Metastasis-associated fibroblasts in peritoneal surface malignancies.

Over decades, peritoneal surface malignancies (PSMs) have been associated with limited treatment options and poor prognosis. However, advancements in perioperative systemic chemotherapy, cytoreductive surgery (CRS), and hyperthermic intraperitoneal chemotherapy (HIPEC) have significantly improved clinical outcomes. PSMs predominantly result from the spread of intra-abdominal neoplasia, which then form secondary peritoneal metastases. Colorectal, ovarian, and gastric cancers are the most common contributors. Despite diverse primary origins, the uniqueness of the peritoneum microenvironment shapes the common features of PSMs. Peritoneal metastization involves complex interactions between tumour cells and the peritoneal microenvironment. Fibroblasts play a crucial role, contributing to tumour development, progression, and therapy resistance. Peritoneal metastasis-associated fibroblasts (MAFs) in PSMs exhibit high heterogeneity. Single-cell RNA sequencing technology has revealed that immune-regulatory cancer-associated fibroblasts (iCAFs) seem to be the most prevalent subtype in PSMs. In addition, other major subtypes as myofibroblastic CAFs (myCAFs) and matrix CAFs (mCAFs) were frequently observed across PSMs studies. Peritoneal MAFs are suggested to originate from mesothelial cells, submesothelial fibroblasts, pericytes, endothelial cells, and omental-resident cells. This plasticity and heterogeneity of CAFs contribute to the complex microenvironment in PSMs, impacting treatment responses. Understanding these interactions is crucial for developing targeted and local therapies to improve PSMs patient outcomes.

Hepatectomy-induced apoptotic extracellular vesicles stimulate neutrophils to secrete regenerative growth factors.

BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value.

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

Alternative activation of human macrophages enhances tissue factor expression and production of extracellular vesicles.

Macrophages are versatile cells that can be polarized by the tissue environment to fulfill required needs. Proinflammatory polarization is associated with increased tissue degradation and propagation of inflammation whereas alternative polarization within a Th2 cytokine environment is associated with wound healing and angiogenesis. To understand if polarization of macrophages can lead to a procoagulant macrophage subset we polarized human monocyte derived macrophages to a proinflammatory and an alternative activation state. Alternative polarization with interleukin-4 and IL-13 led to a macrophage phenotype characterized by increased tissue factor (TF) production and release and by an increase in extracellular vesicle production. In addition, also TF activity was enhanced in extracellular vesicles of alternatively polarized macrophages. This TF induction was dependent on signal transducer and activator of transcription-6 signaling and poly ADP ribose polymerase activity. In contrast to monocytes, human macrophages did not show increased tissue factor expression upon stimulation with lipopolysaccharide and interferon-γ. Previous polarization to either a proinflammatory or an alternative activation subset does not change the subsequent stimulation of TF. The inability of proinflammatory activated macrophages to respond to lipopolysaccharide and interferon-γ with an increase in TF production seems to be due to an increase in TF promoter methylation and was reversible when treating these macrophages with a demethylation agent. In conclusion, we provide evidence that proinflammatory polarization of macrophages does not lead to enhanced procoagulatory function, whereas alternative polarization of macrophages leads to an increased expression of TF and increased production of TF bearing extracellular vesicles by these cells suggesting a procoagulatory phenotype of alternatively polarized macrophages.

Circulating Free Methylated Tumor DNA Markers for Sensitive Assessment of Tumor Burden and Early Response Monitoring in Patients Receiving Systemic Chemotherapy for Colorectal Cancer Liver Metastasis.

