Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products

Combination of phenethyl isothiocyanate and dasatinib inhibits hepatocellular carcinoma metastatic potential through FAK/STAT3/Cadherin signalling and reduction of VEGF secretion

Cancerous cells are characterised by their ability to invade, metastasise and induce angiogenesis. To achieve this, tumour cells use various molecules that can be targeted to reverse these processes. Dasatinib, a potent Src inhibitor, has shown promising results in treating hepatocellular carcinoma (HCC) in vitro and in vivo. However, its effectiveness is limited by FAK activation. Isothiocyanates (ITCs), on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effects of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch wound healing, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac mem-brane (YSM) assays, to evaluate the effect of the drug combination on HCC metastatic potential and angiogenesis in vitro and in vivo. The results showed that the combination inhibited adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Addi-tionally, the combination reduced angiogenesis in vitro, reducing the growth of new vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin ex-pression and reduced VEGF secretion, thus reducing HCC metastatic potential. Therefore, com-bination of PEITC and dasatinib could be a potential therapeutic strategy for the treatment of HCC

Dietary isothiocyanates for cancer combination therapy: Synergistic anticancer activity of phenethyl isothiocyanate and dasatinib

Combination therapies exploit multiple drugs to synergistically target key pathways to reduce tumour growth and metastatic potential. Isothiocyanates (ITCs) have been shown to have anticancer capabilities and may complement the anticancer activity of clinically approved chemotherapeutic agents. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which is second most lethal tumour. Thus, there is a continuous need for the development of new therapeutic solutions, and the use of ITCs in combination therapy seems to be a promising strategy for treatment of HCC progression. The research presented in this thesis aimed to assess the potential of phenethyl isothiocyanate (PEITC) to improve the activity of dasatinib in HCC. A murine syngeneic subcutaneous model was used to assess the combination efficacy in vivo and the MTT cell viability assay was used to assess the anticancer effect in vitro . Furthermore, the effect on HCC metastatic potential and angiogenesis was assessed using adhesion, scratch wound healing, invasion, colony formation, chorioallantoic membrane (CAM), and yolk sac membrane assays. Flow cytometry, RT-qPCR, HPLC and Western blot were used to elucidate the mechanism of action of the drug combination. PEITC and dasatinib showed a synergistic anticancer effect in vitro and in vivo. By inducing oxidative stress, through the production of reactive oxygen species (ROS), PEITC and dasatinib combination promoted the formation of a premature CDK1-Cyclin B1 complex, leading to mitotic catastrophe and activating oxeiptosis, a novel programmed cell death. Furthermore, the inhibition of FAK/STAT3 signalling led to increased E-cadherin expression and diminished secretion of pro-angiogenic factors, reducing HCC metastatic potential. Phenethyl isothiocyanate can complement dasatinib's action in treating HCC, inducing cell cycle arrest and oxeiptosis and decreasing metastatic potential and tumour-induced angiogenesis. This thesis highlights the role of ITCs in cancer therapy to complement clinically approved chemotherapeutic drugs