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The important role of tumor microenvironmental elements in determining tumor progression and metastasis has been firmly established. In particular, the presence and activity profile of tumor-infiltrating immune cells may be associated with the outcome of the disease and may predict responsiveness to (immuno)therapy. Indeed, while some immune cell types, such as macrophages, support cancer cell outgrowth and mediate therapy resistance, the presence of activated CD8+ T cells is usually indicative of a better prognosis. It is therefore of the utmost interest to obtain a full picture of the immune infiltrate in tumors, either as a prognostic test, as a way to stratify patients to maximize therapeutic success, or as therapy follow-up. Hence, the non-invasive imaging of these cells is highly warranted, with biologics being prime candidates to achieve this goal.

Over the past decade, cancer immunotherapy has been steering immune responses toward cancer cell eradication. However, these immunotherapeutic approaches are hampered by the tumor-promoting nature of myeloid cells, including monocytes, macrophages, and neutrophils. Despite the arsenal of defense strategies against foreign invaders, myeloid cells succumb to the instructions of an established tumor. Interestingly, the most primordial defense responses employed by myeloid cells against pathogens, such as complement activation, antibody-dependent cell cytotoxicity and phagocytosis, actually seem to favor cancer progression. In this review, we discuss how rudimentary defense mechanisms deployed by myeloid cells can promote tumor progression.

International audience; PURPOSE OF REVIEW:Tumor stroma is composed of many cellular subtypes, of which the most abundant are fibroblasts, macrophages and endothelial cells. During the process of tissue injury, these three cellular subtypes must coordinate their activity to efficiently contribute to tissue regeneration. In tumor, this mechanism is hijacked by cancer cells, which rewire the interaction of stromal cells to benefit tumor development. The present review aims at summarizing most relevant information concerning both pro-tumorigenic and anti-tumorigenic actions implicating the three stromal cell subtypes as well as their mutual interactions.RECENT FINDINGS:Although stromal cells are generally regarded as tumor-supportive and at will manipulated by cancer cells, several novel studies point at many defaults in cancer cell-mediated stromal reprograming. Indeed, parts of initial tissue-protective and homeostatic functions of the stromal cells remain in place even after tumor development. Both tumor-supportive and tumor-suppressive functions have been well described for macrophages, whereas similar results are emerging for fibroblasts and endothelial cells.SUMMARY:Recent success of immunotherapies have finally brought the long awaited proof that stroma is key for efficient tumor targeting. However, a better understanding of paracrine stromal interactions is needed in order to encourage drug development not only aiming at disruption of tumor-supportive communication but also re-enforcing, existing, tumor-suppressive mechanisms.

© 2017, Springer-Verlag Berlin Heidelberg. In the original version, the left picture in the lower panel in Fig. 2b was accidentally duplicated on the right side. The correct picture is the following

Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) micro-environment is a major contributing factor to drug resistance. Tumor-associated macrophages or TAMs with different polarization states are an important component of this micro-environment. Previous studies revealed a role of TAMs in MM survival and drug resistance; however, the impact of macrophage polarization (anti-tumoral "M1" versus pro-tumoral "M2"-like phenotype) in this process is not yet described. Here the presence of TAMs was confirmed in MM patients on BM sections both at diagnosis and relapse using two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in inflammatory monocytes and increase of M2-oriented TAMs in BM. Co-culture experiments demonstrated that macrophages induce a survival benefit to myeloma cells that is maintained after treatment with several classes of anti-myeloma agents (melphalan and bortezomib) and the biggest effect was observed with M2 polarized macrophages. The pro-survival effect was associated with an activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase-3 and thus less apoptosis. AZD1480, an ATP-competitive JAK2 inhibitor, abrogated the observed TAM mediated MM cell survival and partially inhibited resistance to bortezomib. Despite only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib could significantly reduce tumor load. In conclusion, M2 TAMs are present in the MM micro-environment and contribute to MM cell survival and protection from drug-induced apoptosis. As a result of TAM-induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment.