BACKGROUND: Neoadjuvant chemotherapy (neoCTx) followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CLM). Treatment response is generally assessed using radiologic imaging after several cycles of chemotherapy. However, earlier assessment of response would be desirable since nonresponders could be switched early to an alternative chemotherapy regimen. Recent evidence suggests that circulating free methylated tumor DNA is a highly sensitive biomarker and may more accurately reflect tumor burden and treatment response than conventional markers for CRC. PATIENTS AND METHODS: Thirty-four patients with CLM who received neoCTx prior to intended hepatic resection were included in this prospective nonrandomized study. Peripheral blood plasma was collected at baseline and before each cycle of neoCTx and was then analyzed for aberrant methylation of 48 CRC-associated genes. Methylation marker levels were correlated with baseline tumor volume and treatment response and compared with the standard tumor markers CEA and CA 19-9. RESULTS: The methylation markers SEPT9, DCC, BOLL, and SFRP2 were present in all patients at baseline and displayed a stronger correlation with tumor volume than CEA and CA 19-9. Serial measurement of these methylation markers allowed for discrimination between operated and nonoperated patients already after 1 cycle of neoCTx with high sensitivity and specificity. The early dynamic changes of SEPT9 and DCC also seemed to correlate with pathohistological response. CONCLUSION: Our data suggest that serial measurements of CRC-associated methylation markers could be a particularly valuable tool for early response assessment in patients receiving neoCTx for CLM.

In Situ Characterization of Tissue-Resident Immune Cells by MALDI Mass Spectrometry Imaging.

Tissue-resident immune cells differ from their corresponding blood cells in many functional aspects. Although the proteome of blood immune cells has been well-investigated, there are almost no data on tissue-resident immune cells. Here, we explored the potential of using MALDI-TOF-MS imaging (MSI) to investigate these cells in colon tissue, which exhibits a strong infiltration of immune cells. MSI identified several proteinaceous markers that colocalized with specific structures of the colon, such as mucosa or muscularis mucosae, in six patients. In addition, we showed that certain m/z values have the same spatial distribution as CD3+ T lymphocytes in the lymphoid follicular structures or as CD206+ macrophages in the lamina propria. For further corroboration, blood lymphocytes and monocytes from 10 healthy volunteers were analyzed by intact cell mass spectrometry (ICMS). Furthermore, we analyzed monocyte-derived macrophages that had been polarized in vitro into proinflammatory M1 and anti-inflammatory M2 phenotypes. The mass spectra differed clearly among all immune cell types. Additionally, it was found that distinct signals from ICMS analysis were identical to the m/z values found in the MSI experiment in lymphoid follicular structures. These data show for the first time that MSI is well-suited to visualize the spatial distribution of immune cells in human colon tissue. We consider MALDI mass spectrometry imaging to be a technique with high potential for use in rapid investigations of tissue-specific features of cells.

The immune response to secondary necrotic cells.

When apoptotic cells are not cleared in an efficient and timely manner, they progress to secondary necrosis and lose their membrane integrity. This results in a leakage of immunostimulatory, danger associated molecular patterns (DAMPs), similar to accidental (or primary) necrosis. However, primary necrosis is a sudden event with an inadvertent release of almost unmodified DAMPs. Secondary necrotic cells, in contrast, have gone through various modifications during the process of apoptosis. Recent research revealed that the molecules released from the cytoplasm or exposed on the cell surface differ between primary necrosis, secondary necrosis, and regulated necrosis such as necroptosis. This review gives an overview of these differences and focusses their effects on the immune response. The implications to human physiology and diseases are manifold and will be discussed in the context of cancer, neurodegenerative disorders and autoimmunity.

Serum-dependent processing of late apoptotic cells and their immunogenicity.

The execution phase of apoptosis involves many processes which modify cellular molecules for an efficient and quiet elimination of the dead cell. These include exposure and secretion of "eat-me" signals, to attract phagocytes, as well as degradation of immune-stimulating cell debris. During this phase apoptotic microparticles (MPs) are released from the dying cell. The remaining cell remnant forms large late apoptotic cell-derived membranous vesicles (ACMV(L)) on its surface which remain attached. Phagocytosis is enhanced by cell non-autonomous factors such as complement component C1q and serum DNase I. We studied the formation and retraction of ACMV(L) and the influence of serum on their dynamics. We furthermore investigated the immunogenicity of cell remnants compared to released MPs. ACMV(L) were examined using time-lapse, electron microscopy and confocal microscopy. These blebs were observed on cell remnants with intact and with permeable membrane. This suggests that ACMV(L) remain on the surface by the time the cell remnant enters secondary necrosis. Bleb retraction could also be observed, but was radically enhanced in the presence of serum. Additionally, MPs stimulate peripheral blood mononuclear cells to produce similar IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha levels as LPS. In contrast, cell remnants only induce high levels of IL-8. These data show that cell non-autonomous factors contribute to morphological rearrangements during late apoptosis. In addition, they implicate that apoptotic MPs are released to attract phagocytes, while apoptotic cell remnants further process their potentially immunogenic content to prevent an inflammatory response upon secondary necrosis.

