not only with 1,25[OH]2D3, but also with D3 before inflammatory stimulation, significantly prevented the increase in gene expression and protein secretion of IL-6 and TNF-\u3b1, and significantly increased IL-10 mRNA and protein production compared with adipocytes treated only with lipopolysaccharide (LPS). Biological effects of D3 were still present after inhibition of P450 activity with ketokonazole. LPS determined a decrease in cell area compared with controls, paralleled by a significant increase in optical density (OD) of lipid droplets, whereas 1,25[OH]2D3 and D3 alone significantly increased adipocytes area and decreased OD. Pretreatment with both forms of vitamin D preserved cells from the reduction in their area observed after LPS treatment. LPS decreased more the area of cells grown in a high calcium medium than of adipocytes grown in a low calcium medium. In the presence of a high calcium medium, 1,25(OH)2D3 treatment preserved cell area, maintaining its anti-inflammatory and adipogenic properties. In conclusion our results show that D3, besides 1,25[OH]2D3, presents anti-inflammatory effects on 3T3-L1, as well as that adipocytes have the enzymatic pathways necessary to locally regulate the production of active forms of vitamin D, capable of influencing adipocyte phenotype and function

Borderline ovarian tumors and diagnostic dilemma of intraoperative diagnosis: could preoperative He4 assay and ROMA score assessment increase the frozen section accuracy? A multicenter case-control study.

The aim of our study was to assess the value of a preoperative He4-serum-assay and ROMA-score assessment in improving the accuracy of frozen section histology in the diagnosis of borderline ovarian tumors (BOT). 113 women presenting with a unilateral ovarian mass diagnosed as serous/mucinous BOT at frozen-section-histology (FS) and/or confirmed on final pathology were recruited. Pathologists were informed of the results of preoperative clinical/instrumental assessment of all patients. For Group_A patients, additional information regarding He4, CA125, and ROMA score was available (in Group_B only CA125 was known). The comparison between Group A and Group B in terms of FS accuracy, demonstrated a consensual diagnosis in 62.8% versus 58.6% (P: n.s.), underdiagnosis in 25.6% versus 41.4% (P<0.05), and overdiagnosis in 11.6% versus 0% (P<0.01). Low FS diagnostic accuracy was associated with menopausal status (OR: 2.13), laparoscopic approach (OR: 2.18), mucinous histotype (OR: 2.23), low grading (OR: 1.30), and FIGO stage I (OR: 2.53). Ultrasound detection of papillae (OR: 0.29), septa (OR: 0.39), atypical vascularization (OR: 0.34), serum He4 assay (OR: 0.39), and ROMA score assessment (OR: 0.44) decreased the probability of underdiagnosis. A combined preoperative assessment through serum markers and ultrasonographic features may potentially reduce the risk of underdiagnosis of BOTs on FS while likely increasing the concomitant incidence of false-positive events

Clinical Management of Long-Term Survivors after Classical Hodgkin Lymphoma and Diffuse Large B-Cell Lymphoma

Compared to other patients suffering from hematological malignancies, classical Hodgkin lymphoma (cHL) and diffuse large B-cell lymphoma (DLBCL) patients have a long life expectancy when in complete remission at the end of first, or sometimes second, line treatments [...

Morphological and Functional Changes in the Peritumoral Adipose Tissue of Colorectal Cancer Patients

Objective: The role of peritumoral adipose tissue (AT) has not been extensively studied in colorectal cancer (CRC). Methods: This study was conducted in 20 male subjects undergoing elective surgery for CRC. The differences between the peritumoral visceral adipose tissue (P-VAT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) of the patients were described via immunohistochemistry and molecular biology analyses. The interactions between adipocytes and a colon cancer cell line were also investigated by using an in vitro coculture system. Results: The analyses revealed that adipocytes near the tumor were significantly smaller than the adipocytes from other sites. The P-VAT was preferentially infiltrated by a CD68+/CD163+/IDO- macrophage subset with a prevalent reparative inflammatory response, while the macrophages identified in VAT and SAT mainly presented inflammatory features. Furthermore, the P-VAT presented a higher expression of adiponectin compared with other sites. Morphological analysis in vitro showed that after a few days of coculture, 3T3-L1 adipocytes were reduced in number and size with an increase in lipolysis rate and dedifferentiation phenomena. Conclusions: This study reveals important morphological and functional changes in the AT surrounding the tumor as an increase in lipolysis and in adiponectin-producing adipocytes, preferentially infiltrated by a macrophage subset, with prevalent reparative inflammatory response

Morphological and Functional Changes in the Peritumoral Adipose Tissue of Colorectal Cancer Patients

Objective: The role of peritumoral adipose tissue (AT) has not been extensively studied in colorectal cancer (CRC). Methods: This study was conducted in 20 male subjects undergoing elective surgery for CRC. The differences between the peritumoral visceral adipose tissue (P-VAT), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) of the patients were described via immunohistochemistry and molecular biology analyses. The interactions between adipocytes and a colon cancer cell line were also investigated by using an in vitro coculture system. Results: The analyses revealed that adipocytes near the tumor were significantly smaller than the adipocytes from other sites. The P-VAT was preferentially infiltrated by a CD68+/CD163+/IDO- macrophage subset with a prevalent reparative inflammatory response, while the macrophages identified in VAT and SAT mainly presented inflammatory features. Furthermore, the P-VAT presented a higher expression of adiponectin compared with other sites. Morphological analysis in vitro showed that after a few days of coculture, 3T3-L1 adipocytes were reduced in number and size with an increase in lipolysis rate and dedifferentiation phenomena. Conclusions: This study reveals important morphological and functional changes in the AT surrounding the tumor as an increase in lipolysis and in adiponectin-producing adipocytes, preferentially infiltrated by a macrophage subset, with prevalent reparative inflammatory response