Macrophages as carriers of boron carbide nanoparticles dedicated to boron neutron capture therapy.

BACKGROUND: The use of cells as carriers for the delivery of nanoparticles is a promising approach in anticancer therapy, mainly due to their natural properties, such as biocompatibility and non-immunogenicity. Cellular carriers prevent the rapid degradation of nanoparticles, improve their distribution, reduce cytotoxicity and ensure selective delivery to the tumor microenvironment. Therefore, we propose the use of phagocytic cells as boron carbide nanoparticle carriers for boron delivery to the tumor microenvironment in boron neutron capture therapy. RESULTS: Macrophages originating from cell lines and bone marrow showed a greater ability to interact with boron carbide (B4C) than dendritic cells, especially the preparation containing larger nanoparticles (B4C 2). Consequently, B4C 2 caused greater toxicity and induced the secretion of pro-inflammatory cytokines by these cells. However, migration assays demonstrated that macrophages loaded with B4C 1 migrated more efficiently than with B4C 2. Therefore, smaller nanoparticles (B4C 1) with lower toxicity but similar ability to activate macrophages proved to be more attractive. CONCLUSIONS: Macrophages could be promising cellular carriers for boron carbide nanoparticle delivery, especially B4C 1 to the tumor microenvironment and thus prospective use in boron neutron capture therapy.

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.

Synthesis and Characterization of Boron Carbide Nanoparticles as Potential Boron-Rich Therapeutic Carriers.

Boron carbide is one of the hardest materials in the world which can be synthesized by various methods. The most common one is a carbothermic or magnesiothermic reduction of B2O3 performed at high temperatures, where the obtained powder still requires grinding and purification. The goal of this research is to present the possibility of synthesizing B4C nanoparticles from elements via vapor deposition and modifying the morphology of the obtained powders, particularly those synthesized at high temperatures. B4C nanoparticles were synthesized in the process of direct synthesis from boron and carbon powders heated at the temperature of 1650 °C for 2 h under argon and characterized by using scanning electron microscopy, transmission electron microscopy, atomic force microscopy, X-ray diffraction analysis, and dynamic light scattering measurements. The physicochemical characteristics of B4C nanoparticles were determined, including the diffusion coefficients, hydrodynamic diameter, electrophoretic mobilities, and zeta potentials. An evaluation of the obtained B4C nanoparticles was performed on several human and mouse cell lines, showing the relation between the cytotoxicity effect and the size of the synthesized nanoparticles. Assessing the suitability of the synthesized B4C for further modifications in terms of its applicability in boron neutron capture therapy was the overarching goal of this research.

Inhibition of MC38 colon cancer growth by multicomponent chemoimmunotherapy with anti-IL-10R antibodies, HES-MTX nanoconjugate, depends on application of IL-12, IL-15 or IL-18 secreting dendritic cell vaccines.

Background: The tumor microenvironment (TME) provides a conducive environment for the growth and survival of tumors. Negative factors present in TME, such as IL-10, may limit the effectiveness of cellular vaccines based on dendritic cells, therefore, it is important to control its effect. The influence of IL-10 on immune cells can be abolished e.g., by using antibodies against the receptor for this cytokine - anti-IL-10R. Furthermore, the anticancer activity of cellular vaccines can be enhanced by modifying them to produce proinflammatory cytokines, such as IL-12, IL-15 or IL-18. Additionally, an immunomodulatory dose of methotrexate and hydroxyethyl starch (HES-MTX) nanoconjugate may stimulate effector immune cells and eliminate regulatory T cells, which should enhance the antitumor action of immunotherapy based on DC vaccines. The main aim of our study was to determine whether the HES-MTX administered before immunotherapy with anti-IL-10R antibodies would change the effect of vaccines based on dendritic cells overproducing IL-12, IL-15, or IL-18. Methods: The activity of modified DCs was checked in two therapeutic protocols - immunotherapy with the addition of anti-IL10R antibodies and chemoimmunotherapy with HES-MTX and anti-IL10R antibodies. The inhibition of tumor growth and the effectiveness of the therapy in inducing a specific antitumor response were determined by analyzing lymphoid and myeloid cell populations in tumor nodules, and the activity of restimulated splenocytes. Results and conclusions: Using the HES-MTX nanoconjugate before immunotherapy based on multiple administrations of anti-IL-10R antibodies and cellular vaccines capable of overproducing proinflammatory cytokines IL-12, IL-15 or IL-18 created optimal conditions for the effective action of these vaccines in murine colon carcinoma MC38 model. The applied chemoimmunotherapy caused the highest inhibition of tumor growth in the group receiving DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg at the level of 72.4%. The use of cellular vaccines resulted in cytotoxic activity increase in both immuno- or chemoimmunotherapy. However, the greatest potential was observed both in tumor tissue and splenocytes obtained from mice receiving two- or three-component vaccines in the course of combined application. Thus, the designed treatment schedule may be promising in anticancer therapy.

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.

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.

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.

Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-ß1.

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-ß1 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-ß1 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-ß1) 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-ß1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment.

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

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.

Intratumoral Lentivector-Mediated TGF-ß1 Gene Downregulation As a Potent Strategy for Enhancing the Antitumor Effect of Therapy Composed of Cyclophosphamide and Dendritic Cells.

Vaccination with dendritic cells (DCs) stimulated with tumor antigens can induce specific cellular immune response that recognizes a high spectrum of tumor antigens. However, the ability of cancer cells to produce immunosuppressive factors drastically decreases the antitumor activity of DCs. The main purpose of the study was to improve the effectiveness of DC-based immunotherapy or chemoimmunotherapy composed of cyclophosphamide (CY) and DCs by application of lentivectors (LVs)-encoding short hairpin RNA specific for TGF-ß1 (shTGFß1 LVs). We observed that s.c. inoculation of both MC38 cells with silenced expression of TGF-ß1 (MC38/shTGF-ß1) and direct intratumoral application of shTGFß1 LVs contributed to reduction of suppressor activity of myeloid cells and Tregs in tumor. Contrary to expectations, in mice bearing wild tumor, the application of shTGFß1 LVs prior to vaccination with bone marrow-derived DC stimulated with tumor antigens (BMDC/TAg) did not influence myeloid-derived suppressor cell (MDSC) infiltration into tumor. As a result, we observed only minor MC38 tumor growth inhibition (TGI) accompanied by systemic antitumor response activation comparable to that obtained for negative control (shN). However, when the proposed scheme was complemented by pretreatment with a low dose of CY, we noticed high MC38 TGI together with decreased number of MDSCs in tumor and induction of Th1-type response. Moreover, in both schemes of treatment, LVs (shTGFß1 as well as shN) induced high influx of CTLs into tumor associated probably with the viral antigen introduction into tumor microenvironment. Concluding, the application of shTGFß1 LVs alone or in combination with DC-based vaccines is not sufficient for long-lasting elimination of suppression in tumor. However, simultaneous reduction of TGF-ß1 in tumor microenvironment and its remodeling by pretreatment with a low dose of CY facilitates the settlement of peritumorally inoculated DCs and supports them in restoration and activation of a potent antitumor response.