A platelet protein biochip rapidly detects an Alzheimer's disease-specific phenotype.

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.

Clearance of apoptotic cells by neutrophils in inflammation and cancer.

When a cell dies of apoptosis, it is eliminated either by neighbouring cells or by attracted professional phagocytes. Although it was generally believed that neutrophils also have the ability to perform efferocytosis, their contribution to the clearance of apoptotic cells was considered less important compared with macrophages. Therefore, this ability of neutrophils remained unexplored for a long time. Over the past decade, it has been shown that during inflammation, neutrophils contribute significantly to the clearance of apoptotic neutrophils that accumulate in large numbers at the site of tissue damage. This "neutrophil cannibalism" is accompanied by inhibition of pro-inflammatory activities of these cells, such as respiratory burst and formation of neutrophil extracellular traps (NETs). Furthermore, efferocytosing neutrophils secrete anti-inflammatory mediators and mitogens including hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), vascular endothelial growth factors (VEGF), and transforming growth factor beta (TGFß). Thus, efferocytosis by neutrophils is involved in resolution of inflammation. Recent research indicates that it plays also a role in cancer. Many different solid tumours contain aggregates of dead tumour cells that have undergone spontaneous apoptosis. Their extent correlates with poor clinical outcome in most cancer types. These clusters of apoptotic tumour cells are strongly infiltrated by tumour-associated neutrophils (TANs) that acquired an anti-inflammatory and pro-resolving polarization state. This review summarizes the potential consequences discussed in the current literature. Although the picture of the role of efferocytosis by neutrophils in inflammation and cancer is becoming clearer, many questions are still unexplored.

Reducing Abdominal Aortic Aneurysm Progression by Blocking Neutrophil Extracellular Traps Depends on Thrombus Formation.

Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of abdominal aortic aneurysm (AAA), located in adventitia and intraluminal thrombus. We compared the therapeutic potential of targeting upstream or downstream effector molecules of NET formation in 2 murine AAA models based on angiotensin II or peri-adventitial elastase application. In both models, NETs were detected in formed aneurysms at treatment start. Although NET inhibitors failed in the elastase model, they prevented progression of angiotensin II-induced aneurysms with thrombus, which resembles established human disease (including thrombus development). Blockade of upstream NET mediators was more effective than interference with downstream NET molecules.

Plasma HMGB-1 after the initial dose of epirubicin/docetaxel in cancer.

BACKGROUND: The response of breast cancer patients to neoadjuvant chemotherapy (NCT) is highly heterogeneous, and reliable predictive instruments remain to be defined. High-mobility group box-1 (HMGB-1) protein is a cell death marker, which is easily detectable in plasma. We hypothesized that the initial dose of NCT with epirubicin/docetaxel induces changes in plasma HMGB-1 which could allow for an early prediction of response to therapy. MATERIALS AND METHODS: First, we analysed whether epirubicin/docetaxel releases HMGB-1 from HCC1143 breast cancer cells in vitro. Thereafter, plasma HMGB-1 levels before and 1-4 days after the first dose of epirubicin/docetaxel-based NCT were determined in 41 breast cancer patients and correlated with pathological response to treatment. RESULTS: Treatment of HCC1143 cells with epirubicin/docetaxel resulted in a significant HMGB-1 release in vitro. In vivo, HMGB-1 levels increased significantly only in responders (pathological complete response or partial remission, n = 22) but not in nonresponders (stable or progressive disease, n = 19). CONCLUSION: Our data suggest that early dynamic changes of plasma HMGB1 could be a promising biomarker to predict the final response to NCT in breast cancer patients.

