An in vitro model for osteoarthritis using long-cultured inflammatory human macrophages repeatedly stimulated with TLR agonists.

Osteoarthritis (OA) is characterized by an abundance of inflammatory M1-like macrophages damaging local tissues. The search for new potential drugs for OA suffers from the lack of appropriate methods of long-lasting inflammation. Here we developed and characterized an in vitro protocol of long-lasting culture of primary human monocyte-derived macrophages differentiated with a combination of M-CSF+GM-CSF that optimally supported long-cultured macrophages (LC-Mϕs) for up to 15 days, unlike their single use. Macrophages repeatedly stimulated for 15 days with the TLR2 ligand Pam3CSK4 (LCS-Mϕs), showed sustained levels over time of IL-6, CCL2, and CXCL8, inflammatory mediators that were also detected in the synovial fluids of OA patients. Furthermore, macrophages isolated from the synovia of two OA patients showed an expression profile of inflammation-related genes similar to that of LCS-Mϕs, validating our protocol as a model of chronically activated inflammatory macrophages. Next, to confirm that these LCS-Mϕs could be modulated by anti-inflammatory compounds, we employed dexamethasone and/or celecoxib, two drugs widely used in OA treatment, that significantly inhibited the production of inflammatory mediators. This easy-to-use in vitro protocol of long-lasting inflammation with primary human macrophages could be useful for the screening of new compounds to improve the therapy of inflammatory disorders.

Important functional role of the protein osteopontin in the progression of malignant pleural mesothelioma.

Background: Malignant Pleural Mesothelioma (MPM) is an aggressive cancer of the mesothelial lining associated with exposure to airborne non-degradable asbestos fibers. Its poor response to currently available treatments prompted us to explore the biological mechanisms involved in its progression. MPM is characterized by chronic non-resolving inflammation; in this study we investigated which inflammatory mediators are mostly expressed in biological tumor samples from MPM patients, with a focus on inflammatory cytokines, chemokines and matrix components. Methods: Expression and quantification of Osteopontin (OPN) was detected in tumor and plasma samples of MPM patients by mRNA, immunohistochemistry and ELISA. The functional role of OPN was investigated in mouse MPM cell lines in vivo using an orthotopic syngeneic mouse model. Results: In patients with MPM, the protein OPN was significantly more expressed in tumors than in normal pleural tissues and predominantly produced by mesothelioma cells; plasma levels were elevated in patients and associated with poor prognosis. However, modulation of OPN levels was not significantly different in a series of 18 MPM patients receiving immunotherapy with durvalumab alone or with pembrolizumab in combination with chemotherapy, some of whom achieved a partial clinical response. Two established murine mesothelioma cell lines: AB1 and AB22 of sarcomatoid and epithelioid histology, respectively, spontaneously produced high levels of OPN. Silencing of the OPN gene (Spp1) dramatically inhibited tumor growth in vivo in an orthotopic model, indicating that OPN has an important promoting role in the proliferation of MPM cells. Treatment of mice with anti-CD44 mAb, blocking a major OPN receptor, significantly reduced tumor growth in vivo. Conclusion: These results demonstrate that OPN is an endogenous growth factor for mesothelial cells and inhibition of its signaling may be helpful to restrain tumor progression in vivo. These findings have translational potential to improve the therapeutic response of human MPM.

In Vitro Methods to Evaluate Macrophage Polarization and Function in Cancer.

Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the anti-tumoral efficacy of most treatments currently applied in the clinic. However, a key feature of macrophages is their phenotypical and functional plasticity, which called their attention as promising targets for therapeutic intervention based on their elimination or reprogramming toward M1-like cytotoxic effector cells, with anti-tumor functions. This polarization status of macrophages can be studied in terms of molecular markers and functional activities, using an appropriate combination of experimental methodologies, both in vitro and in vivo. Here we focus on describing in vitro protocols to isolate primary monocytes from buffy coats and to study macrophage phenotype and function, after exposure to new therapies, by a combination of flow cytometry, RT-PCR, and ELISA analysis. We also provide the methodology to evaluate in vitro the cytotoxic activity of treated macrophages toward cancer cells.

In Vivo Analysis of Tumor-Associated Macrophages in the Tumor Microenvironment.

