Tissue localization of tumor antigen-loaded mouse dendritic cells applied as an anti-tumor vaccine and their influence on immune response.

The recognition, internalization and intracellular processing of antigen are the main functions of dendritic cells (DCs). In the course of these processes, DCs differentiate and acquire the ability to produce cytokines responsible for polarization of the immunological response. Therefore, vaccination with tumor antigen-loaded DCs is one of the most promising approaches to induce tumor-specific immune response. The purpose of this study was to analyze the migratory abilities, from an injection site to tumor-draining lymph nodes (tLN), of DCs applied as an anti-tumor vaccine and their capacity for immune response activation. Mouse DCs of the established JAWS II cell line transduced with EGFP gene or ex vivo bone marrow-isolated DCs (BM-DCs) stained with intravital CFDA dye were loaded with MC38 colon carcinoma tumor lysate (TAg) and then administered peritumorally to MC38 tumor-bearing C57BL/6 mice. On the first, third, fifth and seventh days after injection the tumors, tLNs and spleens were examined. The TAg-loaded DCs migrated more effectively to the tLNs than did the unloaded control DCs; however, the majority of them remained in the tumor vicinity. Immunohistological analysis of the tumor tissues demonstrated that only TAg-loaded DCs activated an immune response. Seven days after DCs vaccine administration, numerous necrotic areas and some apoptotic bodies were observed in the tumor tissue. However, the anti-MC38 tumor cytotoxic activity of spleen and tLN cells from mice treated with both TAg-loaded and unloaded DCs reached a maximum on the fifth day after DC injection. Concluding, TAg-loaded DCs migrated more efficiently to tLNs and were more effective activators of local (but not systemic) cellular immune response than were unloaded DCs. We hypothesize that only the application of TAg-loaded DCs to tumor-bearing mice as an adjuvant supporting chemotherapy may activate a more effective anti-tumor response

Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1

Recent developments demonstrate that tumor-derived extracellular vesicles (EVs) could become a highly effective tool for delivery of antitumor factors. The main objective of the study was to determine whether EVs secreted by MC38 colon carcinoma cells genetically engineered for overproduction of interleukin (IL-)12 and/or shRNA targeting TGF-β1 are effectively loaded with these molecules and whether the obtained EVs could be an efficient tool for antitumor therapy. Fractions of EVs released by genetically modified MC38 cells [both modified tumor-derived exosomes (mTEx) and modified microvesicles (mTMv)] and those released by unmodified, wild-type MC38 cells were characterized in terms of loading efficacy, using real-time PCR and ELISA, as well as their antitumor potential. In order to examine the therapeutic potential of mTEx, they were applied in the form of sole treatment as well as in combination with dendritic cell (DC)-based vaccines stimulated with mTMv in the therapy of mice with subcutaneously growing MC38 tumors. The results demonstrated that genetic modification of wild-type MC38 tumor cells is an effective method of loading the molecules of interest into extracellular vesicles secreted by the cells (both TEx and TMv). The results also showed that mTEx secreted by cells engineered for overproduction of IL-12 and/or shRNA for TGF-β1 are able to induce tumor growth inhibition as opposed to TEx from unmodified MC38 cells. Additionally, antitumor therapy composed of mTEx (especially those deprived of TGF-β1) and DC-based vaccines allowed for regeneration of antitumor immunity and induction of the systemic Th1 response responsible for the sustained effect of the therapy. In conclusion, tumor-derived exosomes loaded with IL-12 and/or deprived of TGF-β1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment

Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene

Interleukin (IL-) 2 acts on a number of types of immune cells promoting their effector functions. To replace&lt;br /&gt;systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in&lt;br /&gt;different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral&lt;br /&gt;vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells) alone or combined with tumor antigenstimulated&lt;br /&gt;dendritic cells (JAWS II/TAg) were exploited to treat colon carcinoma MC38-bearing mice. After the&lt;br /&gt;peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating&lt;br /&gt;CD4+ and CD8+ T lymphocytes were noted. A considerable increase in CD4+ cell influx into tumor tissue was observed&lt;br /&gt;when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in&lt;br /&gt;intensity of CD8+ cell infiltration was associated with immune reaction triggered by the same combination of applied&lt;br /&gt;cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell&lt;br /&gt;specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of&lt;br /&gt;various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered&lt;br /&gt;tumor antigen.<br>Interleukin (IL-) 2 acts on a number of types of immune cells promoting their effector functions. To replace&lt;br /&gt;systemic administration of recombinant form of this cytokine, various genetically modified cells have been used in&lt;br /&gt;different preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral&lt;br /&gt;vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells) alone or combined with tumor antigenstimulated&lt;br /&gt;dendritic cells (JAWS II/TAg) were exploited to treat colon carcinoma MC38-bearing mice. After the&lt;br /&gt;peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating&lt;br /&gt;CD4+ and CD8+ T lymphocytes were noted. A considerable increase in CD4+ cell influx into tumor tissue was observed&lt;br /&gt;when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in&lt;br /&gt;intensity of CD8+ cell infiltration was associated with immune reaction triggered by the same combination of applied&lt;br /&gt;cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell&lt;br /&gt;specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of&lt;br /&gt;various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered&lt;br /&gt;tumor antigen

Reprogramming the murine colon cancer microenvironment using lentivectors encoding shRNA against IL-10 as a component of a potent DC-based chemoimmunotherapy

