Diabetic patients display increased risk of periodontitis and failure in bone augmentation procedures. Mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) represent a relevant advantage in tissue repair process and regenerative medicine. We isolated MSCs from Bichat's buccal fat pad (BFP) and measured the effects of glucose and PRP on cell number and osteogenic differentiation potential. Cells were cultured in the presence of 5.5-mM glucose (low glucose [LG]) or 25-mM glucose (high glucose [HG]). BFP-MSC number was significantly lower when cells were cultured in HG compared with those in LG. Following osteogenic differentiation procedures, calcium accumulation, alkaline phosphatase activity, and expression of osteogenic markers were significantly lower in HG compared with LG. Exposure of BFP-MSC to PRP significantly increased cell number and osteogenic differentiation potential, reaching comparable levels in LG and in HG. Thus, high-glucose concentrations impair BFP-MSC growth and osteogenic differentiation. However, these detrimental effects are largely counteracted by PRP. K E Y W O R D S Bichat's buccal fat pad, bone reconstruction, diabetes, mesenchymal stem cells, platelet-rich plasma Vittoria D'Esposito and Manuela Lecce equally contributed to this work.

Background Serotonin (or 5-Hydroxytryptamine, 5-HT) signals in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis, and angiogenesis. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. Methods Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; mRNA levels of CTGF and ABC transporters have been further measured by qPCR. Protein levels of 5-HT2C receptors have been analyzed by Western blot. All data were statistically analyzed using GraphPad Prism 7. Results We found that treatment with SER for 72 h reduced viability of BC cells. SER were more effective on MCF7 ER+ cells (IC50 range 10.2 μM - 99.2 μM) compared to SKBR3 (IC50 range 43.3 μM - 260 μM) and MDA-MB231 BC cells (IC50 range 91.3 μM - 306 μM). This was paralleled by accumulation of cells in G0/G1 phase of cell cycle. Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 cells and modulated the expression of CTGF, without affecting viability of MCF10A non-cancer breast epithelial cells. In a cell model of Tamoxifen resistance, SER68 also restored drug effect independently of CTGF. Conclusions These results identified serotoninergic receptor ligands potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

Adiposity and diabetes affect breast cancer (BC) progression. We addressed whether glucose may affect the interaction between mammary adipose tissue-derived mesenchymal stromal/stem cells (MAT-MSCs) and BC cells. Two-dimensional co-cultures and spheroids were established in 25 mM or 5.5 mM glucose (High Glucose-HG or Low Glucose-LG) by using MAT-MSCs and MCF7 or MDA-MB231 BC cells. Gene expression was measured by qPCR, while protein levels were measured by cytofluorimetry and ELISA. CD44high/CD24low BC stem-like sub-population was quantified by cytofluorimetry. An in vivo zebrafish model was assessed by injecting spheroid-derived labeled cells. MAT-MSCs co-cultured with BC cells showed an inflammatory/senescent phenotype with increased abundance of IL-6, IL-8, VEGF and p16INK4a, accompanied by altered levels of CDKN2A and LMNB1. BC cells reduced multipotency and increased fibrotic features modulating OCT4, SOX2, NANOG, αSMA and FAP in MAT-MSCs. Of note, these co-culture-mediated changes in MAT-MSCs were partially reverted in LG. Only in HG, MAT-MSCs increased CD44high/CD24low MCF7 sub-population and promoted their ability to form mammospheres. Injection in zebrafish embryos of HG spheroid-derived MCF7 and MAT-MSCs was followed by a significant cellular migration and caudal dissemination. Thus, MAT-MSCs enhance the aggressiveness of BC cells in a HG environment.

