An In Vitro Approach for Investigating the Safety of Lipotransfer after Breast-Conserving Therapy.

The application of lipotransfer after breast-conserving therapy (BCT) and irradiation in breast cancer patients is an already widespread procedure for reconstructing volume deficits of the diseased breast. Nevertheless, the safety of lipotransfer has still not been clarified yet due to contradictory data. The goal of this in vitro study was to further elucidate the potential effects of lipotransfer on the irradiated remaining breast tissue. The mammary epithelial cell line MCF-10A was co-cultured with the fibroblast cell line MRC-5 and irradiated with 2 and 5 Gy. Afterwards, cells were treated with conditioned medium (CM) from adipose-derived stem cells (ADSC), and the effects on the cellular functions of MCF-10A cells and on gene expression at the mRNA level in MCF-10A and MRC-5 cells were analyzed. Treatment with ADSC CM stimulated transmigration and invasion and decreased the surviving fraction of MCF-10A cells. Further, the expression of cytokines, extracellular, and mesenchymal markers was enhanced in mammary epithelial cells. Only an effect of ADSC CM on irradiated fibroblasts could be observed. The present data suggest epithelial-mesenchymal transition-like changes in the epithelial mammary breast cell line. Thus, the benefits of lipotransfer after BCT should be critically weighed against its possible risks for the affected patients.

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines.

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

An inhibitor of RORγ for chronic pulmonary obstructive disease treatment.

The role of RORγ as a transcription factor for Th17 cell differentiation and thereby regulation of IL-17 levels is well known. Increased RORγ expression along with IL-17A levels was observed in animal models, immune cells and BAL fluid of COPD patients. Increased IL-17A levels in severe COPD patients are positively correlated with decreased lung functions and increased severity symptoms and emphysema, supporting an urgency to develop novel therapies modulating IL-17 or RORγ for COPD treatment. We identified a potent RORγ inhibitor, PCCR-1 using hit to lead identification followed by extensive lead optimization by structure-activity relationship. PCCR-1 resulted in RORγ inhibition with a high degree of specificity in a biochemical assay, with > 300-fold selectivity over other isoforms of ROR. Our data suggest promising potency for IL-17A inhibition in human and canine PBMCs and mouse splenocytes with no significant impact on Th1 and Th2 cytokines. In vivo, PCCR-1 exhibited significant efficacy in the acute CS model with dose-dependent inhibition of the PD biomarkers that correlated well with the drug concentration in lung and BAL fluid, demonstrating an acceptable safety profile. This inhibitor effectively inhibited IL-17A release in whole blood and BALf samples from COPD patients. Overall, we identified a selective inhibitor of RORγ to pursue further development of novel scaffolds for COPD treatment.

Curcumin reverses diabetes-induced endothelial progenitor cell dysfunction by enhancing MnSOD expression and activity in vitro and in vivo.

Diabetes mellitus (DM) causes dysfunction of endothelial progenitor cells (EPCs), resulting in impaired wound healing. EPC therapy is a potential substitute to the current treatments of chronic wounds. Because EPCs isolated from diabetic patients are dysfunctional and therefore pose an obstacle in their efficacious employment in autologous cell therapy, a strategy to rescue them prior to transplantation would be expected to improve the efficacy of autologous cell therapy multifold. Compromised reactive oxygen species scavenging ability being the main cause of EPC dysfunction (EPCD), reactive oxygen species scavengers are likely to reverse or rescue EPCD. Therefore, in this study, we evaluated the potential of curcumin in reversing DM-induced EPCD. We found that in vitro treatment of bone marrow EPCs from diabetic mice (D-EPC) with curcumin restored their functionality, as judged by colony formation, tubule formation, and migration assays. Most importantly, autologous transplantation of curcumin-treated D-EPCs onto diabetic wounds also resulted in accelerated wound healing. Furthermore, curcumin-treated diabetic mice exhibited improved wound healing, as compared with their vehicle-treated diabetic counterparts, underscoring the efficacy of curcumin in vivo as well. The levels and activity of manganese superoxide dismutase (MnSOD) in D-EPCs treated in vitro with curcumin or those isolated from curcumin-treated diabetic mice were comparable with those in non-diabetic EPCs. Addition of methyl mercury chloride to inhibit MnSOD activity during curcumin treatment abolished the salutary effects of curcumin. Our data demonstrate that curcumin reverses DM-induced EPCD by boosting MnSOD expression and activity and emphasizes its potential for use in autologous cell therapy for diabetic wound management.

Matrix-entrapped cellular secretome rescues diabetes-induced EPC dysfunction and accelerates wound healing in diabetic mice.

Cellular secretory products have infinite potential, which is only recently explored for research and therapeutic applications. The present study elaborated on the formation of a unique matrix-entrapped cellular secretome (MCS), a hydrogel-like secretome produced by bone marrow-derived mononuclear cells when cultured on a three-dimensional electrospun nanofiber matrix under specific conditions. These culture conditions support the growth of a mixed population predominantly comprising of endothelial precursor cells (EPCs), along with mesenchymal stromal cells and pericytes. Interestingly, such secretome is not formed in a pure culture of EPCs on the similarly formulated matrix, suggesting that a heterotypic cell-cell interaction is essential for the formation of MCS. In addition, the specific composition of the matrix was found to be a critical necessity for the formation of MCS. Furthermore, the application of the MCS as a substrate promotes the growth of EPCs in culture. It also rescues the diabetes-induced EPC dysfunction as assessed based on the parameters, such as viability, proliferation, colony formation, cellular adhesion, chemotactic migration, and tubule formation. MCS augments the levels of eNOS-specific mRNA (Nos3) and also promotes the restoration of the SDF1/CXCR4 axis in diabetic EPCs. Notably, a topical application of MCS on diabetic wounds leads to an accelerated wound closure. Thus, the current data showed that MCS forms an excellent cell-free biomaterial in the treatment of diabetic wounds and non-healing ulcers.