Circulating caspase-cleaved cytokeratin 18 correlates with tumour burden and response to therapy in patients with colorectal cancer liver metastasis.

BACKGROUND AND AIMS: Systemic chemotherapy followed by hepatic resection is the treatment of choice for patients with colorectal cancer liver metastasis (CRCLM) but reliable biomarkers predicting response to therapy are needed. Spontaneous apoptosis of single tumour cells is common in CRCLM. We explored the potential of circulating apoptosis markers to predict treatment response. MATERIALS AND METHODS: Fifty-eight patients with CRCLM or hepatocellular carcinoma (HCC) were included in this study. Tumour tissue and blood samples were obtained before and after initiation of chemotherapy. Immunohistochemistry and ELISA assays were utilized to quantify the apoptosis marker caspase-cleaved cytokeratin 18 (M30) in tissue and circulation. RESULTS: CRCLM tissues showed more apoptotic tumour cells than HCC, or healthy liver. This was associated with elevated levels of circulating M30 (median = 244 U/l vs. 37 U/l in healthy controls, p = 0.009) which correlated with tumour volume (r2 = 0.92). Patients with progressive disease during chemotherapy showed higher M30 levels before therapy than responders (745 U/l vs. 136 U/l, p = 0.016). The predictive potential of M30 was higher than that of the tumour markers CA19-9 or CEA (AUC: 0.93, 0.63, and 0.78, respectively). CONCLUSIONS: Apoptotic tumour cells release cellular debris into the circulation, which provides information about tumour size and vitality.

Differential immunomodulatory effects of epirubicin/cyclophosphamide and docetaxel in breast cancer patients.

BACKGROUND: Epirubicin/cyclophosphamide (EC) and docetaxel (D) are commonly used in a sequential regimen in the neoadjuvant treatment of early, high-risk or locally advanced breast cancer (BC). Novel approaches to increase the response rate combine this treatment with immunotherapies such as PD-1 inhibition. However, the expected stimulatory effect on lymphocytes may depend on the chemotherapy backbone. Therefore, we separately compared the immunomodulatory effects of EC and D in the setting of a randomized clinical trial. METHODS: Tumor and blood samples of 154 patients from the ABCSG-34 trial were available (76 patients received four cycles of EC followed by four cycles of D; 78 patients get the reverse treatment sequence). Tumor-infiltrating lymphocytes, circulating lymphocytes and 14 soluble immune mediators were determined at baseline and at drug change. Furthermore, six BC cell lines were treated with E, C or D and co-cultured with immune cells. RESULTS: Initial treatment with four cycles of EC reduced circulating B and T cells by 94% and 45%, respectively. In contrast, no comparable effects on lymphocytes were observed in patients treated with initial four cycles of D. Most immune mediators decreased under EC whereas D-treatment resulted in elevated levels of CXCL10, urokinase-type plasminogen activator (uPA) and its soluble receptor (suPAR). Accordingly, only the exposure of BC cell lines to D induced similar increases as compared to E. While treatment of BC cells with E was associated with cell shrinkage and apoptosis, D induced cell swelling and accumulation of cells in G2 phase. CONCLUSION: The deleterious effect of EC on lymphocytes indicates strong immunosuppressive properties of this combination therapy. D, in contrast, has no effect on lymphocytes, but triggers the secretion of stimulatory proteins in vivo and in vitro, indicating a supportive effect on the immune system. Underlying differences in the induced cell death might be causal. These divergent immunomodulatory effects of epirubicin/cyclophosphamide and docetaxel should be considered when planning future combinations with immunotherapies in breast cancer.

Tumour cell apoptosis modulates the colorectal cancer immune microenvironment via interleukin-8-dependent neutrophil recruitment.