Background: The tightly regulated pattern of serotonin (or 5-Hydroxytryptamine, 5-HT) in mammary gland becomes dysregulated in cancer, also contributing to proliferation, metastasis and angiogenesis. Antagonists of serotonin receptors or inhibitors of serotonin reuptake/synthesis may be co-prescribed with Tamoxifen in estrogen receptor-positive (ER+) tumors, and may modulate its efficacy. Thus, the discovery of novel compounds targeting serotonin signaling may contribute to tailor new therapeutic strategies usable in combination with endocrine therapies. We have previously synthesized serotoninergic receptor ligands (SER) with high affinity and selectivity towards 5-HT2A and 5-HT2C receptors, the main mediators of mitogenic effect of serotonin in breast cancer (BC). Here, we investigated the effect of 10 SER on viability of MCF7, SKBR3 and MDA-MB231 BC cells and focused on their potential ability to affect Tamoxifen responsiveness in ER+ cells. Methods: Cell viability has been assessed by sulforhodamine B assay. Cell cycle has been analyzed by flow cytometry. Gene expression of 5-HT receptors and Connective Tissue Growth Factor (CTGF) has been checked by RT-PCR; CTGF mRNA levels have been measured by qPCR. All data were statistically analyzed using GraphPad Prism 7. Results: We found that SER were more effective on MCF7 ER+ cells (IC50 range 10.2µM - 99.2µM) compared to SKBR3 (IC50 range 43.3µM - 260µM) and MDA-MB231 BC cells (IC50 range 91.3µM - 306µM). Next, we provided evidence that two ligands, SER79 and SER68, improved the effectiveness of Tamoxifen treatment in MCF7 BC cells and modulated the expression of CTGF. Moreover, in a cell model of Tamoxifen-resistance, SER79 and SER68 restored drug responsiveness and reduced CTGF levels. Conclusions: These results identified new compounds potentially usable in combination with Tamoxifen to improve its effectiveness on ER+ BC patients.

ConclusionIn conclusion, exerts an important antitumor effect by inhibiting c-Src and up-regulating PTEN with the subsequent reduction in FAK phosphorylation required to establish appropriate focal adhesions for migration, proliferation and survival. This inhibition is mediated by the recruitment of the c-Src intrinsic inhibitors, PTEN and Csk. All together, these results reinforce the relevance of this sequence of Cx43 that interacts with c-Src for the development of new therapies against glioblastoma. PO-292BREAST CANCER STEM CELL REPROGRAMMING: DECIPHERING THE IMPACT OF GLUCOSE AND THE CONTRIBUTE OF TUMOUR MICROENVIRONMENT Introduction Diabetes-associated hyperglycemia is linked to poorer prognosis and survival in breast cancer (BC). Indeed, glucose can affect both tumour and tumour-surrounding cells. BC cells are embedded in an adipocyte-rich microenvironment, which, beside adipocytes, contains Stromal-Vascular Fraction Cells (SVFCs). In this scenario, epithelial and stromal compartments communicate through the release of soluble factors and establish an intricate crosstalk. Here, we analysed whether glucose could directly affect the phenotype of ER + MCF7 BC cells and interfere with their interaction with adipose-derived SVFCs, thereby promoting tumour progression. Material and methods MCF7 cell stemness markers were measured by qReal-Time PCR. Adipose-derived (Ad-)SVFCs were obtained by mammary adipose tissue specimens of women undergoing plastic surgery. The trascriptome of MCF7 exposed to either low (LG-5.5 mM) or high glucose concentration (HG-25 mM) was obtained by RNA-Sequencing (Illumina HiSeq3000). Results and discussions HG exposure of MCF7 determined a significant increase of SOX2 mRNA levels as compared to LG, suggesting the induction of stemness programming. Coculture with Ad-SVFCs in HG increased SOX2, NANOG and OCT4 mRNA levels in MCF7, as compared to isolated culture, indicating the involvement of SVF-produced soluble factors in BC stem cell reprogramming. Moreover, in presence of Ad-SVFCs and HG, MCF7 produced a higher number of mammospheres, which also displayed larger size. However, both in LG and in HG, conditioned media (CM) obtained from Ad-SVFCs produced no relevant effect on MCF7 stemness. Nevertheless, when Ad-SVFCs were pre-incubated with CM obtained from HG-treated MCF7, their CM very effectively increased OCT4, NANOG and SOX2 mRNA levels in MCF7. Thus, HG likely perturbs MCF7, which produce soluble factors leading Ad-SVFCs to release, in turn, reprogramming factors for BC cell stemness. In this regard, we have observed that HG modification of MCF7 transcriptome includes deregulation of 17 genes (pval=0.05) encoding for secreted proteins involved in cancer progression-related pathways, which may potentially play a role in tumour-stroma interactions.