Background and aims: Pannexins constitute a relative new family of transmembrane proteins that form channels linking the cytoplasmic compartment with the extracellular environ- ment. Recent research showed that pannexin1 channel inhibi- tion alleviates acetaminophen-induced hepatotoxicity in mice. However, the mechanisms underlying this observation are not entirely clear. On the other hand, monocyte migration has been reported to be mediated in part by pannexin1-depentent ade- nosine triphosphate release in vitro and fine-tuned chemotactic responses of neutrophils seem to require pannexin1 signaling. To check if this also holds true in vivo, in casu in a model of acetaminophen-induced hepatotoxicity, leukocyte recruitment was monitored following inhibition of pannexin1 channel opening. Methods: C57BL/6J mice were overdosed with acet- aminophen followed by treatment with the pannexin1 channel inhibitor 10Panx1 after 1.5 hours. Sampling was performed 6 and 24 hours after acetaminophen administration. In addition to the evaluation of clinically relevant read-outs, such as serum aminotransaminase and cytokine levels, hepatic quantities of inflammatory monocytes (CD45+CD11bhiLy6ChiF4/80lo cells), neutrophils (CD45+CD11bhiLy6G+ cells) and Kupffer cells (CD45+Ly6G-F4/80hiCD11bint cells) were measured using flow cytometric analysis. Tumor necrosis factor α content of these particular cell populations was assessed in parallel. Results: Compared to untreated animals, acetaminophen-overdosed mice showed less Kupffer cells, but increased numbers of liver neutrophils and monocytes. Upon blockage of pannexin1 channels, a significantly reduced amount of neutrophils were recruited to the liver, while no differences were observed in the number of monocytes and Kupffer cells. Concomitantly, diminished tumor necrosis factor α production by monocytes was measured following pannexin1 channel inhibition. Conclusion: These results suggest the involvement of pannexin1 channels in neutrophil recruitment and tumor necrosis factor α production by monocytes during acetaminophen-induced hepatotoxicity in mice.

Pancreas injury by partial duct ligation (PDL) activates a healing response, encompassing β-cell neogenesis and proliferation. Macrophages were recently shown to promote β-cell proliferation after PDL, but they remain poorly characterized. We assessed myeloid cell diversity and the factors driving myeloid cell dynamics following acute pancreas injury by PDL. In naive and sham-operated pancreas, the myeloid cell compartment consisted mainly of two distinct tissue-resident macrophage types, designated MHC-II(lo) and MHC-II(hi) macrophages, the latter being predominant. MHC-II(lo) and MHC-II(hi) pancreas macrophages differed at the molecular level, with MHC-II(lo) macrophages being more M2-activated. After PDL, there was an early surge of Ly6C(hi) monocyte infiltration in the pancreas, followed by a transient MHC-II(lo) macrophage peak and ultimately a restoration of the MHC-II(hi) macrophage-dominated steady-state equilibrium. These intricate macrophage dynamics in PDL pancreas depended on monocyte recruitment by CCR2 and M-CSFR as well as on M-CSFR-dependent local macrophage proliferation. Functionally, MHC-II(lo) macrophages were more angiogenic. We further demonstrated that, at least in CCR2-KO mice, tissue macrophages, rather than Ly6C(hi) monocyte-derived macrophages, contributed to β-cell proliferation. Together, our study fully characterizes the macrophage subsets in the pancreas and clarifies the complex dynamics of macrophages after PDL injury. This article is protected by copyright. All rights reserved.

Multiple Myeloma (MM) is an incurable malignancy of terminally differentiated plasma cells, which are predominantly localized in the bone marrow. The presence of an immunosuppressive bone marrow microenvironment and dysfunction of immune cells are both described in MM patients; however the mechanisms controlling this immunosuppression are not well defined. Recently, a heterogeneous population of immature myeloid cells, the so called myeloid-derived suppressor cells (MDSC), were identified to be present and active in MM. MDSC are thought to promote cancer progression by their T-cell suppressive capacity, however little is known about the generation and activation of MDSC by MM cells. Also, it is not known whether MDSC are involved in the development of drug resistance. In this study we investigated the effects of myeloma cells on MDSC induction using the 5T33MM mouse model and healthy donor blood samples. In a first instance, myeloid cells (CD11b+) were isolated from the bone marrow of naïve C57BL/KaLwRij mice and cultured in conditioned medium (CM) derived from 5T33MMvt cells to measure survival with CellTiterGlo Assay and AnnexinV staining. Increased viability and reduced apoptosis of CD11b+ cells cultured in MM-CM compared to control medium were observed. In literature GM-CSF is described as a major survival factor for MDSC. The pro-survival effect induced by MM-CM could be abrogated by the use of a GM-CSF blocking antibody. In addition, we analyzed the effect of CM of human myeloma cell lines (HMCL) RPMI8226, LP-1 and OPM2 on the viability of peripheral blood mononuclear cells (PBMC) of healthy individuals. Human myeloma CM increased the viability of PBMC compared to control medium. When we analyzed the phenotype of PBMC using MDSC markers, we did not detect differences in the total CD11b+CD33+ percentage between control condition and HMCL-CM. However, in the CD11b+CD33+ population, the percentages of mononuclear MDSC (identified as CD14+HLA-DRlow) and polymorphonuclear MDSC (identified as CD11b+CD33+CD15+)were increased compared to control conditions. The immunosuppressive capacity of the generated population was analyzed. For mouse as well as for human experiments, we could observe a decreased proliferation of T-cells in CM compared to control medium. To unravel the mechanism of MM-induced MDSC survival, the expression of apoptosis regulating proteins from the Bcl-2 family (bcl-2,bcl-xl,mcl-1) and survival proteins (pSTAT3,STAT3) were investigated by western blot. Mouse CD11b+ cells incubated with 5T33MMvt CM had a clear increase in pSTAT3 compared to control. Moreover the anti-apoptotic proteins mcl-1 and bcl-xl were increased in 5T33MMvt-CM compared to control, while bcl-2 was not affected. We were able to confirm the results of pSTAT3, bcl-2 and mcl-1 using human CD11b+ cells incubated in control or RPMI8226-CM. In addition, we investigated the sensitivity of MM cells to drugs currently used in the clinic (melphalan and bortezomib) in the presence of MDSC. 5T33MMvv cells incubated with MDSC could survive better than 5T33MMvv cells cultured without MDSC. This phenomenon was also observed when drugs were added, indicating a pro-survival effect of MDSC on MM cells. From our experiments we can conclude that soluble factor(s) from myeloma cells are able to reduce T-cell proliferation. Since we could observe a better survival of CD11b+ cells and the appearance of MDSC phenotypes in myeloma CM, this immunosuppression is likely caused by the generation and increased survival of MDSC in myeloma CM with an increased STAT3 phosphorylation, mcl-1 and bcl-xl expression as underlying mechanisms.