In most solid cancers, tumor-associated macrophages (TAMs) infiltrating the tumor microenvironment (TME) represent a major population of immunosuppressive cells. This correlates with poor prognosis and resistance to antitumoral therapies, including immune checkpoint inhibitors. Although initial preclinical studies were primarily meant to deplete macrophages in the TME or prevent their recruitment at tumor sites, recent evidence has indicated that the reprogramming of macrophages into cytotoxic effectors might be more beneficial in eliciting an effective antitumor immune response. Taking this into consideration, the comprehensive analysis of the phenotype and function of macrophages in the TME, and their interaction with cancer cells or other immune cells, has become of paramount importance in oncological research. Accordingly, here we explain the experimental procedures for the in vivo evaluation of tumor progression and response to therapy, with a particular focus on the detailed analysis of TAMs and related immune cells in the TME by flow cytometry, RNA analysis, and multiplex immunophenotyping. The output generated through these experiments allow researchers to test the efficacy of new therapeutic strategies on targeting.

Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

Background: In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. Methods: NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. Results: We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Conclusion: Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

Innate and Adaptive Responses of Intratumoral Immunotherapy with Endosomal Toll-Like Receptor Agonists.

Toll-like receptors (TLRs) are natural initial triggers of innate and adaptive immune responses. With the advent of cancer immunotherapy, nucleic acids engineered as ligands of endosomal TLRs have been investigated for the treatment of solid tumors. Despite promising results, their systemic administration, similarly to other immunotherapies, raises safety issues. To overcome these problems, recent studies have applied the direct injection of endosomal TLR agonists in the tumor and/or draining lymph nodes, achieving high local drug exposure and strong antitumor response. Importantly, intratumoral delivery of TLR agonists showed powerful effects not only against the injected tumors but also often against uninjected lesions (abscopal effects), resulting in some cases in cure and antitumoral immunological memory. Herein, we describe the structure and function of TLRs and their role in the tumor microenvironment. Then, we provide our vision on the potential of intratumor versus systemic delivery or vaccination approaches using TLR agonists, also considering the use of nanoparticles to improve their targeting properties. Finally, we collect the preclinical and clinical studies applying intratumoral injection of TLR agonists as monotherapies or in combination with: (a) other TLR or STING agonists; (b) other immunotherapies; (c) radiotherapy or chemotherapy; (d) targeted therapies.

Intratumoral combination therapy with poly(I:C) and resiquimod synergistically triggers tumor-associated macrophages for effective systemic antitumoral immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer and hinder the antitumoral efficacy of most treatments currently applied in the clinic. Previous studies have evaluated the antitumoral immune response triggered by (TLR) agonists, such as poly(I:C), imiquimod (R837) or resiquimod (R848) as monotherapies; however, their combination for the treatment of cancer has not been explored. This study investigates the antitumoral efficacy and the macrophage reprogramming triggered by poly(I:C) combined with R848 or with R837, versus single treatments. METHODS: TLR agonist treatments were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry using primary human and murine M-CSF-differentiated macrophages. Cytotoxic activity of TLR-treated macrophages toward cancer cells was evaluated with an in vitro functional assay by flow cytometry. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry, RT-qPCR, multispectral immunophenotyping, quantitative proteomic experiments, and protein-protein interaction analysis. RESULTS: Results demonstrated the higher efficacy of poly(I:C) combined with R848 versus single treatments or combined with R837 to polarize macrophages toward M1-like antitumor effectors in vitro. In vivo, the intratumoral synergistic combination of poly(I:C)+R848 significantly prevented tumor growth and metastasis in lung cancer and fibrosarcoma immunocompetent murine models. Regressing tumors showed increased infiltration of macrophages with a higher M1:M2 ratio, recruitment of CD4+ and CD8+ T cells, accompanied by a reduction of immunosuppressive CD206+ TAMs and FOXP3+/CD4+ T cells. The depletion of both CD4+ and CD8+ T cells resulted in complete loss of treatment efficacy. Treated mice acquired systemic antitumoral response and resistance to tumor rechallenge mediated by boosted macrophage cytotoxic activity and T-cell proliferation. Proteomic experiments validate the superior activation of innate immunity by poly(I:C)+R848 combination versus single treatments or poly(I:C)+R837, and protein-protein-interaction network analysis reveal the key activation of the STAT1 pathway. DISCUSSION: These findings demonstrate the antitumor immune responses mediated by macrophage activation on local administration of poly(I:C)+R848 combination and support the intratumoral application of this therapy to patients with solid tumors in the clinic.

Cellular and Molecular Mechanisms Underlying Glioblastoma and Zebrafish Models for the Discovery of New Treatments.

Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood-brain-barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBM.

Therapeutic Manipulation of Tumor-associated Macrophages: Facts and Hopes from a Clinical and Translational Perspective.