Sporadic apoptosis of tumour cells is a commonly observed feature of colorectal cancer (CRC) and strongly correlates with adverse patient prognosis. The uptake of apoptotic cell debris by neutrophils induces a non-inflammatory, pro-regenerative, and hence potentially pro-tumorigenic phenotype. In this study, we therefore sought to investigate the impact of apoptotic CRC cells on neutrophils and its consequence on other immune cells of the tumour microenvironment. Apoptosis induced by combined TNFα-treatment and UV-C irradiation, as well as various chemotherapeutic agents, led to a substantial release of neutrophil-attracting chemokines, most importantly interleukin-8 (IL-8), in both primary patient-derived and established CRC cells. Accordingly, conditioned media of apoptotic tumour cells selectively stimulated chemotaxis of neutrophils, but not T cells or monocytes. Notably, caspase-inhibition partially reduced IL-8 secretion, suggesting that caspase activity might be required for apoptosis-induced IL-8 release. Moreover, apoptotic tumour cell-conditioned media considerably prolonged neutrophil lifespan and induced an activated CD66bhighCD11bhighCD62Llow phenotype, comparable to that of tumour-associated neutrophils in CRC patients, as assessed by flow cytometry of dissociated CRC tissues. Immunohistochemical analyses of 35 CRC patients further revealed a preferential accumulation of neutrophils at sites of apoptotic tumour cells defined by the expression of epithelial cell-specific caspase-cleaved cytokeratin-18. The same areas were also highly infiltrated by macrophages, while T cells were virtually absent. Notably, neutrophils induced an M2-like CD86lowCD163+CD206+ phenotype in co-cultured monocyte-derived macrophages and suppressed LPS-induced pro-inflammatory cytokine release. In an in vitro transwell model, IL-8 blockade efficiently prevented neutrophil-induced anti-inflammatory macrophage polarisation by inhibiting neutrophil migration towards IL-8 gradients generated by apoptotic CRC cells. To conclude, our data suggest that apoptotic cancer cells release chemotactic factors that attract neutrophils into the tumour, where their interaction with neighbouring macrophages might promote an immunologically unfavourable tumour microenvironment. This effect may contribute to tumour recurrence after chemotherapy-induced apoptosis.

Metastatic colorectal carcinoma-associated fibroblasts have immunosuppressive properties related to increased IGFBP2 expression.

Fibroblasts are the most abundant stromal constituents of the tumour microenvironment in primary as well as metastatic colorectal cancer (CRC). Their supportive effect on tumour cells is well established. There is growing evidence that stromal fibroblasts also modulate the immune microenvironment in tumours. Here, we demonstrate a difference in fibroblast-mediated immune modulation between primary CRC and peritoneal metastasis. Cancer-associated fibroblasts (CAFs) were isolated from primary cancer and from peritoneal metastases (MAFs) from a total of 17 patients. The ectoenzyme CD38 was consistently expressed on the surface of all MAFs, while it was absent from CAFs. Furthermore, MAFs secreted higher levels of IGFBP2, CXCL2, CXCL6, CXCL12, PDGF-AA, FGFb, and IL-6. This was associated with a decreased activation of macrophages and a suppression of CD25 expression and proliferation of co-cultivated T-cells. Downregulation of IGFBP2 abolished these immunosuppressive effects of MAFs. Taken together, these results show that MAFs contribute to an immunosuppressive tumour microenvironment in CRC metastases by modulating the phenotype of immune cells through an IGFBP2-dependent mechanism.

Stromal fibroblasts shape the myeloid phenotype in normal colon and colorectal cancer and induce CD163 and CCL2 expression in macrophages.