Macrophages are often found to be present in tumors in high numbers; and this is correlated with a shorter survival of the patients. Tumor-associated macrophages (TAM) are associated with an M2 "alternatively activated" profile (CD206+, CD163+) and function as promoters of tumor growth by inducing angiogenesis, invasion and metastasis; protecting tumor cells from therapy-induced apoptosis; and suppressing the immune system mainly in hypoxic regions. Macrophages with an M1 "classically activated" profile on the contrary stimulate immune responses in normoxic regions. The aim of our study was to investigate the characteristics of macrophages in multiple myeloma (MM) looking at phenotype and drug resistance. We identified TAM on bone marrow sections of MM patients by immunostainings for CD163. A higher number of CD163+ cells was noticed in the MM samples compared to normal samples. Further analysis of the number CD163+cells in healthy areas and MM infiltrated areas confirmed that the higher amount of CD163+ originates from MM infiltrated areas. In addition, in the 5T33MM mouse model the phenotype of bone marrow located macrophages was investigated. Macrophages isolated from both 5T33MM and naive mice revealed a different expression of several macrophage surface markers: a higher expression of CD206, CD80 and CD115; a lower expression of CD86, CCR7, Ly6G and MHCII on the membrane of 5T33MM macrophages compared to naive macrophages. According to these data, macrophages isolated from 5T33MM mice had more frequently an M2 profile (CD206+) compared to macrophages isolated from naive mice (pNext, we generated TAMs in vitro. CD11b+ cells, isolated from naive mice, were incubated with conditioned medium (CM) from 5T33MMvitro cells. After 1 week culture, the phenotype of surviving cells was analyzed. The bulk population was positive for the macrophage marker F4/80. Also a higher expression of CD206 was observed on generated TAMs compared to the starting population. 5T33MMvitro CM contains Interleukin-10 (IL-10) which isdescribed as a M2 macrophage differentiator. Addition of IL-10 blocking antibodies reduced CD206 upregulation on generated TAMs with an average of 30% compared to control. The involvement of TAMs in drug resistance was investigated. In vitro generated TAMs were cocultured with 5T33MMvt cells with or without bortezomib or melphalan. TAMs increased the survival of 5T33MMvt cells compared to control (pPreliminary results performed with ex vivo isolated macrophages from 5T33MM mice, also demonstrated a protection from apoptosis of 5T33MMvt cells when co-cultured with ex vivo isolated macrophages. This protective effect remains when bortezomib 5-10nM or Melphalan 15µM were added. In conclusion, we analyzed the presence and characteristics of macrophages in MM. The number of macrophages is higher in bone marrow samples from MM patients compared to healthy persons. Also, a higher percentage of M2 macrophages (CD11b+/F4/80+/CD206+) was observed in the bone marrow of 5T33MM mice compared to naive mice. In vitro generation of M2 macrophages with 5T33MMvt CM is at least partially IL-10 mediated. Furthermore, MM-associated macrophages contribute to a better survival of 5T33MMvt cells and protect them against several anti-myeloma drugs.