The stroma of most solid tumors is populated by myeloid cells, which mostly represent macrophages. Tumor-associated macrophages (TAMs), strongly influenced by cancer cell-derived factors, are key drivers of immunosuppression and support tumor growth and spread to distant sites. Their accurate quantification and characterization in the tumor microenvironment are gaining prognostic value: increasing evidence demonstrates their ability to hamper cancer patients' response to chemotherapy, as well as to immunotherapies based on checkpoint inhibition. Therefore, strategies to counteract their negative effects are nowadays gaining momentum at preclinical, translational, and clinical levels. Our knowledge of the biology of TAMs has greatly advanced in the last years; several strategies to target and reprogram their functions to become antitumor effectors have proven successful in experimental preclinical tumor models; on the other hand, few approaches have so far been effectively translated into clinic practice. A growing interest in the therapeutic manipulation of TAMs is evidenced by numerous early-phase clinical trials, which are continuously fueled by new discoveries from basic research. This gives us hope that the targeting and sustained reprogramming of TAMs will be more specific to synergize with current therapies and maximize antitumor responses in patients.

Targeting Tumor-Associated Macrophages in Anti-Cancer Therapies: Convincing the Traitors to Do the Right Thing.

In the last decade, it has been well-established that tumor-infiltrating myeloid cells fuel not only the process of carcinogenesis through cancer-related inflammation mechanisms, but also tumor progression, invasion, and metastasis. In particular, tumor-associated macrophages (TAMs) are the most abundant leucocyte subset in many cancers and play a major role in the creation of a protective niche for tumor cells. Their ability to generate an immune-suppressive environment is crucial to escape the immune system and to allow the tumor to proliferate and metastasize to distant sites. Conventional therapies, including chemotherapy and radiotherapy, are often not able to limit cancer growth due to the presence of pro-tumoral TAMs; these are also responsible for the failure of novel immunotherapies based on immune-checkpoint inhibition. Several novel therapeutic strategies have been implemented to deplete TAMs; however, more recent approaches aim to use TAMs themselves as weapons to fight cancer. Exploiting their functional plasticity, the reprogramming of TAMs aims to convert immunosuppressive and pro-tumoral macrophages into immunostimulatory and anti-tumor cytotoxic effector cells. This shift eventually leads to the reconstitution of a reactive immune landscape able to destroy the tumor. In this review, we summarize the current knowledge on strategies able to reprogram TAMs with single as well as combination therapies.

Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors.

Background: Tumor-associated macrophages (TAMs), with M2-like immunosuppressive profiles, are key players in the development and dissemination of tumors. Hence, the induction of M1 pro-inflammatory and anti-tumoral states is critical to fight against cancer cells. The activation of the endosomal toll-like receptor 3 by its agonist poly(I:C) has shown to efficiently drive this polarization process. Unfortunately, poly(I:C) presents significant systemic toxicity, and its clinical use is restricted to a local administration. Therefore, the objective of this work has been to facilitate the delivery of poly(I:C) to macrophages through the use of nanotechnology, that will ultimately drive their phenotype toward pro-inflammatory states. Methods: Poly(I:C) was complexed to arginine-rich polypeptides, and then further enveloped with an anionic polymeric layer either by film hydration or incubation. Physicochemical characterization of the nanocomplexes was conducted by dynamic light scattering and transmission electron microscopy, and poly(I:C) association efficiency by gel electrophoresis. Primary human-derived macrophages were used as relevant in vitro cell model. Alamar Blue assay, ELISA, PCR and flow cytometry were used to determine macrophage viability, polarization, chemokine secretion and uptake of nanocomplexes. The cytotoxic activity of pre-treated macrophages against PANC-1 cancer cells was assessed by flow cytometry. Results: The final poly(I:C) nanocomplexes presented sizes lower than 200 nm, with surface charges ranging from +40 to -20 mV, depending on the envelopment. They all presented high poly(I:C) loading values, from 12 to 50%, and great stability in cell culture media. In vitro, poly(I:C) nanocomplexes were highly taken up by macrophages, in comparison to the free molecule. Macrophage treatment with these nanocomplexes did not reduce their viability and efficiently stimulated the secretion of the T-cell recruiter chemokines CXCL10 and CCL5, of great importance for an effective anti-tumor immune response. Finally, poly(I:C) nanocomplexes significantly increased the ability of treated macrophages to directly kill cancer cells. Conclusion: Overall, these enveloped poly(I:C) nanocomplexes might represent a therapeutic option to fight cancer through the induction of cytotoxic M1-polarized macrophages.

Nanosized zeolites as a gas delivery platform in a glioblastoma model.

