Understanding iron homeostasis in MDS: the role of erythroferrone.

Myelodysplastic neoplasms (MDS) are a heterogenous group of clonal stem cell disorders characterized by dysplasia and cytopenia in one or more cell lineages. Anemia is a very common symptom that is often treated with blood transfusions and/or erythropoiesis stimulating factors. Iron overload results from a combination of these factors together with the disease-associated ineffective erythropoiesis, that is seen especially in MDS cases with SF3B1 mutations. A growing body of research has shown that erythroferrone is an important regulator of hepcidin, the master regulator of systemic iron homeostasis. Consequently, it is of interest to understand how this molecule contributes to regulating the iron balance in MDS patients. This short review evaluates our current understanding of erythroferrone in general, but more specifically in MDS and seeks to place in context how the current knowledge could be utilized for prognostication and therapy.

Titanium induces pro-inflammatory and tissue-destructive responses in primary human macrophages.

Implants and medical devices are efficient and practical therapeutic solutions for a multitude of pathologies. Titanium and titanium alloys are used in orthopedics, dentistry, and cardiology. Despite very good mechanical properties, and corrosion resistance titanium implants can fail due to inflammatory or tissue-degradation related complications. Macrophages are major immune cells that control acceptance of failure of the implant. In this study, for the first time, we have performed a systematic analysis of the response of differentially activated human macrophages (M(Control), M(IFNγ) and M(IL-4)) to the polished and porous titanium surfaces in order to identify the detrimental effect of titanium leading to the tissue destruction and chronic inflammation. Transcriptome analysis revealed that the highest number of differences between titanium and control settings are found in M(IL-4) that model healing type of macrophages. RT-qPCR analysis confirmed that both polished and porous titanium affected expression of cytokines, chitinases/chitinase-like proteins and matrix metalloproteinases. Titanium-induced release and activation of MMP7 by macrophages was enhanced by fibroblasts in both juxtacrine and paracrine cell interaction models. Production of titanium-induced MMPs and cytokines associated with chronic inflammation were independent of the presence of Staphylococcus aureus. MMP7, one of the most pronounced tissue-destroying factor and chitinase-like protein YKL-40 were expressed in CD68+ macrophages in peri-implant tissues of patients with orthopedic implants. In summary, we demonstrated that titanium induces pro-inflammatory and tissue-destructing responses mainly in healing macrophages, and the detrimental effects of titanium surfaces on implant-adjacent macrophages are independent on the bacterial contamination.

Treatment with the apoptosis inhibitor Asunercept reduces clone sizes in patients with lower risk Myelodysplastic Neoplasms.

In low-risk Myelodysplastic Neoplasms (MDS), increased activity of apoptosis-promoting factors such as tumor necrosis factor (TNFα) and pro-apoptotic Fas ligand (CD95L) have been described as possible pathomechanisms leading to impaired erythropoiesis. Asunercept (APG101) is a novel therapeutic fusion protein blocking CD95, which has previously shown partial efficacy in reducing transfusion requirement in a clinical phase I trial for low-risk MDS patients (NCT01736436; 2012-11-26). In the current study we aimed to evaluate the effect of Asunercept therapy on the clonal bone marrow composition to identify potential biomarkers to predict response. Bone marrow samples of n = 12 low-risk MDS patients from the above referenced clinical trial were analyzed by serial deep whole exome sequencing in a total of n = 58 time points. We could distinguish a mean of 3.5 molecularly defined subclones per patient (range 2-6). We observed a molecular response defined as reductions of dominant clone sizes by a variant allele frequency (VAF) decrease of at least 10% (mean 20%, range: 10.5-39.2%) in dependency of Asunercept treatment in 9 of 12 (75%) patients. Most of this decline in clonal populations was observed after completion of 12 weeks treatment. Particularly early and pronounced reductions of clone sizes were found in subclones driven by mutations in genes involved in regulation of methylation (n = 1 DNMT3A, n = 1 IDH2, n = 1 TET2). Our results suggest that APG101 could be efficacious in reducing clone sizes of mutated hematopoietic cells in the bone marrow of Myelodysplastic Neoplasms, which warrants further investigation.

ASXL1 mutations are associated with a response to alvocidib and 5-azacytidine combination in myelodysplastic neoplasms.

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMAs) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5- AZA could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in n=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene-expression of the pro-apoptotic factor NOXA in ASXL1 mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Significant improvement of bone marrow-derived MSC expansion from MDS patients by defined xeno-free medium.