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The lack of a crucial metabolic enzyme can lead to accumulating substrate concentrations in the bloodstream and severe human enzyme deficiency diseases. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is such a fatal genetic disorder, caused by a thymidine phosphorylase deficiency. Enzyme replacement therapy is a strategy where the deficient enzyme is administered intravenously in order to decrease the toxic substrate concentrations. Such a therapy is however not very efficient due to the fast elimination of the native enzyme from the circulation. In this study we evaluate the potential of using polymeric enzyme-loaded nanoparticles to improve the delivery of therapeutic enzymes. We constructed new 200-nanometer PMOXA-PDMS-PMOXA polymeric nanoparticles that encapsulate the enzyme thymidine phosphorylase. These particles are permeabilised for substrates and products by the reconstitution of the nucleoside-specific porin Tsx in their polymeric wall. We show that the obtained 'nanoreactors' are enzymatically active and stable in blood serum at 37 degrees C. Moreover, they do not provoke cytotoxicity when incubated with hepatocytes for 4 days, nor do they induce a macrophage-mediated inflammatory response ex vivo and in vivo. All data highlight the potential of such nanoreactors for their application in enzyme replacement therapy of MNGIE.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous mix of myeloid cells in different maturation stages generated in the BM. The role of MDSCs in cancer is to suppress T-cell responses, thereby likely regulating tumor progression. In mice, MDSCs are identified by expression of surface markers CD11b and Gr-1. The role of MDSCs in immune suppression in MM is not yet described. We investigated the immunosuppressive activity and mechanism of MDSC subsets in the syngeneic, immunocompetent 5TMM mouse model (5T2 and 5T33 models). CD11b+Ly6G-/low and CD11b+Ly6Ghi myeloid MDSC subsets were detected in 5TMM-diseased BM. These subsets were analyzed for T-cell suppressive activity. Both MDSC subsets from 5TMM BM were able to suppress antigen-specific T-cell responses at a higher level compared to purified MDSC subsets from normal BM. On average, Ly6G-/low MDSCs were more suppressive than Ly6Ghi MDSCs. To unravel the suppressive mechanism of MDSC subsets, inhibitors were used in ovalbumin-stimulated cocultures. Ly6G-/low MDSC-mediated suppression was partially reversed by the iNOS inhibitor L-NMMA and the COX-2 inhibitor sc-791, both of which lower the NO concentration in culture. In contrast, superoxide dismutase and especially catalase enhance NO concentrations, resulting in enhanced T-cell suppression. None of these inhibitors had any impact on the Ly6Ghi MDSC-mediated suppression. The Ly6G-/low population was further analysed and could be subdivided into 3 subpopulations based on the expression of Ly6C and SSC. These subpopulations show a different expression of surface markers, including SiglecF (eosinophil marker) and CD115/CCR2 (inflammatory monocyte markers). These subpopulations were sorted and analysed for their T-cell suppressive activity. Only the inflammatory monocytes have suppressive capacity. In conclusion, these data reveal the presence of MDSCs as a novel immune suppressive strategy employed by MM cells in the BM.

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Background Chemoradiotherapy (CRT) remains one of the most common cancer treatment modalities, and recent data suggest that CRT is maximally effective when there is generation of an anti-tumoral immune response. However, CRT has also been shown to promote immunosuppressive mechanisms which must be blocked or reversed to maximize its immune stimulating effects. Methods Therefore, using a preclinical model of human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), we developed a clinically relevant therapy combining CRT and two existing immunomodulatory drugs: cyclophosphamide (CTX) and the small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). In this model, we treated the syngeneic HPV-HNSCC mEER tumor-bearing mice with fractionated (10 fractions of 3 Gy) tumor-directed radiation and weekly cisplatin administration. We compared the immune responses induced by CRT and those induced by combinatory treatment (CRT + CTX/L-NIL) with flow cytometry, quantitative multiplex immunofluorescence and by profiling immune-related gene expression changes. Results We show that combination treatment favorably remodels the tumor myeloid immune microenvironment including an increase in anti-tumor immune cell types (inflammatory monocytes and M1-like macrophages) and a decrease in immunosuppressive granulocytic myeloid-derived suppressor cells (MDSCs). Intratumoral T cell infiltration and tumor antigen specificity of T cells were also improved, including a 31.8-fold increase in the CD8+ T cell/ regulatory T cell ratio and a significant increase in tumor antigen-specific CD8+ T cells compared to CRT alone. CTX/LNIL immunomodulation was also shown to significantly improve CRT efficacy, leading to rejection of 21% established tumors in a CD8-dependent manner. Conclusions Overall, these data show that modulation of the tumor immune microenvironment with CTX/L-NIL enhances susceptibility of treatment-refractory tumors to CRT. The combination of tumor immune microenvironment modulation with CRT constitutes a translationally relevant approach to enhance CRT efficacy through enhanced immune activation.

In this work, we were interested in studying the effect of two main metabolic factors, hypoxia and extracellular ATP metabolism, on the effector function of T helper subsets. The major oxygen sensor is HIF-1α which is continuously degraded in the presence of oxygen but is stabilized in hypoxia, leading to transcription of genes involved in cellular adaptation to low oxygen level. Our data show that the proportion of IFN-&, Doctorat en Sciences, info:eu-repo/semantics/nonPublished