Approaches able to counteract, at least temporarily, hypoxia, a well-known factor of resistance to treatment in solid tumors are highly desirable. Herein, we report the use of nanosized zeolite crystals as hyperoxic/hypercapnic gas carriers for glioblastoma. First, the non-toxic profile of nanosized zeolite crystals in living animals (mice, rats and non-human primates) and in various cell types is presented. Second, the ability of the nanosized zeolites to act as a vasoactive agent for a targeted re-oxygenation of the tumor after intravenous injection is shown. As attested by an MRI protocol, the zeolites were able to increase oxygenation and blood volume specifically within the brain tumor whilst no changes in the healthy-non tumoral brain-were observed. The first proof of concept for the use of metal-containing nanosized zeolites as a tool for vectorization of hyperoxic/hypercapnic gases in glioblastoma is revealed.

Impact of Hypoxia on Carbon Ion Therapy in Glioblastoma Cells: Modulation by LET and Hypoxia-Dependent Genes.

Tumor hypoxia is known to limit the efficacy of ionizing radiations, a concept called oxygen enhancement ratio (OER). OER depends on physical factors such as pO2 and linear energy transfer (LET). Biological pathways, such as the hypoxia-inducible transcription factors (HIF), might also modulate the influence of LET on OER. Glioblastoma (GB) is resistant to low-LET radiation (X-rays), due in part to the hypoxic environment in this brain tumor. Here, we aim to evaluate in vitro whether high-LET particles, especially carbon ion radiotherapy (CIRT), can overcome the contribution of hypoxia to radioresistance, and whether HIF-dependent genes, such as erythropoietin (EPO), influence GB sensitivity to CIRT. Hypoxia-induced radioresistance was studied in two human GB cells (U251, GL15) exposed to X-rays or to carbon ion beams with various LET (28, 50, 100 keV/µm), and in genetically-modified GB cells with downregulated EPO signaling. Cell survival, radiobiological parameters, cell cycle, and ERK activation were assessed under those conditions. The results demonstrate that, although CIRT is more efficient than X-rays in GB cells, hypoxia can limit CIRT efficacy in a cell-type manner that may involve differences in ERK activation. Using high-LET carbon beams, or targeting hypoxia-dependent genes such as EPO might reduce the effects of hypoxia.

Incorporation of trivalent cations in NaX zeolite nanocrystals for the adsorption of O2 in the presence of CO2.

The O2 and CO2 sorption properties of nanosized zeolite X with faujasite type structure through a partial ionic exchange of sodium (Na+) by trivalent cations (Gd3+ and Ce3+) were evaluated. Three faujasite samples were studied, the as-synthesized Na-X possessing Na+ solely, and the modified samples Na-Gd-X and Na-Ce-X containing Gd3+ (1.8 wt%) and Ce3+ (0.82 wt%), respectively. Incorporating scarce amounts of trivalent cations modified the adsorption affinity of zeolites towards O2 and CO2 as demonstrated by in situ Fourier-transform infrared spectroscopy (FTIR). While Na-Ce-X encounters the highest O2 physisorption capacity, the Na-Gd-X is adsorbing the highest quantities of molecular CO2. All three samples exhibit the chemisorbed CO2 in the form of carbonates, while the Na-X stores carbonates in monodentate and polydentate forms, the Na-Gd-X and Na-Ce-X allow the formation of polydentate carbonates only. Density functional theory (DFT) calculations revealed that trivalent cations tend to adsorb gases through two cations simultaneously which explains the presence of polydentate carbonates exclusively in the corresponding modified zeolites. The DFT results confirmed the higher affinity of Na-Gd-X and Na-Ce-X nanocrystals towards O2 in the presence of CO2. The affinity of Na-Gd-X and Na-Ce-X nanocrystals towards O2 opens the door of their use as oxygen transporters for medical applications where CO2 is constantly present. The toxicity of the nanosized zeolites and their performance in O2 release are reported too.

Current Strategies to Target Tumor-Associated-Macrophages to Improve Anti-Tumor Immune Responses.

: Established evidence demonstrates that tumor-infiltrating myeloid cells promote rather than stop-cancer progression. Tumor-associated macrophages (TAMs) are abundantly present at tumor sites, and here they support cancer proliferation and distant spreading, as well as contribute to an immune-suppressive milieu. Their pro-tumor activities hamper the response of cancer patients to conventional therapies, such as chemotherapy or radiotherapy, and also to immunotherapies based on checkpoint inhibition. Active research frontlines of the last years have investigated novel therapeutic strategies aimed at depleting TAMs and/or at reprogramming their tumor-promoting effects, with the goal of re-establishing a favorable immunological anti-tumor response within the tumor tissue. In recent years, numerous clinical trials have included pharmacological strategies to target TAMs alone or in combination with other therapies. This review summarizes the past and current knowledge available on experimental tumor models and human clinical studies targeting TAMs for cancer treatment.