BACKGROUND: Robust and reliable in vitro and in vivo models of primary cells are necessary to study the pathomechanisms of Myelodysplastic Neoplasms (MDS) and identify novel therapeutic strategies. MDS-derived hematopoietic stem and progenitor cells (HSPCs) are reliant on the support of bone marrow (BM) derived mesenchymal stroma cells (MSCs). Therefore, isolation and expansion of MCSs are essential for successfully modeling this disease. For the clinical use of healthy MSCs isolated from human BM, umbilical cord blood or adipose tissue, several studies showed that xeno-free (XF) culture conditions resulted in superior growth kinetics compared to MSCs cultured in the presence of fetal bovine serum (FBS). In this present study, we investigate, whether the replacement of a commercially available MSC expansion medium containing FBS with a XF medium is beneficial for the expansion of MSCs derived from BM of MDS patients which are often difficult to cultivate. METHODS: MSCs isolated from BM of MDS patients were cultured and expanded in MSC expansion medium with FBS or XF supplement. Subsequently, the impact of culture media on growth kinetics, morphology, immunophenotype, clonogenic potential, differentiation capacity, gene expression profiles and ability to engraft in immunodeficient mouse models was evaluated. RESULTS: Significant higher cell numbers with an increase in clonogenic potential were observed during culture of MDS MSCs with XF medium compared to medium containing FBS. Differential gene expression showed an increase in transcripts associated with MSC stemness after expansion with XF. Furthermore, immunophenotypes of the MSCs and their ability to differentiate into osteoblasts, adipocytes or chondroblasts remained stable. MSCs expanded with XF media were similarly supportive for creating MDS xenografts in vivo as MSCs expanded with FBS. CONCLUSION: Our data indicate that with XF media, higher cell numbers of MDS MSCs can be obtained with overall improved characteristics in in vitro and in vivo experimental models.

Inhibition of lysyl oxidases synergizes with 5-azacytidine to restore erythropoiesis in myelodysplastic and myeloid malignancies.

Limited response rates and frequent relapses during standard of care with hypomethylating agents in myelodysplastic neoplasms (MN) require urgent improvement of this treatment indication. Here, by combining 5-azacytidine (5-AZA) with the pan-lysyl oxidase inhibitor PXS-5505, we demonstrate superior restoration of erythroid differentiation in hematopoietic stem and progenitor cells (HSPCs) of MN patients in 20/31 cases (65%) versus 9/31 cases (29%) treated with 5-AZA alone. This effect requires direct contact of HSPCs with bone marrow stroma components and is dependent on integrin signaling. We further confirm these results in vivo using a bone marrow niche-dependent MN xenograft model in female NSG mice, in which we additionally demonstrate an enforced reduction of dominant clones as well as significant attenuation of disease expansion and normalization of spleen sizes. Overall, these results lay out a strong pre-clinical rationale for efficacy of combination treatment of 5-AZA with PXS-5505 especially for anemic MN.

Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF.

Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified. Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer. Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

In Silico Pan-Cancer Analysis Reveals Prognostic Role of the Erythroferrone (ERFE) Gene in Human Malignancies.

The erythroferrone gene (ERFE), also termed CTRP15, belongs to the C1q tumor necrosis factor-related protein (CTRP) family. Despite multiple reports about the involvement of CTRPs in cancer, the role of ERFE in cancer progression is largely unknown. We previously found that ERFE was upregulated in erythroid progenitors in myelodysplastic syndromes and strongly predicted overall survival. To understand the potential molecular interactions and identify cues for further functional investigation and the prognostic impact of ERFE in other malignancies, we performed a pan-cancer in silico analysis utilizing the Cancer Genome Atlas datasets. Our analysis shows that the ERFE mRNA is significantly overexpressed in 22 tumors and affects the prognosis in 11 cancer types. In certain tumors such as breast cancer and adrenocortical carcinoma, ERFE overexpression has been associated with the presence of oncogenic mutations and a higher tumor mutational burden. The expression of ERFE is co-regulated with the factors and pathways involved in cancer progression and metastasis, including activated pathways of the cell cycle, extracellular matrix/tumor microenvironment, G protein-coupled receptor, NOTCH, WNT, and PI3 kinase-AKT. Moreover, ERFE expression influences intratumoral immune cell infiltration. Conclusively, ERFE is aberrantly expressed in pan-cancer and can potentially function as a prognostic biomarker based on its putative functions during tumorigenesis and tumor development.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Humanized three-dimensional scaffold xenotransplantation models for myelodysplastic syndromes.

Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34+ hematopoietic stem and progenitor cells (HSPCs). Our study included13 primary MDS patient samples transplanted in parallel according to these five different conditions. Engraftment of MDS samples was assessed by flow cytometry, immunohistological staining, and molecular validation. We determined that three-dimensional ossicle-based methods achieved higher relative rates of engraftment and enabled long-term retrievability of patient-derived MSCs from implanted ossicles. In summary, HSPCs and MSCs derived from MDS BM, which did not significantly engraft in NSG mice after intrafemoral injection, were able to colonize humanized scaffold models. Therefore, these models are promising new xenotransplantation techniques for addressing preclinical and functional questions of the interaction between hematopoiesis and the BM niche in MDS.

Preclinical evaluation of eltrombopag in a PDX model of myelodysplastic syndromes.

Preclinical research of myelodysplastic syndromes (MDSs) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here we demonstrate for the first time that this model is applicable as a preclinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our preclinical study included n = 49 xenografts generated from n = 9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification and clonal bone marrow evolution was reconstructed by serial whole-exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient-individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients' clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.

Bone marrow derived stromal cells from myelodysplastic syndromes are altered but not clonally mutated in vivo.

The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients.

