The modulation of local and systemic anti-tumor immune response induced by methotrexate nanoconjugate in murine MC38 colon carcinoma and B16 F0 melanoma tumor models.

Methotrexate (MTX) which is one of the longest-used cytostatics, belongs to the group of antimetabolites and is used for treatment in different types of cancer as well as during autoimmune diseases. MTX can act as a modulator enable to create the optimal environment to generate the specific anti-tumor immune response. A novel system for MTX delivery is its conjugation with high-molecular-weight carriers such as hydroxyethyl starch (HES), a modified amylopectin-based polymer applied in medicine as a colloidal plasma volume expander. Such modification prolongs the plasma half-life of the HES-MTX nanoconjugate and improves the distribution of the drug in the body. In the current study, we focused on evaluating the dose-dependent therapeutic efficacy of chemotherapy with HES-MTX nanoconjugate compared to the free form of MTX, and examining the time-dependent changes in the local and systemic anti-tumor immune response induced by this therapy. To confirm the higher effectiveness of HES-MTX in comparison to MTX, we analyzed its action using murine MC38 colon carcinoma and B16 F0 melanoma tumor models. It was noted that HES-MTX at a dose of 20 mg/kg b.w. was more effective in tumor growth inhibition than MTX in both tumor models. One of the main differences between the two analyzed tumor models concerned the kinetics of the appearance of the immunomodulation. In MC38 tumors, the beneficial change in the tumor microenvironment (TME) landscape, manifested by the depletion of pro-tumor immune cells, and increased influx of cells with strong anti-tumor activity was noted already 3 days after HES-MTX administration, while in B16 F0 model, these changes occurred 10 days after the start of therapy. Thus, the immunomodulatory potential of the HES-MTX nanoconjugate may be closely related to the specific immune cell composition of the TME, which combined with additional treatment such as immunotherapies, would enhance the therapeutic potential of the nanoconjugate.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Background: Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. Methods: With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. Results and conclusions: In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

The Beneficial Effect of IL-12 and IL-18 Transduced Dendritic Cells Stimulated with Tumor Antigens on Generation of an Antitumor Response in a Mouse Colon Carcinoma Model.

The main purpose of our study was to determine the effect of dendritic cell (DC) transduction with lentiviral vectors carrying sequences of il18 and/or il12 genes on the level of antitumor activity in vitro and in vivo. We examined the ability of DCs to migrate to the tumor-draining lymph nodes and infiltrate tumor tissue and to activate the local and systemic antitumor response. On the 15th day, DCs genetically modified for production of IL-12 and/or IL-18 were administered peritumorally to C57BL/6 female mice with established MC38 tumors. Lymphoid organs and tumor tissue were collected from mice on the 3rd, 5th, and 7th days after a single administration of DCs for further analysis. Administration of DCs transduced for production of IL-12 alone and in combination with IL-18 increased the inflow and activity of CD4+ and CD8+ T lymphocytes in the tumor microenvironment and tumor-draining lymph nodes. We also found that even a single administration of such modified DCs could trigger a systemic antitumor response as well as inhibit tumor growth. Application of the developed DC-based vaccines may exert a favorable impact on stimulation of an antitumor immune response, especially if these DC vaccines are administered repeatedly.

Boron-Rich Boron Carbide Nanoparticles as a Carrier in Boron Neutron Capture Therapy: Their Influence on Tumor and Immune Phagocytic Cells.

The aim of the work was to study the interaction between boron-rich boron carbide nanoparticles and selected tumor and immune phagocytic cells. Experiments were performed to investigate the feasibility of the application of boron carbide nanoparticles as a boron carrier in boron neutron capture therapy. Boron carbide powder was prepared by the direct reaction between boron and soot using the transport of reagents through the gas phase. The powder was ground, and a population of nanoparticles with an average particle size about 80 nm was selected by centrifugation. The aqueous suspension of the nanoparticles was functionalized with human immunoglobulins or FITC-labeled human immunoglobulins and was then added to the MC38 murine colon carcinoma and to the RAW 264.7 cell line of mouse macrophages. Flow cytometry analysis was used to determine interactions between the functionalized boron carbide nanoparticles and respective cells. It was shown that B4C-IgG nanoconjugates may bind to phagocytic cells to be internalized by them, at least partially, whereas such nanoconjugates can only slightly interact with molecules on the cancer cells' surface.