Colorectal cancer (CRC) accounts for about 10% of cancer deaths worldwide. Colon carcinogenesis is critically influenced by the tumor microenvironment. Cancer associated fibroblasts (CAFs) and tumor associated macrophages (TAMs) represent the major components of the tumor microenvironment. TAMs promote tumor progression, angiogenesis and tissue remodeling. However, the impact of the molecular crosstalk of tumor cells (TCs) with CAFs and macrophages on monocyte recruitment and their phenotypic conversion is not known in detail so far. In a 3D human organotypic CRC model, we show that CAFs and normal colonic fibroblasts are critically involved in monocyte recruitment and for the establishment of a macrophage phenotype, characterized by high CD163 expression. This is in line with the steady recruitment and differentiation of monocytes to immunosuppressive macrophages in the normal colon. Cytokine profiling revealed that CAFs produce M-CSF, and IL6, IL8, HGF and CCL2 secretion was specifically induced by CAFs in co-cultures with macrophages. Moreover, macrophage/CAF/TCs co-cultures increased TC invasion. We demonstrate that CAFs and macrophages are the major producers of CCL2 and, upon co-culture, increase their CCL2 production twofold and 40-fold, respectively. CAFs and macrophages expressing high CCL2 were also found in vivo in CRC, strongly supporting our findings. CCL2, CCR2, CSF1R and CD163 expression in macrophages was dependent on active MCSFR signaling as shown by M-CSFR inhibition. These results indicate that colon fibroblasts and not TCs are the major cellular component, recruiting and dictating the fate of infiltrated monocytes towards a specific macrophage population, characterized by high CD163 expression and CCL2 production.

Identification of Non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.

The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.

Mass spectrometrical analysis of the mitochondrial carrier Aralar1 from mouse hippocampus.

Aralar1 is a mitochondrial aspartate/glutamate carrier and a key component of the malate-aspartate NADH shuttle system. An analytical approach to obtain high sequence coverage is important to predict conformation, identify splice variants and binding partners or generate specific antibodies. Moreover, a method allowing determination of Aralar1 from brain samples is a prerequisite for evaluating a biological role. Sucrose gradient ultracentrifugation was applied to enrich native membrane protein fractions and these were run on blue-native PAGE, followed by multidimensional gel electrophoresis. Spots from the third-dimensional gel electrophoresis were in-gel digested with trypsin, chymotrypsin and subtilisin. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using collision-induced dissociation and electron transfer dissociation modes. Modiro v1.1 along with Mascot v2.2 software was used for data handling. Aralar1 could be clearly separated, unambiguously identified and characterized from protein extracts of mouse hippocampus by the use of the multidimensional gel electrophoretic steps. The combined sequence coverage of Aralar1 from trypsin, chymotrypsin and subtilisin digestions was 99.85%. The results provide the basis for future studies of Aralar1 at the protein chemical rather than at the immunochemical level in the brain and thus challenge and enable determination of Aralar1 levels required for understanding biological functions in health and disease.

The role of neutrophil death in chronic inflammation and cancer.

The lifespan of a neutrophil is short and limited by programmed cell death, followed by efferocytosis. When activated or exposed to insult, neutrophil death may be delayed to support neutrophil effector functions such as phagocytosis, cytokine release, and pathogen destruction by degranulation. However, neutrophils may also alter the type of cell death and thereby affect inflammatory responses and tissue remodeling. This review briefly introduces the various forms of neutrophil death including apoptosis, necrosis/necroptosis, and the formation of so-called "neutrophil extracellular traps" (NETs), and it summarizes the clearance of dead cells by efferocytosis. Importantly, distinct types of neutrophil death have been found to drive chronic inflammatory disorders and cancer. Thus, the tumor and its microenvironment can delay neutrophil apoptosis to exploit their pro-angiogenic and pro-metastatic properties. Conversely, neutrophils may enter rapid and suicidal cell death by forming extracellular traps, which are expelled DNA strands with neutrophil proteins. Components of these DNA-protein complexes such as histones, high-mobility group protein B1, or neutrophil elastase have been found to promote cancer cell proliferation, adhesion, migration, invasion, and thereby tumor metastasis. In other settings of chronic inflammatory disease such as gout, NETs have been found protective rather than detrimental, as they promoted the local degradation of pro-inflammatory cytokines by neutrophil proteases. Thus, the interaction of neutrophils with the tissue environment extends beyond the stage of the living cell and the type of neutrophil death shapes immune responses and tissue remodeling in health and disease.