High erythroferrone expression in CD71+ erythroid progenitors predicts superior survival in myelodysplastic syndromes.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g. SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with Myelodysplastic Syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease therefore making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation-associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association of R-loops and ATR sensitivity with the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Epigenetic Regulation of S100A9 and S100A12 Expression in Monocyte-Macrophage System in Hyperglycemic Conditions.

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions.

Accumulation of stabilin-1 positive macrophages in the early stage of gastric cancer is associated with short cumulative survival.

The scavenger receptor stabilin-1 has been reported to be expressed by tumor-associated macrophages (TAMs) and to facilitate tumor growth and metastasis in mouse models of breast carcinoma and melanoma. However, to the best of our knowledge, its expression and association with prognosis in human gastric cancer has not been evaluated. The present study investigated the expression of stabilin-1 and its association with clinicopathological parameters in patients with gastric cancer. The expression of stabilin-1 was evaluated by immunohistochemical staining of gastric cancer tissue samples of 371 Chinese patients with primary gastric adenocarcinoma. Confocal laser scanning microscopy was used to determine the cellular source of stabilin-1 in the gastric cancer tissues using anti-CD68, anti-CD163, anti-stabilin-1 and anti-secreted protein acidic and rich in cysteine antibodies. A higher number of stabilin-1-positive cells were observed in the cancer tissues of primary gastric adenocarcinoma compared with adjacent non-cancerous tissues of primary gastric adenocarcinoma (P<0.001); the majority of stabilin-1-positve cells were CD68+/CD163+ macrophages. Poorly-differentiated gastric cancer tissue had fewer stabilin-1-positive cells compared with medium- and well-differentiated gastric cancer (P=0.030). A higher number of stabilin-1-positive cells were found in the early Tumor-Node-Metastasis (TNM) stage (TNM I stage) of primary gastric adenocarcinoma (P=0.038) compared with TNM stage IV. For patients with TNM stage I disease, a higher number of stabilin-1-positive TAMs was associated with shorter cumulative survival (P<0.05). Overall, stabilin-1 was found to be expressed by CD68+ TAMs in human gastric cancer. The high expression of stabilin-1 in TNM stage I disease was associated with poor patient survival, indicating the clinical significance of stabilin-1 in gastric cancer.

SI-CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor-associated macrophages.

Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.

Reactive oxygen species (ROS) in macrophage activation and function in diabetes.

In a diabetic milieu high levels of reactive oxygen species (ROS) are induced. This contributes to the vascular complications of diabetes. Recent studies have shown that ROS formation is exacerbated in diabetic monocytes and macrophages due to a glycolytic metabolic shift. Macrophages are important players in the progression of diabetes and promote inflammation through the release of pro-inflammatory cytokines and proteases. Because ROS is an important mediator for the activation of pro-inflammatory signaling pathways, obesity and hyperglycemia-induced ROS production may favor induction of M1-like pro-inflammatory macrophages during diabetes onset and progression. ROS induces MAPK, STAT1, STAT6 and NFκB signaling, and interferes with macrophage differentiation via epigenetic (re)programming. Therefore, a comprehensive understanding of the impact of ROS on macrophage phenotype and function is needed in order to improve treatment of diabetes and its vascular complications. In the current comprehensive review, we dissect the role of ROS in macrophage polarization, and analyze how ROS production links metabolism and inflammation in diabetes and its complications. Finally, we discuss the contribution of ROS to the crosstalk between macrophages and endothelial cells in diabetic complications.

The effect of healing phenotype-inducing cytokine formulations within soft hydrogels on encapsulated monocytes and incoming immune cells.

The adverse immune responses to implantable biomedical devices is a general problem with important consequences for the functionality of implants. Immunomodulatory soft hydrogel-based interfaces between the implant and the host can attenuate these reactions. Moreover, encapsulation of the patient's own immune cells into these interfaces can lead to the personalisation of implants from the immune reaction point of view. Herein, we described a co-crosslinkable composite hydrogel (composed of gelatin and hyaluronic acid), which could be used for the encapsulation of macrophages in the presence of an anti-inflammatory phenotype-fixing cytokine cocktail. To mimick the incoming immune cells on the coating surface in vivo, peripheral blood mononuclear cells were seeded on the hydrogels. The encapsulation of monocytic cells into the composite hydrogels in the presence of cytokine cocktails at 5× or 10× concentrations led to the spreading of the encapsulated cells instead of the formation of clusters. Moreover, the secretion of the anti-inflammatory cytokines IL-1RA and CCL-18 was significantly increased. The attachment of PBMC to the surface of the hydrogel is dependent on the hydrogel composition and also significantly increased in the presence of the cytokine cocktail together with the number of CD68+ cells on the hydrogel surface. Our study demonstrates that the delivery of a polarisation cocktail with biocompatible hydrogels can control the initial response by the incoming immune cells. This effect can be improved by the encapsulation of autologous monocytes that are also polarised by the cytokine cocktail and secrete additional anti-inflammatory cytokines. This interface can fine tune the initial immune response to an implanted biomaterial in a personalised manner.

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy.

In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.