A Facile Route toward the Increase of Oxygen Content in Nanosized Zeolite by Insertion of Cerium and Fluorinated Compounds.

Enriching oxygen content within nanosized zeolite X (as synthesized Na-X) by insertion of cerium (ion exchanged Ce-X) and functionalization with bromoperfluoro-n-octane (fluorinated F-X) is reported. The materials were fully characterized by powder X-ray diffraction (XRD), dynamic light scattering (DLS), zeta potential, thermogravimetric analysis (TGA), nitrogen adsorption, and nuclear magnetic resonance (19F NMR). The O2 adsorption in the zeolite samples at various concentrations (0 to 165 Torr) at -196 °C was studied by in situ FTIR. The modification of nanosized zeolites did not alter their colloidal stability, crystallinity, porosity, and particle size distribution. The inclusion of cerium and bromoperfluoro-n-octane considerably increase the oxygen capacity by 33% for samples Ce-X and F-X in comparison to the as-synthesized Na-X zeolite. Further, toxicity tests revealed that these materials are safe, which opens the door for their implementation in medical applications, where controlled delivery of oxygen is highly desirable.

M2 macrophages are more resistant than M1 macrophages following radiation therapy in the context of glioblastoma.

In some highly inflammatory tumors, such as glioblastoma (GB), macrophages (MΦ) represent the most abundant population of reactive cells. MΦ, initially denoted as M0 MΦ, can be polarized into two further phenotypes: the antitumor M1 MΦ, and the protumor M2 MΦ. The three phenotypes can reside simultaneously in the tumor mass and various external factors may influence MΦ polarization. Radiotherapy is a common modality of cancer treatment aiming to target tumor cells. However, the specific effects of X-ray radiation on the inflammatory cells are, so far, controversial and not fully understood. In the present investigation, we have first analyzed, in vivo, the effect of X-ray radiation on MΦ present in GB tumors. We have observed a decrease in MΦ number paralleled by an increase in the proportion of M2 MΦ. To understand this phenomenon, we then evaluated, in vitro, the effects of X-rays on the MΦ phenotypes and survival. We have found that X-ray radiation failed to modify the phenotype of the different MΦ. However, M1 MΦ were more sensitive to ionizing radiation than M2 MΦ, both in normoxia and in hypoxia, which could explain the in vivo observations. To conclude, M2 MΦ are more radioresistant than M0 and M1 MΦ and the present study allows us to propose that X-ray radiotherapy could contribute, along with other phenomena, to the increased density in the protumor M2 MΦ in GB.

Acute Toxicity of Silver Free and Encapsulated in Nanosized Zeolite for Eukaryotic Cells.

The potential toxicity of encapsulated silver in EMT-type nanosized zeolites on prokaryotic cells, human tumor cell lines from various origins, and primary cultures of neurons and astrocytes was investigated. Silver in cationic form (Ag+) was encapsulated in EMT-type nanosized zeolites via an ion exchange process (Ag+-EMT) and compared with the reduced silver (Ag0) in the zeolite (Ag0-EMT). As reference samples for the toxicity measurements, pure EMT-type zeolite and silver perchlorate were used. Cells were exposed to silver-containing zeolites (50, 100, and 400 µg/mL) for 24 and 48 h. After exposure to Ag+-EMT (50 µg/mL) for 24 h, a loss in cell viability independent of the cell type was observed, ranging from -34.37 ± 23.90% for astrocytes to -99.5 ± 0.24% for U87-MG cells. These results were comparable with the toxicity for silver perchlorate. The Ag0-EMT sample showed lower toxicity on human cell lines in comparison to that of Ag+-EMT. A decrease in cell viability, i.e., -73.46 ± 20.78% and -62.3 ± 17.96% for U87-MG and HEK 293 cells, respectively, under exposure only to high concentration of Ag0-EMT (400 µg/mL) for 24 h was measured. However, the Ag0-EMT was as toxic as the Ag+-EMT for astrocytes and neurons (-97.95 ± 3.31% and -100 ± 1.11%, respectively, after exposure to 50 µg/mL for 24 h). No decrease in cell viability exposed to pure EMT zeolite was found. The results demonstrate the severe toxicity of silver cations, either free or encapsulated, in comparison to reduced silver encapsulated in zeolite nanocrystals. Therefore, silver cations, either free or encapsulated, must be used with great caution regarding their toxicity on eukaryotic cells.