Treatment with lentiviral vectors encoding shRNA against interleukin 10 modulates the immunosuppressive activity of murine colon carcinoma-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are potent suppressors of antitumor immunity and their accumulation is often associated with poor prognosis. The aim of the present study was to determine the mechanisms of action of lentiviral vectors encoding short hairpin (sh)RNA against interleukin-10 (IL-10), with particular emphasis on their influence on the activity of tumor-derived MDSCs. Lentiviral vectors encoding shRNA against IL-10 (shIL-10 LVs) were utilized to silence the expression of IL-10 either in MDSCs that were generated ex vivo from bone marrow cells cultured in the presence of supernatant from MC38 colon carcinoma cells, or in situ in the MC38 murine colon carcinoma environment. Although monocytic MDSCs (M-MDSCs) transduced with shIL-10 LVs exhibited increased suppressor activity, transduction of polymorphonuclear MDSCs (PMN-MDSCs) appeared to reduce their ability to inhibit T lymphocyte functions. Analysis of EGFP expression in MC38 tumors revealed that intratumorally inoculated shIL-10 LVs transduced tumor-infiltrating myeloid cells with the highest efficiency and, led to a decreased IL-10 level in the tumor microenvironment. However, the effect was accompanied by increased influx of PMN-MDSCs into tumors observed both on the 6th and on the 10th day after shIL-10 LV injections. Nevertheless, it was noted that suppressor activity of myeloid cells isolated from tumors was dependent on the efficiency of tumor-derived PMN-MDSC transduction with shIL-10 LVs. The increased percentage of transduced PMN-MDSCs on the 10th day was associated with diminished immunosuppressive activity of tumor-derived myeloid cells and an elevated ratio of cytotoxic T lymphocytes to M-MDSCs. The obtained data indicated that treatment with shIL-10 LVs may result in modulation of the immunosuppressive activity of MC38 colon carcinoma-derived MDSCs.

Immunomodulatory potential of anticancer therapy composed of methotrexate nanoconjugate and dendritic cell­based vaccines in murine colon carcinoma.

Chemotherapy with low­molecular weight compounds, despite elimination of cancer cells, entails adverse effects. To overcome this disadvantage, innovative drug delivery systems are being developed, including conjugation of macromolecular carriers with therapeutics, e.g. a nanoconjugate of hydroxyethyl starch and methotrexate (HES­MTX). The purpose of the present study was to determine whether HES­MTX, applied as a chemotherapeutic, is able to modulate the immune response and support the antitumor response generated by dendritic cells (DCs) used subsequently as immunotherapeutic vaccines. Therefore, MTX or HES­MTX was administered, as sole treatment or combined with DC­based vaccines, to MC38 colon carcinoma tumor­bearing mice. Alterations in antitumor immune response were evaluated by multiparameter flow cytometry analyses and functional assays. The results demonstrated that the nanoconjugate possesses greater immunomodulatory potential than MTX as reflected by changes in the landscape of immune cells infiltrating the tumor and increased cytotoxicity of splenic lymphocytes. In contrast to MTX, therapy with HES­MTX as sole treatment or combined with DC­based vaccines, contributed to significant tumor growth inhibition. However, only treatment with HES­MTX and DC­based vaccines activated the systemic specific antitumor response. In conclusion, due to its immunomodulatory properties, the HES­MTX nanoconjugate could become a potent anticancer agent used in both chemo­ and chemoimmunotherapeutic treatment schemes.

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.

Halogenated aromatic thiosemicarbazones as potent inhibitors of tyrosinase and melanogenesis.

A set of 21 halogenated thiosemicarbazones (TSCs) have been synthesized and its inhibitory properties toward activity diphenolase of mushroom tyrosinase and their ability to inhibition of melanogenesis in B16F10 murine, melanoma cell line have been investigated. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. The obtained outcomes allowed us to perform SAR analysis. TSC 6, 12 and 21 exhibited the most potent inhibitory properties showing IC50 of 0.5, 0.9 and 0.8 µM, respectively. They revealed reversible and competitive manner of tyrosinase inhibition. According to SAR analysis, para-substituted acetophenone derivatives of thiosemicarbazones have the highest affinity to the enzyme among the investigated compounds. Melanin production in B16F10 cells was inhibited by all investigated compounds at the micromolar level. Suggested inhibition mechanism is based on the interaction between a sulfur atom of thiourea moiety of the thiosemicarbazones, and copper ions in the active site of the enzyme. These results might be useful in searching novel inhibitors of melanogenesis which could be used in the cosmetic and food industry.

De­O­acylated lipooligosaccharide of E. coli B reduces the number of metastatic foci via downregulation of myeloid cell activity.

Lipopolysaccharides are the main surface antigens and virulence factors of gram­negative bacteria. Removal of four ester­bound fatty acid residues from hexaacyl lipid A of Escherichia coli lipooligosaccharide (LOS) resulted in the de­O­acylated derivative E. coli LOS­OH (LOS­OH). This procedure caused a significant reduction in the toxicity of this compound compared to the native molecule. We investigated the effect of such a structural LOS modification on its biological activity using in vitro assays with monocytic cells of the RAW264.7 line, dendritic cells of the JAWS II line, bone marrow­derived dendritic cells (BM­DCs), and spleen cells. Furthermore, in in vivo experiments with a melanoma B16 metastasis model, the anti­metastatic activity of the compounds and spleen cell reactivity mediated by them representing a systemic response were analyzed. The results revealed that LOS­OH demonstrated weaker ability than LOS to stimulate and polarize an immune response both in vitro and in vivo. It induced lower cytokine production by cells of myeloid lines. Multiple applications of LOS­OH into mice injected intravenously with B16 cells significantly (P<0.05; P<0.01) reduced the number of metastatic foci in the lungs, presumably via silencing of myeloid cell reactivity as well as the inability to stimulate lymphoid cells both directly and indirectly. These findings suggest that LOS­OH maintained in the body of metastasis­bearing mice appears to modulate or downregulate the innate response, leading to the inability of blood myeloid cells to support the migration of melanoma cells to lung tissue.