Abstract Background The excessive amounts of immunosuppressive factors present in a tumor microenvironment (TME) reduce the effectiveness of cancer vaccines. The main objective of our research was to improve the effectiveness of dendritic cell (DC)-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors encoding shRNA specific to IL-10 (shIL10 LVs) in murine colon carcinoma MC38 model. Methods The efficacy of shIL10 LVs in silencing of IL-10 expression was measured both in vitro and in vivo using Real-Time PCR and ELISA assays. In addition, the influence of intratumorally inoculated lentivectors on MC38 tumor microenvironment was examined using flow cytometry method. The effect of applied therapeutic schemes was determined by measurement of tumor growth inhibition and activation state of local and systemic immune response. Results We observed that intratumorally inoculated shIL10 LVs transduced tumor and TME-infiltrating cells and reduced the secretion of IL-10. Application of shIL10 LVs for three consecutive weeks initiated tumor growth inhibition, whereas treatment with shIL10 LVs and BMDC/TAg did not enhance the antitumor effect. However, when pretreatment with CY was introduced to the proposed scheme, we noticed high MC38 tumor growth inhibition accompanied by reduction of MDSCs and Tregs in TME, as well as activation of potent local and systemic Th1-type antitumor response. Conclusions The obtained data shows that remodeling of TME by shIL10 LVs and CY enhances DC activity and supports them during regeneration and actuation of a potent antitumor response. Therefore, therapeutic strategies aimed at local IL-10 elimination using lentiviral vectors should be further investigated in context of combined chemoimmunotherapies

Monosubstituted Acetophenone Thiosemicarbazones as Potent Inhibitors of Tyrosinase: Synthesis, Inhibitory Studies, and Molecular Docking

A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC50) below 1 &micro;M. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The para-substituted TSCs had higher affinity for the enzyme as compared to their ortho- and meta-analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries

Monosubstituted Acetophenone Thiosemicarbazones as Potent Inhibitors of Tyrosinase: Synthesis, Inhibitory Studies, and Molecular Docking

A set of 12 monosubstituted acetophenone thiosemicarbazone derivatives (TSCs) were synthesized and their inhibitory properties toward tyrosinase activity were tested. Moreover, their ability to inhibit melanogenesis in the B16F10 murine melanoma cell line was studied. In order to investigate the nature of interactions between the enzyme and the inhibitors, molecular docking to the active site was performed. TSCs 5, 6, 8, and 9 revealed a half maximal inhibitory concentration (IC50) below 1 µM. Compound 6 turned out to be the most potent tyrosinase inhibitor. All investigated compounds showed reversible inhibition of competitive or mixed type. The para-substituted TSCs had higher affinity for the enzyme as compared to their ortho- and meta-analogues. All investigated compounds inhibited melanin production in B16F10 cells at the micromolar level. Molecular docking showed that the sulfur atom of the thiourea moiety penetrates the active site and interacts with copper ions. The above outcomes might be helpful in the design of new tyrosinase inhibitors in the food and cosmetic industries

Electroporation with Cisplatin against Metastatic Pancreatic Cancer: In Vitro Study on Human Primary Cell Culture

Despite the rapid progression of cancer pharmacotherapy, the high drug resistance of pancreatic ductal adenocarcinoma (PDA) makes it one of the most lethal malignancies. Therefore, there are high expectations associated with experimental therapies, such as electrochemotherapy (ECT). This technique involves the application of short electric pulses to induce transitional permeabilization of the cellular membrane, thus enhancing drug molecules influx. The aim of the study was to investigate the influence of electroporation with cisplatin (CisEP) on the primary culture of human PDA cells from lung metastases—their survival and stress response. Considering the growing importance of various research models, two established human PDA cell lines, EPP85-181P (sensitive to daunorubicin) and EPP85-181RDB (resistant to daunorubicin), were utilized as a reference control. Cisplatin revealed higher cytotoxicity towards established cell lines. Following CisEP application, we observed a significant decrease of cells viability in the primary culture model. After CisEP therapy, an increased immunoreactivity with SOD-2 and Casp-3 antibodies was noticed. In conclusion, we discovered that electroporation can enhance the cytotoxic effect of cisplatin in pancreatic cancer cells in vitro. This effect was evident for cells from the primary culture. The obtained results confirm the importance of primary cells models in studies on the efficacy of experimental cancer therapies

The favourable effect of catechin in electrochemotherapy in human pancreatic cancer cells

Until recently, green tea polyphenols were considered strong antioxidants. However, the latest reports have revealed that bioflavonoids can play a multiple role in anticancer therapy, including the inhibition of cell proliferation and generation of the oxidative stress in a dose-dependent manner. The presented research was designed to examine the potential of the green tea (±)-catechin as a reinforcement of the electrochemotherapy (ECT) with cisplatin in pancreatic cancer in vitro. The study was performed on two cell lines of the pancreatic ductal adenocarcinoma (PDA) - parental EPP85-181P and multidrug-resistant EPP85-181RNOV. Prior to the ECT protocol the cells were preincubated with high or low concentration of catechin for 2 or 24 hours, respectively. We assessed the influence of preincubation on the cisplatin toxicity with and without electroporation (EP), the electrosensitivity of PDA cell lines and the uptake of the daunorubicin and propidium iodide. Additionally, we evaluated the antioxidative properties of catechin by the measurement of the ROS-related fluorescence and the immunoreactivity of the oxidative stress-related enzymes superoxide dismutase (SOD2) and glutathione S-transferase (GST). We found that co-treatment with catechin can firmly enhance the efficacy of electroporation with cisplatin in vitro. More favorable effect was obtained for 2-hour incubation, which indicates the involvement of the transcriptional-independent mechanisms of catechin action. The effect may be partially explained by the increased oxidative stress level, which was higher in multidrug-resistant cells. However, further studies on cisplatin-catechin interplay and the thorough examination of the catechin-cell membrane interaction need to be performed