Therapeutic Targeting Cancer-Initiating Cell Markers by Exosome miRNA: Efficacy and Functional Consequences Exemplified for claudin7 and EpCAM

AIM: Transfer of exosomes (Exo) miRNA was described interfering with tumor progression. We here explored for claudin7 (cld7) and EpCAM (EpC), cancer-initiating-cell markers in colorectal and pancreatic cancer, the efficacy of Exo loading with miRNA and miRNA transfer. METHODS: Exo were collected from nontransformed mouse (NIH3T3) and rat lung fibroblasts (rFb), which were transfected with Tspan8 cDNA (NIH3T3-Tspan8, rFb-Tspan8). Exo were loaded by electroporation with miRNA. The transfer of Exo-miRNA was evaluated in vitro and in vivo in a rat pancreatic (ASML) and a human colon (SW948) cancer line. RESULTS: NIH3T3-Tspan8- or rFb-Tspan8-Exo were efficiently loaded with cld7- or EpC-miRNA. Exo targeting in vivo was strongly improved by tailoring with Tspan8. Exo-miRNA transfer into tumor targets promoted cld7, respectively, EpC downregulation by 33%-60%. Cld7 silencing was accompanied by reduced expression of additional cancer-initiating cell markers and NOTCH. EpC silencing reduced vimentin, N-cadherin, and Nanog expression. The Exo-miRNA transfer affected anchorage-independent growth, motility, and invasion. CONCLUSIONS: Exo are efficiently loaded with miRNA, miRNA-delivery being supported by Exo tailoring. Partial cld7 and EpC silencing by Exo miRNA affects metastasis-promoting tumor cell activities. The findings suggest miRNA loading of tailored Exo as an easy approachable and efficient adjuvant therapy

Low efficiency of leucocyte plugging-based drug delivery to cancer in mice

Cells of the immune system were proposed for use as Trojan horse for tumour-specific drug delivery. The efficacy of such cell-based drug delivery depends on the site-specific cell homing. This present study was aimed to investigate the potential of leucocytes for intratumoural site-specific enrichment using a locoregional application route in experimental liver tumours. Human neutrophils were isolated from peripheral blood and directly labelled with calcein AM or loaded with doxorubicin. The neutrophil loading and release of doxorubicin and the migration and adhesion to ICAM-1 were analysed in vitro. Macrophages were isolated and activated in vitro. Leucocyte plugging and the distribution pattern in the liver microvasculature were studied ex vivo, and the efficacy of leucocyte plugging in tumour blood vessels was analysed in vivo after superselective intra-arterial injection in mouse liver tumour models. Neutrophils were characterised by the high dose-dependent uptake and rapid release of doxorubicin. Doxorubicin loading did not affect neutrophil migration function. Neutrophil plugging in liver microvasculature was very high (> 90%), both after ex vivo perfusion and after injection in vivo. However, neutrophils as well as activated macrophages plugged insufficiently in tumour blood vessels and passed through the tumour microvasculture with a very low sequestration rate in vivo. Neutrophils possess several properties to function as potentially effective drug carriers; however, the tumour site-specific drug delivery after selective locoregional injection was observed to be insufficient owing to low intratumoural microvascular plugging

Blockade of leukocyte haptokinesis and haptotaxis by ketoprofen, diclofenac and SC-560

Abstract Background Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. The potential direct effect of NSAID on leukocyte migration was poorly investigated. Using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different NSAID, ketoprofen, diclofenac and SC-560, on leukocyte haptokinesis and haptotaxis in vivo and in vitro. Results NSAID induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. This effect was dose-dependent and was not restricted to a specific type of leukocytes. The inhibition of leukocyte migration by NSAID was partially re-stored after removal of inhibiting agent. Only complete blockade of leukocyte migration was accompanied by a strong reduction of [Ca2+]i. Conclusions NSAID strongly supress leukocyte migration. The results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of NSAID.</p